Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
DSpace
UBCV
Bahreyni, Amirhossein
Doctor of Philosophy - PhD
University of British Columbia
Breast cancer poses a significant global challenge, impacting millions worldwide. Despite significant progress in understanding and treating it, there is still a critical need for more effective approaches due to the heterogeneity of breast cancer. Our lab previously developed a safe variant of Coxsackievirus B3 (CVB3), namely microRNA-modified CVB3 (miR-CVB3), with remarkable antitumor activity against lung cancer. In this thesis, I aimed to assess the safety and efficacy of miR-CVB3 in breast cancer therapy. Additionally, I sought to develop a safe platform capable of delivering multiple therapeutic agents, including miR-CVB3, to breast cancer cells. My hypothesis is that miR-CVB3, encapsulated into modified extracellular vesicles, is an excellent candidate for development into a novel oncolytic virotherapy for breast cancer. When applied, miR-CVB3 utilized miR sequences to selectively target cancerous cells while sparing healthy tissue. In vivo experiments revealed promising outcomes, with miR-CVB3 effectively reducing tumor size in mice bearing 4T1 breast tumors. Moreover, miR-CVB3 treatment led to increased immune cell infiltration within the tumor microenvironment, all while maintaining superior safety compared to its parent virus, CVB3. Furthermore, combining miR-CVB3 with additional therapeutic elements such as melittin and CpG sequences significantly enhanced immunogenic cell death in tumor cells. This combination therapy not only enhanced immune cell infiltration but also exhibited efficacy against both primary and distant tumors. Additionally, I showed that miR-CVB3 was able to modify proteins within cancer cells and exosomes, transforming them into immunostimulatory molecules and generating a pro-immune environment within the tumor. To optimize delivery, I harnessed exosomes as carriers for miR-CVB3. These exosomes, released from miR-CVB3-infected cancer cells, contained miR-CVB3 and efficiently transported it to other breast cancer cells. Further modification of miR-CVB3-encapsulating exosomes with the AS1411 aptamer and doxorubicin significantly improved treatment safety and efficacy in 4T1-tumor bearing mice. In summary, my thesis suggests that combining miR-CVB3 with immune enhancers and advanced delivery methods could reduce breast tumor growth and improve survival in 4T1 tumor-bearing mice. This approach represents a significant step forward in breast cancer treatment, potentially leading to more effective therapies.
DEVELOPMENT OF A SAFE TREATMENT FOR BREAST CANCER USING ONCOLYTIC COXSACKIEVIRUS B3 by Amirhossein Bahreyni B.Sc., Mashhad Azad University, 2014 M.Sc., Mazandaran University of Medical Sciences, 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2024 © Amirhossein Bahreyni, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Development of a safe treatment for breast cancer using an oncolytic Coxsackievirus B3 submitted by Amirhossein Bahreyni in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pathology and Laboratory Medicine Examining Committee: Dr. Honglin Luo, Professor, Pathology and Laboratory Medicine, UBC Supervisor Dr. Jayachandran Kizhakkedathu, Professor, Pathology and Laboratory Medicine, UBC Supervisory Committee Member Dr. Pascal Bernatchez, Associate Professor, Anesthesiology, Pharmacology and Therapeutics, UBC Supervisory Committee Member Dr. Xiaoyan Jiang , Professor , Department of Medical Genetics, UBC University Examiner Dr. Christopher Maxwell , Professor, Department of Pediatrics, UBC University Examiner Additional Supervisory Committee Members: Dr. Xuesen Dong , Associate Professor, Department of Urologic Sciences, UBC Supervisory Committee Member iii Abstract Breast cancer poses a significant global challenge, impacting millions worldwide. Despite significant progress in understanding and treating it, there is still a critical need for more effective approaches due to the heterogeneity of breast cancer. Our lab previously developed a safe variant of Coxsackievirus B3 (CVB3), namely microRNA-modified CVB3 (miR-CVB3), with remarkable antitumor activity against lung cancer. In this thesis, I aimed to assess the safety and efficacy of miR-CVB3 in breast cancer therapy. Additionally, I sought to develop a safe platform capable of delivering multiple therapeutic agents, including miR-CVB3, to breast cancer cells. My hypothesis is that miR-CVB3, encapsulated into modified extracellular vesicles, is an excellent candidate for development into a novel oncolytic virotherapy for breast cancer. When applied, miR-CVB3 utilized miR sequences to selectively target cancerous cells while sparing healthy tissue. In vivo experiments revealed promising outcomes, with miR-CVB3 effectively reducing tumor size in mice bearing 4T1 breast tumors. Moreover, miR-CVB3 treatment led to increased immune cell infiltration within the tumor microenvironment, all while maintaining superior safety compared to its parent virus, CVB3. Furthermore, combining miR-CVB3 with additional therapeutic elements such as melittin and CpG sequences significantly enhanced immunogenic cell death in tumor cells. This combination therapy not only enhanced immune cell infiltration but also exhibited efficacy against both primary and distant tumors. Additionally, I showed that miR-CVB3 was able to modify proteins within cancer cells and exosomes, transforming them into immunostimulatory molecules and generating a pro-immune environment within the tumor. To optimize delivery, I harnessed exosomes as carriers for miR-CVB3. These exosomes, released from miR-CVB3-infected cancer cells, contained miR-CVB3 and efficiently transported it to other breast cancer cells. Further modification of miR-CVB3-encapsulating exosomes with the AS1411 iv aptamer and doxorubicin significantly improved treatment safety and efficacy in 4T1-tumor bearing mice. In summary, my thesis suggests that combining miR-CVB3 with immune enhancers and advanced delivery methods could reduce breast tumor growth and improve survival in 4T1 tumor-bearing mice. This approach represents a significant step forward in breast cancer treatment, potentially leading to more effective therapies. v Lay Summary Breast cancer, a widespread challenge, drives the ongoing quest for improved treatments. A promising development is the coxsackievirus B3 (CVB3), which has been modified to combat cancer. My research focused on assessing safety and efficacy of this modified CVB3 in breast cancer therapy, emphasizing targeted delivery to cancer cells while sparing healthy ones. Results demonstrated the virus's precision in selectively targeting cancer cells and leaving healthy cells unharmed. In mice with breast tumors, modified CVB3 not only reduced tumor size but also enhanced the immune response. Combining the virus with other therapeutics amplified the immune attack on cancer, significantly suppressing tumor growth. Additionally, modified CVB3 altered cancer cell proteins, stimulating the immune system. Utilizing vesicles as carriers for the virus facilitated efficient delivery, with further refinements enhancing safety and efficacy in mice. This innovative approach holds promise for advancing breast cancer treatments, offering potential for improved outcomes in the future. vi Preface In this dissertation, I presented the culmination of my original research endeavors conducted during my doctoral training under the expert guidance of Dr. Honglin Luo. The insightful input from my excellent supervisory committee members further enriched the outcomes. The body of work encapsulates two review articles and three original research papers, all crafted during my tenure as either the first or co-first author at UBC. In Chapter 1, the foundation is derived from two review articles: [Bahreyni A, Mohamud Y, Luo H. Emerging nanomedicines for effective breast cancer immunotherapy. Journal of Nanobiotechnology. 2020; doi: 10.1186/s12951-020-00741-z] and [Bahreyni A, Mohamud Y, Luo H. Oncolytic virus-based combination therapy in breast cancer. Cancer Letters. 2024; doi: 10.1016/j.canlet.2024.216634]. I conducted an extensive literature review and crafted the manuscript, contributing to about 90% of the writing. YM drafted the autophagy section. Notably, the manuscript received invaluable input from Dr. Honglin Luo, who meticulously edited it. Chapter 2 is based on a manuscript [Bahreyni A#, Liu H# (# co-first author), Xue YC, Mohamud Y, Zhang J, Luo H. A new miRNA-modified coxsackievirus B3 inhibits triple-negative breast cancer growth with improved safety profile in immunocompetent mice. Cancer Letters. 2022, doi: 10.1016/j.canlet.2022.215849]. I contributed approximately 50% of the research presented in this paper, with all data discussed in this chapter originating from my work on this manuscript. HLiu participated in the animal studies, while YM, YCX and JZ were involved in evaluating the efficacy of miR-CVB3 in vitro. I drafted the original manuscript. All authors contributed to the discussion, analyzed the results, and approved the final manuscript. HLuo conceptualized the project with me and edited the manuscript. vii Chapter 3 is derived from the manuscript [Bahreyni A, Liu H, Mohamud Y, Xue YC, Fan YM, Zhang YL, Luo H. A combination of genetically engineered oncolytic virus and melittin-CpG for cancer viro-chemo-immunotherapy. BMC Medicine 2023, doi: 10.1186/s12916-023-02901-y]. I performed the majority of the experiments and analyzed the data. HLiu contributed to the animal studies, while YM, YCX, YMF, and YLZ were involved in evaluating the efficacy of the developed treatment in vitro. I drafted the original manuscript and all authors participated in the discussion, analyzed the results, and approved the final manuscript. HLuo conceptualized the project with me and edited the manuscript. Chapter 4 is built upon a manuscript [Bahreyni A, Mohamud Y, Ahraf SN, Zhang YL, Luo H. Synergistic viro-chemoimmunotherapy in breast cancer enabled by bioengineered immunostimulatory exosomes and dual-targeted coxsackievirus B3. ACS Nano. https://doi.org/10.1021/acsnano.3c09491]. I conducted most of the experiments and analyzed the data. YM, SAN, and YLZ contributed to the in vitro studies. I drafted the original manuscript and all authors participated in the discussion, analyzed the results, and approved the final manuscript. HL conceptualized the project with me and edited the manuscript. All animal protocols were strictly adhered to the highest ethical standards for the treatment and use of laboratory animals. These protocols were approved by the Animal Care Committee at the University of British Columbia (A18-0275 and A22-0237). All experiments with live viruses were performed in compliance with Biosafety Protocols B14-0190 and B19-0145, approved by the University of British Columbia's Research Ethics Board. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................... xxi Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ................................................................................................................1 1.1 Breast cancer......................................................................................................................... 1 1.1.1 Classification ................................................................................................................. 1 1.1.2 Current treatments and limitations ................................................................................ 3 1.2 Cancer cells and host immune responses: mechanism of immunosurveillance ................... 4 1.3 Oncolytic viruses .................................................................................................................. 5 1.3.1 Mechanisms of actions .................................................................................................. 6 1.3.2 Oncolytic virus in breast cancer .................................................................................... 9 1.3.3 Oncolytic virus-based combination therapy in breast cancer, preclinical studies ...... 11 1.3.3.1 Vaccinia virus ...................................................................................................... 15 1.3.3.2 Herpes simplex virus type 1 ................................................................................ 17 1.3.3.3 Human adenovirus ............................................................................................... 18 1.3.3.4 Newcastle disease virus ....................................................................................... 19 ix 1.3.3.5 Vesicular stomatitis virus .................................................................................... 20 1.3.3.6 Reovirus ............................................................................................................... 20 1.3.3.7 Measles virus ....................................................................................................... 21 1.3.3.8 Coxsackievirus..................................................................................................... 22 1.4 Exosome… ......................................................................................................................... 27 1.4.1 Exosome biosynthesis ................................................................................................. 27 1.4.2 Exosomes in drug delivery .......................................................................................... 28 1.5 Rationale, Hypothesis and Specific Aims .......................................................................... 29 Chapter 2: A new miRNA-modified coxsackievirus B3 inhibits triple-negative breast cancer growth with improved safety profile in immunocompetent mice ............................................33 2.1 Background ......................................................................................................................... 33 2.2 Materials and methods ........................................................................................................ 33 2.2.1 Cell lines and culture ................................................................................................... 33 2.2.2 Virus propagation ........................................................................................................ 34 2.2.3 Cell viability assay ...................................................................................................... 34 2.2.4 Reverse transcription- quantitative polymerase chain reaction .................................. 35 2.2.5 Western blot analysis .................................................................................................. 36 2.2.6 Animal studies ............................................................................................................. 36 2.2.7 Immunohistochemistry ................................................................................................ 37 2.2.8 Hematoxylin and Eosin (H&E) staining ..................................................................... 37 2.2.9 Statistical analysis ....................................................................................................... 38 2.3 Results….. .......................................................................................................................... 38 2.3.1 miR-CVB3 shows strong lytic potency towards various breast cancer cells .............. 38 x 2.3.2 MCF-7 cells express a low level of coxsackievirus and adenovirus receptor ............ 42 2.3.3 Intratumoral injection of miRNA-CVB3 impedes tumor growth in TNBC-bearing mice with high safety profile .................................................................................................... 45 2.3.4 miR-CVB3 promotes immune cell infiltration into the tumor microenvironment and prevents tumor metastasis when given intratumorally ............................................................. 48 2.4 Discussion.. ......................................................................................................................... 51 Chapter 3: Combination of miR-CVB3 with chemo and immunotherapeutic drugs improves breast tumor suppression ............................................................................................................55 3.1 Background ......................................................................................................................... 55 3.2 Materials and methods ........................................................................................................ 57 3.2.1 Cell culture .................................................................................................................. 57 3.2.2 Virus propagation ........................................................................................................ 58 3.2.3 Preparation and characterization of CpGMel .............................................................. 58 3.2.4 Cellular uptake of miR-CVB3 and CpGMel ............................................................... 58 3.2.5 In vitro anti-cancer study ............................................................................................ 59 3.2.5.1 Cell viability assay............................................................................................... 59 3.2.5.2 Apoptosis detection ............................................................................................. 60 3.2.5.3 Detection of danger-associated molecular patterns ............................................. 60 3.2.5.4 Reverse transcription-quantitative polymerase chain reaction ............................ 61 3.2.5.5 Macrophage activation ........................................................................................ 61 3.2.6 In vivo anti-tumor study .............................................................................................. 62 3.2.6.1 Animals ................................................................................................................ 62 3.2.6.2 Therapeutic effects in a murine breast cancer model .......................................... 62 xi 3.2.6.3 Safety analysis ..................................................................................................... 63 3.2.6.4 Immune cell infiltration ....................................................................................... 63 3.2.6.5 Inhibition of distant metastatic tumor .................................................................. 64 3.2.7 Statistical analysis ....................................................................................................... 64 3.3 Results….. .......................................................................................................................... 65 3.3.1 Characterization of CpGMel and miR-CVB3+CpGMel ............................................ 65 3.3.2 Viral replication and uptake of CpG ........................................................................... 65 3.3.3 Cell viability and apoptosis induction ......................................................................... 69 3.3.4 In vitro induction of DAMP release and macrophage maturation .............................. 69 3.3.5 In vivo anti-tumor therapy and safety analysis ............................................................ 74 3.3.6 In vivo assessment of inflammatory response and immune cell infiltration ............... 78 3.3.7 Growth inhibition of distant and metastatic tumor ..................................................... 78 3.4 Discussion …………………………………………………………………..……………..82 Chapter 4: Synergistic viro-chemoimmunotherapy in breast cancer enabled by bioengineered immunostimulatory exosomes and dual-targeted coxsackievirus B3 ............85 4.1 Background ......................................................................................................................... 85 4.2 Materials and methods ........................................................................................................ 87 4.2.1 Cell culture .................................................................................................................. 87 4.2.2 Animals.. ..................................................................................................................... 87 4.2.3 Virus propagation ........................................................................................................ 87 4.2.4 Isolation of Exo and ExomiR-CVB3 .......................................................................... 87 4.2.5 Generation of ExomiR-CVB3/Dox ............................................................................. 88 4.2.6 Generation of ExomiR-CVB3/DoxApt ....................................................................... 89 xii 4.2.7 Assessment of the impact of miR-CVB3 on immunological proteins in breast cancer cells and exosomes released by breast cancer cells .................................................................. 89 4.2.8 Analysis of the effect of miR-CVB3 on immune-related proteins in the tumor microenvironment and exosomes secreted by tumor cells in mice .......................................... 91 4.2.9 MiR-CVB3 and ExomiR-CVB3 comparison ............................................................. 92 4.2.9.1 Cell internalization .............................................................................................. 92 4.2.9.2 Cell viability ........................................................................................................ 92 4.2.9.3 Stimulation of immune cells ................................................................................ 93 4.2.9.4 Antitumor activity................................................................................................ 94 4.2.10 Examination of cellular uptake and in vitro cytotoxicity of ExomiR-CVB3/DoxApt…. ..................................................................................................................... 96 4.2.11 Assessment of the performance of ExomiR-CVB3/DoxApt .................................... 96 4.2.12 Statistical analysis ..................................................................................................... 97 4.3 Results…… ........................................................................................................................ 97 4.3.1 Infection with miR-CVB3 leads to a reduction in immune checkpoints and 'don't eat me' signals, while concurrently boosting 'eat me' and 'find me' signals ................................... 97 4.3.2 MiR-CVB3 infection not only amplifies the production and release of Exo from cancer cells but also augments their immunostimulatory potential ......................................... 98 4.3.3 Exosomes released by cancer cells after miR-CVB3 infection can serve as a potential carrier for miR-CVB3 .............................................................................................................. 99 4.3.4 ExomiR-CVB3 is able to infect cells that lack the coxsackievirus and adenovirus receptor and the decay-accelerating factor ............................................................................. 103 xiii 4.3.5 ExomiR-CVB3 triggers a more pronounced activation of immune cells in comparison to unbound miR-CVB3 .......................................................................................................... 104 4.3.6 ExomiR-CVB3 exhibits remarkable antitumor efficacy and effectively mobilizes immune cells to infiltrate the tumor microenvironment ......................................................... 108 4.3.7 Exosomes provide a protective shield for miR-CVB3, mitigating its swift clearance by immune cells following systemic administration .............................................................. 112 4.3.8 Further modification of ExomiR-CVB3 increases its specificity and potency against cancer cells………………………………………………………………………..…………116 4.3.9 ExomiR-CVB3/DoxApt exhibits potent tumor-targeting efficacy in mice bearing breast tumors…… .................................................................................................................. 120 4.4 Discussion.. ....................................................................................................................... 124 Chapter 5: Closing Remarks.....................................................................................................127 5.1 Research summary and conclusions ................................................................................. 127 5.2 Conclusion ........................................................................................................................ 128 5.3 Limitation and future direction ......................................................................................... 129 5.3.1 Animal model specificity and diversity .................................................................... 129 5.3.2 Translational challenges and human complexity ...................................................... 129 5.3.3 Long-term effects and relapse dynamics................................................................... 129 5.3.4 Immunological considerations and the tumor microenvironment ............................ 130 5.4 Significance ...................................................................................................................... 131 Bibliography ...............................................................................................................................133 xiv List of Tables Table 1-1. Synergistic Approaches in Breast Cancer Treatment using Oncolytic Viruses with Multimodal Therapies ................................................................................................................... 13 Table 2-1. The primer pairs used for viral RNA, Human IFNB1 and Murine Ifnb1 .................... 35 Table 3-1. Designed primers for RT-qPCR .................................................................................. 61 Table 4-1. Primers used for the RT-qPCR analysis ...................................................................... 94 xv List of Figures Figure 1-1. Four common subtypes of breast cancer. ..................................................................... 2 Figure 1-2. Dual mechanisms of oncolytic virus action in cancer treatment. ................................. 8 Figure 1-3. The importance of combination therapy in addressing the challenge of heterogeneous breast tumors. ................................................................................................................................ 12 Figure 1-4. microRNA-detargeting strategy to improve safety profile of oncolytic virus. .......... 26 Figure 2-1. miR-CVB3 potently kills various breast cancer cells through replication ................. 41 Figure 2-2. Human breast cancer MCF-7 cells express low protein level of viral receptor CAR but display the highest Ifnb1 gene expression in response to polyIC treatment. ................................ 44 Figure 2-3. miR-CVB3 limits breast cancer growth in 4T1 tumor-bearing Balb/c mice with high safety profile ................................................................................................................................. 47 Figure 2-4. miR-CVB3 replicates in tumor cells, facilitates immune cell infiltration into the tumor microenvironment, and prevents tumor metastasis into the lung. ................................................ 50 Figure 3-1. CpGMel does not affect miR-CVB3 internalization and replication in cancer cells, while miR-CVB3 enhances internalization of CpGMel to the cancer cells. ................................ 68 Figure 3-2. miR-CVB3+CpGMel significantly increases apoptosis, DAMPs release and macrophage activation compared with miR-CVB3 and CpGMel. ............................................... 72 Figure 3-3. miR-CVB3+CpGMel significantly inhibits tumor growth in 4T1 breast tumor-bearing Balb/c mice. .................................................................................................................................. 77 Figure 3-4. miR-CVB3+CpGMel increases immune cell infiltration into the tumor microenvironment and reduces distant tumor growth and tumor metastasis into lungs. .............. 81 Figure 4-1. miR-CVB3 infection of breast cancer cells leads to altered expression levels of immunomodulatory proteins in both cell lysates and released exosomes. ................................. 102 xvi Figure 4-2. ExomiR-CVB3 outperforms free miR-CVB3, efficiently infecting cells with low viral receptor CAR and DAF expression, and stimulating more effective immune cell activations. . 107 Figure 4-3. ExomiR-CVB3 demonstrates enhanced antitumor efficacy compared to free miR-CVB3 and effectively recruits immune cells to infiltrate the tumor microenvironment. ........... 111 Figure 4-4. Exosomes provide a protective shield for miR-CVB3, preventing its interaction with neutralizing antibodies and subsequent elimination by immune cells. ....................................... 115 Figure 4-5. Further modification of ExomiR-CVB3 with the AS1411 aptamer and Doxorubicin enhances its specificity and cytotoxic potential. ......................................................................... 119 Figure 4-6. The systemic administration of ExomiR-CVB3/DoxApt markedly diminishes tumor growth, enhances survival rates, and amplifies the infiltration of immune cells into the tumor microenvironment. ...................................................................................................................... 123 xvii List of Abbreviations Ad: Adenovirus ALT: Alanine Aminotransferase ANOVA: Analysis of Variance AST: Aspartate Aminotransferase ATCC: American Type Culture Collection ATP: Adenosine Triphosphate BW: Body Weight CAR-T: Chimeric Antigen Receptor-T cell CAR: Coxsackievirus and Adenovirus Receptor CD4: Cluster of Differentiation 4 CD8: Cluster of Differentiation 8 CTLA-4: Cytotoxic T-Lymphocyte-Associated Protein 4 CVA21: Coxsackievirus A21 CVB3: Coxsackievirus B3 CXCR: C-X-C Chemokine Receptor DAMPs: Danger-Associated Molecular Patterns DMEM: Dulbecco's Modified Eagle Medium DNA: Deoxyribonucleic Acid DCs: Dendritic Cells ESCRT: Endosomal Sorting Complex Required for Transport EGFR: Epidermal Growth Factor Receptor xviii ER: Estrogen Receptor Exo: Exosomes FDA: U.S. Food and Drug Administration FITC: Fluorescein Isothiocyanate GFRα1: Glial Cell-Derived Neurotrophic Factor Receptor Alpha 1 GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor H&E: Hematoxylin and Eosin HMGB1: High-Mobility Group Box 1 HSV: Herpes Simplex Virus HER2: Human Epidermal Growth Factor Receptor 2 HR+: Hormone Receptor Positive ICD: Immunogenic Cell Death IFN-γ: Interferon Gamma IFNB1: Interferon Beta-1 IFN-I: Type I interferon IL-12: Interleukin 12 IL-15: Interleukin 15 IL-15Rα: Interleukin 15 Receptor Subunit Alpha IL-24: Interleukin 24 ILV: Intraluminal Vesicles IHC: Immunohistochemistry miR: MicroRNA miR-CVB3: MicroRNA-Modified Coxsackievirus B3 xix MOI: Multiplicity of Infection MTS:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium MVB: Multivesicular Body NDV: Newcastle Disease Virus NK: Natural Killer NT: Nucleotide ODN: Oligodeoxynucleotides OVs: Oncolytic Viruses PAMPs: Pathogen-Associated Molecular Patterns PBS: Phosphate-buffered saline PD-1: Programmed Cell Death Protein 1 PFU: Plaque Forming Units PR: Progesterone Receptor PRRs: Pattern Recognition Receptors RNA: Ribonucleic Acid SD: Standard Deviation SEM: Standard Error of the Mean STAT3: Signal Transducer and Activator of Transcription 3 T-VEC: Talimogene Laherparepvec TAA: Tumor-Associated Antigen TAN: Tumor Antigen Neoantigen TLR9: Toll-Like Receptor 9 xx TNBC: Triple-Negative Breast Cancer TNF-α: Tumor Necrosis Factor Alpha TME: Tumor Microenvironment TSR: Tumor Suppression Rate TUNEL: Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling UTR: Untranslated Region VSV: Vesicular Stomatitis Virus WT: Wild-Type xxi Acknowledgements I am profoundly grateful to everyone who has contributed to the completion of this thesis. Their guidance, support, and encouragement have been invaluable throughout this journey. First and foremost, I extend my deepest gratitude to my supervisor, Dr. Honglin Luo. Her exceptional mentorship, dedication, and expertise have been the cornerstone of my academic and research endeavors. Dr. Luo's insightful guidance not only provided direction for my research but also instilled in me a deep sense of curiosity and critical thinking. Her unwavering support and encouragement empowered me to overcome challenges and pursue excellence in my work. Beyond her role as a supervisor, Dr. Luo has been a source of inspiration and a role model for me. Her passion for research, commitment to academic rigor, and integrity in scholarly pursuits have left an indelible mark on my academic journey. Her mentorship has shaped not only my research skills but also my professional development and personal growth. I would like to express my appreciation to the members of my thesis committee, Dr. Chun Seow, Dr. Jayachandran Kizhakkedathu, Dr. Pascal Bernatchez and Dr. Xuesen Dong for their insightful feedback and constructive criticism. Their expertise and dedication have played a significant role in refining this work and ensuring its quality. I am indebted to my lab members, Dr. Yasir Mohamud, Dr. Yuan Chao Xue, Dr. Huitao Liu, Dr. John Zhang, Sanaz Ashraf Nouhegar, Lyanne Zhang, Michelle Fan, Cathy Fu, Carly Lin, Wendy Hwang, Razieh Sadat Banijamali, Claire Wang and Bob Lin for their camaraderie, collaboration, and willingness to share their knowledge and expertise. Their contributions have enriched my research experience and made the process both enjoyable and rewarding. xxii I am also grateful to the individuals at the Heart Lung Innovation Center for their support and collaboration throughout this project. Their expertise and resources have been invaluable in advancing my research and expanding its impact. Finally, I would like to express my deepest gratitude to my parents for their unwavering love, encouragement, and sacrifices. Their endless support and belief in my abilities have been my source of strength throughout this journey. I am forever grateful for their guidance and inspiration. To all those mentioned above and to those who have supported me in ways beyond words, thank you for being part of this journey and for helping me reach this milestone in my academic career. xxiii Dedication To my lovely family1 Chapter 1: Introduction 1.1 Breast cancer Breast cancer is a type of cancer that develops in the breast tissue, typically starting from the cells in the milk ducts or lobules. This disease is marked by the unchecked proliferation of abnormal cells within the breast, which may infiltrate neighboring tissues and have the potential to metastasize to distant parts of the body (1). Breast cancer continues to be the most frequently diagnosed malignancy, representing approximately 12% of all cancer diagnoses (2). It accounts as the second leading cause of cancer-related fatalities among women, claiming over 600,000 lives worldwide each year (3). Notably, high-income countries exhibit a higher incidence of this disease compared to low- to middle-income countries (4). It is projected that by 2030, the global number of new cases will reach 3.9 million, while fatalities are expected to increase to 766,000 (5). 1.1.1 Classification As a heterogeneous disease, breast cancer exhibits a notable degree of diversity both in their phenotypic characteristics and molecular profiles (6). It is characterized by a widely employed clinical classification based on the expression of hormone receptors (estrogen receptor, ER, and progesterone receptor, PR) and the oncogenic receptor tyrosine kinase ERBB2, erythroblastic oncogene B, (also known as HER2) (6). Four common subtypes of breast cancer are (Fig. 1-1): Luminal A: This subtype is characterized by the presence of hormone receptors (ER+ and/or PR+), and it lacks HER2 overexpression (HER2-). Luminal A tumors tend to grow slowly and often have a more favorable prognosis. Luminal B: Similar to Luminal A, Luminal B breast cancer is ER and/or PR positive, but it also shows HER2 overexpression (HER2+). It is generally more aggressive than Luminal A. 2 HER2-enriched (HER2+): In this subtype, breast cancer cells overexpress HER2 but do not express ER or PR. HER2+ breast cancers are typically aggressive but can be targeted effectively with therapies like trastuzumab. Basal-like: This subtype, known as triple-negative breast cancer (TNBC), lacks expression of ER, PR, and HER2. TNBC is aggressive and presents challenges due to limited targeted treatment options (7, 8). Figure 1-1. Four common subtypes of breast cancer. Breast cancerBasal like(15-20%)HER2 enriched(10-15%)Luminal B(~20%)Luminal A(~40%)HR + HER2 - HR + HER2 + HR - HER2 + HR - HER2 - 3 Each subtype of breast cancer has its distinct clinical characteristics and associated risk factors. These differences extend to treatment responses and long-term patient survival, which can vary significantly among the subtypes. This complexity introduces additional challenges when it comes to accurately diagnosing and effectively treating breast cancer (4). 1.1.2 Current treatments and limitations Early detection greatly enhances the curability of breast cancer. In the preoperative phase, systemic endocrine or immunotherapies are employed, primarily for cases positive for ER, PR, or HER2. Preoperative chemotherapy becomes the only option when tumors lack all three receptors. Following surgery, the postoperative phase incorporates a comprehensive approach encompassing radiation, endocrine therapy, immunotherapy, and chemotherapy (9, 10). Traditional chemotherapy and immunotherapy, as the main options for cancer treatment, have some significant drawbacks (11). One key issue is that they lack precision, which means they can harm healthy cells along with cancer cells. This imprecision can lead to a range of undesirable side effects like nausea, hair loss, fatigue, and a weakened immune system (12, 13). Another problem is the narrow therapeutic window, where the difference between an effective dose and a harmful one is very small, making it difficult for doctors to manage. Additionally, the development of drug resistance is a notable issue, particularly with conventional chemotherapy (14). Over time, cancer cells can adapt and become less responsive to chemotherapy drugs, rendering these treatments less effective (15). This not only poses a hurdle in the management of the disease but also limits the options available to patients. Given these challenges, ongoing research and advancements in cancer treatment focus on developing more targeted therapies with improved specificity, reduced side effects, and enhanced efficacy (16, 17). These innovative approaches aim to not only address the shortcomings of 4 conventional chemotherapy but also provide patients with more effective and tolerable treatment options, ultimately improving their quality of life and long-term outcomes. 1.2 Cancer cells and host immune responses: mechanism of immunosurveillance Immune editing within the context of cancer is an intricate process that characterizes the dynamic interplay between an individual's immune system and cancerous cells. It contains three phases: elimination, equilibrium, and escape. Elimination Phase (also called immunosurveillance): In this initial stage, the immune system actively identifies and eradicates cancer cells. Specialized immune cells, such as cytotoxic T cells and natural killer (NK) cells, discern cancer cells as aberrant due to the presence of specific antigens on the surface of cancerous cells. This recognition triggers a robust immune response aimed at eliminating the tumor. The elimination phase is often successful, leading to the eradication of many incipient cancers before they manifest clinically. Equilibrium Phase: Subsequently, certain cancer cells may evade elimination and enter a phase referred to as equilibrium. Within this stage, a delicate balance forms between the immune system and the cancer cells, restraining further tumor growth. Nevertheless, the immune system continues to exert selective pressure on the cancer cells, potentially fostering the development of immune-resistant variants. Escape Phase: In the final stage, escape, cancer cells deploy various tactics to circumvent the immune system's detection. These strategies encompass the downregulation of antigen presentation, the creation of an immunosuppressive microenvironment within the tumor, and even disguising themselves as normal, healthy cells. Consequently, the immune system becomes less effective in recognizing and combating these cancer cells, ultimately enabling the tumor to proliferate and metastasize (18, 19). Cancer immunotherapy is a revolutionary treatment approach that mobilizes the body's own immune system to combat cancer. It includes various strategies, including immune checkpoint inhibitors, 5 adoptive cell therapy, and cancer vaccines, which have shown promise in treating different types of cancer (20). However, not all cancers respond equally to immunotherapy, with some, like breast cancer, being considered "cold" tumors and less responsive (21). In addressing this challenge, oncolytic viruses (OVs) have emerged as a promising approach, as they can selectively infect and destroy cancer cells while activating the immune system. In breast cancer, OVs have the potential to transform "cold" tumors into "hot" ones, making them more responsive to immunotherapy (22). This combination approach represents an exciting frontier in enhancing the effectiveness of cancer immunotherapy. 1.3 Oncolytic viruses OV therapy has emerged as an innovative and promising avenue in cancer treatment (23). An OV is characterized as a virus, whether genetically engineered or occurring naturally, with the unique capability to selectively kill cancer cells while leaving normal tissues unharmed (24). This sets OV therapy apart from conventional gene therapy, where viruses primarily function as carriers for delivering therapeutic genes. In OV therapy, the virus takes on an active role as a therapeutic agent, directly targeting and eliminating cancer cells. The initial discovery of the interaction between viruses and tumor cells dates back to the early 1800s when patients experienced tumor regression following viral infection (25) Up to the present, three OVs have received global approvals for the treatment of cancers. The first instance occurred in 2004 when Rigvir, an RNA virus derived from the native ECHO-7 strain of a picornavirus, gained approval for melanoma treatment specifically in Latvia. Subsequently, in 2005, China granted approval for H101, a genetically modified adenovirus (Ad), as a treatment for nasopharyngeal carcinoma in conjunction with cytotoxic chemotherapy. In 2015, Talimogene laherparepvec (T-VEC), an attenuated herpes simplex virus type 1 (HSV-1) encoding granulocyte-macrophage colony-stimulating factor (GM-6 CSF), received the endorsement from the U.S. Food and Drug Administration (FDA) for the localized treatment of patients with melanoma (26). 1.3.1 Mechanisms of actions OVs employ two primary mechanisms for cancer destruction: direct cell lysis and the induction of anti-tumor immunity (27) (Fig. 1-2). The first mechanism harnesses the virus's natural life cycle. When the virus infects and replicates within the tumor, it triggers cell lysis or apoptosis. Following replication and cell lysis, the newly formed viral particles continue the lytic cycle by infecting adjacent cells, creating a self-amplifying therapeutic effect localized to the tumor site (28). This continuous cycle lasts until the immune responses either quell virus replication or the pool of susceptible host cells is depleted (28). The second mechanism of OVs is the induction antitumor immunity. OVs induce the lysis of tumor cells, releasing tumor-associated antigens (TAAs) and neoantigens (TANs) (29). These released antigens become accessible to tumor-infiltrating antigen-presenting cells, particularly dendritic cells (DCs) and macrophages. The captured TAAs and TANs are processed by DCs and presented to T cells, initiating a tumor-specific T cell response. This response is directed against a wide spectrum of the released antigens, enhancing the immune attack on the tumor (30). Furthermore, OVs possess a remarkable ability to trigger immunogenic cell death (ICD), initiating a robust immune response (31). Once ICD is induced by OVs, a sequence of events occurs (32). This includes the release of vital danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). The exposure of calreticulin on the surface of tumor cells, coupled with the release of high-mobility group box 1 (HMGB1) and the secretion of adenosine triphosphate (ATP), are regarded as the key markers indicating ICD. When calreticulin becomes exposed, it acts as a ‘eat me’ signal for immune cells to engulf the tumor cells, thereby enhancing 7 the tumor's immunogenicity. Simultaneously, the released ATP serves as a signal that guides immune cells to locate and infiltrate the tumor microenvironment (TME), facilitating their presence within the tumor. Finally, the extracellular release of HMGB1 induces an inflammatory response, leading to the recruitment of additional immune cells into the TME (33). The combined release of these molecules, collectively known as DAMPs, alongside the release of viral components like nucleic acids (such as DNA, double-stranded RNA, single-stranded RNA, and 5′-triphosphate RNA), proteins, and capsid components, known as PAMPs, which are recognized by pattern recognition receptors (PRRs), leads to increased recruitment and maturation of tumor-specific T cells within the TME (34). In the field of breast cancer research, a multitude of viruses have undergone thorough preclinical and clinical evaluations to assess their effectiveness as oncolytic agents either alone or in combination with other treatments. 8 Figure 1-2. Dual mechanisms of oncolytic virus action in cancer treatment. (1) Direct lysis of cancer cells. Oncolytic viruses (OVs) selectively infect and replicate within cancer cells, leading to the eventual destruction of the infected cells. This direct lysis mechanism reduces the tumor burden by causing the rupture and elimination of cancerous cells. (2) Virus-mediated release of damage-associated molecular patterns (DAMPs), tumor antigens, and PAMPs. OVs trigger the release of key signaling molecules, including DAMPs, tumor antigens, and pathogen-associated molecular patterns (PAMPs), into the TME. These released molecules activate the immune system against the tumor. 9 1.3.2 Oncolytic virus in breast cancer Numerous OVs have been employed as monotherapies for treating breast cancer (35). Among these, only a few naturally possess the ability to target breast cancer cells, also known as wild-type (WT) viruses. Cancerous cells often exhibit characteristics that render them more susceptible to specific OVs. For instance, research has shown that many cancer cells have compromised signaling pathways, like those responsible for producing type I interferon (IFN-I), making them more receptive to viral infections (36, 37). Furthermore, breast cancer cells have been found to overexpress receptors such as intercellular adhesion molecule-1 and CD46. These receptors are utilized by Coxsackievirus A21 (CVA21) and the measles virus for cellular internalization, thus rendering breast cancer cells more susceptible to infection by these particular viruses (38, 39). Furthermore, it has been demonstrated that CD155 is upregulated in breast cancer cells and serves as the preferred entry receptor for poliovirus. Studies have revealed that the introduction of poliovirus into breast cancer cells leads to a more pronounced cytopathic effect and increased apoptosis response in these cancer cells compared to normal breast cells (40). Nevertheless, it is important to note that most OVs require genetic or surface modifications to either reduce their off-target effects, for example, by incorporating microRNA (miR) targeting sequences into the virus genome, or to enhance their toxicity towards breast cancer cells (41). These enhancements may involve the addition of immune-stimulatory factors and apoptosis inducers (42-45). As an example, researchers modified oncolytic HSVs to express glial cell-derived neurotrophic factor, enabling them to bind specifically to Glial Cell-Derived Neurotrophic Factor Receptor Alpha 1 (GFRα1). These receptors are abundantly expressed in breast cancer cells while having limited expression in normal cells. This modification demonstrated the recombinant HSV's remarkable selectivity for breast cancer cells, effectively inhibiting tumor growth upon 10 intratumoral injection (46). Similarly, the development of recombinant Ads specifically designed to target the C-X-C Chemokine Receptors (CXCR4 and CXCR7), which are known to be overexpressed in breast cancer cells and associated with tumor progression and metastasis, results in significantly increased infection rates and cytotoxicity compared to the WT-viruses (47). Scientists have also developed various recombinant OVs equipped with additional genes, with the aim of achieving more favorable clinical outcomes. Notably, one significant advancement involves the utilization of a recombinant measles virus vaccine engineered to express Helicobacter pylori heat shock protein A. This innovative approach has demonstrated remarkable potential, exhibiting superior tumor-killing capabilities in vitro across a spectrum of human solid tumor cell lines, including sarcoma, ovarian cancer, and breast cancer. Furthermore, this modified virus has been found to induce an immunogenic cytopathic effect, an essential facet of its anti-tumor activity. Remarkably, this modification also serves to prevent the upregulation of immune checkpoint proteins, further enhancing its therapeutic potential (48). Similarly, studies have demonstrated that Ad encoding interleukin (IL)-12 not only effectively reduces tumor growth in the 4T1 syngeneic TNBC model but also elicits abscopal effects. This is primarily attributed to the activation of immune cells and the inhibition of tumor angiogenesis (49). Comparable results were observed with a vaccinia virus (VV) expressing the IL-24 gene. The recombinant virus, VG9-IL-24, demonstrated efficient infection and selective elimination of breast cancer cells with minimal cytotoxicity towards normal cells. In vivo administration of VG9-IL-24 results in the delayed growth of tumors and improved overall survival (50). While OVs hold promise as a potential treatment option for breast cancer, it is increasingly clear that relying solely on OVs may not be sufficient for comprehensive and effective treatment (51, 52). Breast cancer is a complex and heterogeneous disease, and its progression often involves 11 various mechanisms and pathways that can limit the efficacy of OVs alone (53). Therefore, combining OVs with other therapeutic modalities, such as chemotherapy, immunotherapy, or targeted therapies, may offer a more robust and comprehensive approach to treating breast cancer. These combination therapies have the potential to target multiple aspects of the disease, enhance the immune response, and overcome resistance mechanisms, ultimately improving the overall outcomes for patients with breast cancer (54). 1.3.3 Oncolytic virus-based combination therapy in breast cancer, preclinical studies Combination therapy utilizing OVs has shown great promise and satisfaction in breast cancer treatment. This approach capitalizes on the unique strengths of OVs, which can selectively target and destroy cancer cells, while also leveraging the synergistic benefits of other therapeutic strategies. Integrating OVs with conventional treatments such as chemotherapy, immunotherapies, or targeted therapies enables the development of personalized and diverse treatment strategies to address the complexity and heterogeneity of breast cancer (Fig. 1-3). 12 Figure 1-3. The importance of combination therapy in addressing the challenge of heterogeneous breast tumors. Distinguishing oncolytic virotherapy as monotherapy (Left) versus combination therapy (Right). In numerous preclinical and clinical studies, OVs have exhibited impressive therapeutic efficacy when used along with other treatments (Table 1-1). 13 Table 1-1. Synergistic Approaches in Breast Cancer Treatment using Oncolytic Viruses with Multimodal Therapies Oncolytic virus Supplementary treatments Main findings Ref Vaccinia virus PD-1/ CTLA-4 antibody Enhancing the transformation of the tumor microenvironment, resulting in heightened immune cell infiltration and the stimulation of immune-related genes within the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) transgenic mouse model (55) Herpes simplex virus type 1 Paclitaxel Elevating immune cell infiltration accompanied by a substantial reduction in lung metastasis of breast cancer (52) Mitoxantrone Increasing intratumoral recruitment of both neutrophils and T cells within the tumor microenvironment of BALB/c mice hosting HER2/neu TUBO-derived tumors (56) STAT3 inhibitors Potentiates robust T-cell-mediated antitumor responses, thereby augmenting the efficacy of immune checkpoint blockade therapy against local recurrences and pulmonary metastasis in preclinical breast cancer models (57) EGFR-CAR NK cells Inducing tumor regression and significantly extending survival in mice harboring tumors (58) Integrin β1 inhibitors Enhancing the HSV-1-mediated antitumor response in xenograft models of TNBC (59) 14 Human adenovirus PD-L1/ CTLA-4 antibody Inhibiting TNBC tumor growth, extending survival, and reshaping the TME to counter immune evasion after oncolytic virus treatment (60) Mesothelin-CAR T cells Outperforming individual treatments in inhibiting tumor growth in animal models (61) G-quadruplex binders Enhancing cytotoxicity and elevated cell accumulation in the subG0/G1 phase (62) Cyclophosphamide Elevating apoptosis rates in breast cancer cells, consequently extending the survival of nude mice (63) EGFR inhibitor Overcoming drug resistance in breast cancer cells, amplifying cellular apoptosis (64) Paclitaxel Modifying cytokine network within the tumor site, heightening tumor cell apoptosis, and reducing neovascularization in tumors (65) Histone deacetylase inhibitors Improving virus replication and its capacity to induce cell death (66) Newcastle disease virus Alpha-glucosidase inhibitor Escalating oxidative stress and prompting apoptosis in a breast cancer mouse model (67) Hexokinase inhibitor Augmenting the oncolytic potential of the virus, increasing its efficacy in killing breast cancer cells (68) 15 Vesicular stomatitis virus NKT cell-based immunotherapy Boosting ICD in tumor cells, extending overall survival, and reducing metastatic burden significantly (69) Reovirus Doxorubicin Shrinking the primary tumor and reducing the metastatic burden (70) Topoisomerase inhibitors Amplifying reovirus infection and enhancing TNBC cell eradication (71) PD-1 antibody Reducing tumor burden, enhancing survival rates, and prompting the development of a systemic adaptive antitumor immune response (72) Measles virus Camptothecin Enhancing apoptosis within breast cancer cells (73) Paclitaxel Inducing robust cell death (74) Coxsackievirus B3 Melittin/CpG sequence Promoting ICD and facilitating immune cell infiltration into the TME, leading to the regression of both primary and distant tumors. (51) 1.3.3.1 Vaccinia virus The VV emerges as a standout candidate among double-stranded DNA viruses for oncolytic viral therapy. Its unique cytoplasmic replication, which avoids the cell nucleus, enhances safety by eliminating the risk of genome integration and underscores its potential as a potent oncolytic agent (75). VV's natural affinity for tumor cells, particularly evident in breast cancer research, showcases its exceptional tumor tropism and capacity to induce tumor regression. 16 Moreover, VVs possess the versatility to efficiently express foreign genes, further solidifying their promise as valuable contributors to the future of cancer therapy (76). It has been shown that a combination of the oncolytic VV LIVP strain expressing IL-15 or its receptor subunit alpha (IL-15Rα) promoted the formation of the IL-15/IL-15Rα complex, leading to significant tumor regression, increased lymphocyte recruitment at the tumor site, and activation of cytotoxic T cells and macrophages in 4T1 tumor-bearing mice (77). Moreover, the combination of oncolytic VV armed with GM-CSF with anti-programmed cell death protein 1 (PD-1) and/or anti- Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) significantly changed the TME, leading to increased immune cell infiltration and immune-related gene activation in the MMTV-PyMT transgenic mouse model (55). In ongoing clinical research, VV is currently being evaluated in various combinations to enhance its therapeutic potential. One such study is a Phase I/IIa multicenter clinical trial (NCT04725331) exploring the use of VV in combination with Pembrolizumab, which targets PD-1, in patients with metastatic/advanced solid tumors, including breast cancer. Additionally, another Phase II trial (NCT02630368) is investigating VV in combination with cyclophosphamide and Avelumab for patients with advanced soft-tissue sarcoma and advanced breast cancer. Moreover, VV has been a subject of interest in Phase I clinical trials (NCT02977156) when used in combination with an anti-CTLA-4 monoclonal antibody, with a focus on patients dealing with advanced/metastatic tumors, including breast cancer. These trials imply that VV is a compelling candidate in the realm of cancer treatment. Multiple clinical trials are actively investigating its potential when combined with diverse therapies, demonstrating its adaptability across solid tumor types, including breast cancer. 17 1.3.3.2 Herpes simplex virus type 1 HSV-1, a large enveloped double-stranded DNA virus, employs a unique fusion mechanism by merging its envelope with host cells and releasing its virion inside the cell (78). Over time, researchers have developed several OVs derived from HSV-1, with a particular focus on breast cancer treatment. These OVs have been investigated both as single treatment and in combination with other therapeutic agents, highlighting their potential in cancer treatment (79). In one study, the potential of combining a novel replicable recombinant oncolytic HSV-1, VG161, with paclitaxel for breast cancer treatment was examined. While a single administration of VG161 effectively suppressed breast cancer growth and triggers a robust antitumor immune response in a breast tumor mouse model. The combination with paclitaxel amplified these effects, leading to increased immune cell infiltration and a significant reduction in breast cancer lung metastasis (52). Similarly, combining the treatment with ICD-inducing chemotherapeutic drug mitoxantrone led to increased intratumoral infiltration of neutrophils and T cells to the TME in BALB/c mice bearing HER2/neu TUBO-derived tumors (56). Furthermore, it has been demonstrated that the combination of HSV-1 with Signal Transducer and Activator of Transcription 3 (STAT3) inhibitors induces pyroptosis in cancer cells and transforms "cold" tumors into "hot" ones. This strategy effectively elevated robust T-cell-dependent antitumor responses, enhancing the effectiveness of immune checkpoint blockade therapy against local recurrence and pulmonary metastasis in preclinical breast cancer models (57). Moreover, the synergy between HSV-1 and other immunotherapeutic strategies, such as chimeric antigen receptor-modified immune cells, has demonstrated enhanced efficacy in the elimination of MDA-MB-231 tumor cells. This combined approach has also led to significantly prolonged survival in mice bearing tumors, surpassing the outcomes achieved through monotherapies (58). Furthermore, research has demonstrated that 18 certain pharmaceutical compounds, such as OS2966, which inhibits integrin β1, notably augment the replication of oncolytic HSV-1. This enhancement has translated into a significantly improved HSV-mediated antitumor response in xenograft models of TNBC, achieved through the downregulation of IFN signaling and the mitigation of proinflammatory cytokine induction (59). These findings demonstrate strong potential of HSV-1 as a valuable candidate for breast cancer treatment, particularly when combined with complementary therapies. It has to be mentioned that the safety and effectiveness of HSV-1 are currently under evaluation in a Phase II clinical trial. This trial investigates the combination of HSV-1 with Pembrolizumab in patients diagnosed with TNBC (NCT03004183). 1.3.3.3 Human adenovirus Human Ad is a prominent OV platform in breast cancer research. Ad serotype 5 (Ad5) constructs utilize the human Coxsackievirus and adenovirus receptor (CAR) present on the surfaces of cells for precise targeting. Following receptor binding, Ad5 enters cells through endocytosis and delivers its genome to the host cell nucleus. Within the nucleus, transcription and replication occur, enabling viral protein production and DNA packaging (80). Combining oncolytic Ad with anti-PD-L1 and anti-CTLA-4 significantly inhibited tumor growth and extends survival in a TNBC model. This combination therapy boosted the antitumor effect by enhancing CD8+ T and T memory cell recruitment, reducing regulatory T cells and tumor-associated macrophages, and shifting macrophages from an M2 to M1 phenotype, thus reshaping the TME. Interestingly, this combination therapy effectively countered immune evasion following OV treatment by suppressing the expression of immune checkpoint molecules (60, 81). Similarly, the combination of Ads with Meso-CAR-T cells, which specifically target mesothelin (overexpressed in breast cancer patient tumor tissues), demonstrated superior inhibitory effects on tumor tissues 19 in animal models compared to individual treatments (61). Moreover, combining Ads with G-quadruplex binders, known for their ability to induce DNA damage and promote cancer cell death, led to increased cytotoxicity and a higher accumulation of cells in the subG0/G1 phase (62). The combination of Ads with cyclophosphamide, a chemotherapy agent, significantly heightened the apoptosis rate in breast cancer cells and extends the survival rate of nude mice. Interestingly, this combination also mitigated the toxicity typically associated with cyclophosphamide in normal cells (63). Combining Ads with an epidermal growth factor receptor (EGFR) inhibitor also significantly intensified cellular apoptosis, arrests the cell cycle in vitro, and reduced the tumor burden in xenografts of nude mice. This effect was achieved by upregulating caspase-9 and cleaved caspase-3. Notably, Ads also reversed drug resistance in breast cancer cells to the EGFR inhibitor by deactivating the phosphatidylinositol 3-kinase/Akt pathway (64). Ads showed better therapeutic efficacy once combined with paclitaxel (65). Additionally, studies have shown that histone deacetylase inhibitors can significantly enhance the replication and cell-killing capacity of Ad5, both in laboratory settings and in mouse models of breast tumors (66). 1.3.3.4 Newcastle disease virus Newcastle disease virus (NDV) belongs to the category of enveloped, non-segmented, negative-sense RNA viruses (82). NDV triggers apoptosis through both caspase-dependent and caspase-independent pathways (82). Studies have revealed that employing an alpha-glucosidase inhibitor to induce glucose deprivation, in conjunction with NDV, can substantially elevate oxidative stress and induce apoptosis in a mouse model of breast cancer featuring mammary adenocarcinoma tumor cells, surpassing the effects of single treatments (67). Similar findings were observed after combining NDV with a hexokinase inhibitor to inhibit glycolysis (68). These promising results highlight NDV's potential as a valuable candidate for combinational therapy 20 against breast cancer. Nevertheless, it is imperative to note that further investigations are required to comprehensively assess its safety, efficacy, and potential side effects in clinical settings. 1.3.3.5 Vesicular stomatitis virus Vesicular stomatitis virus (VSV) belongs to the Rhabdoviridae family and is characterized as an attenuated negative-strand RNA virus. It enters cells by interacting with the widely expressed low-density lipoprotein receptor. Notably, its replication is sensitive to IFN-I, which restricts its ability to replicate in normal cells. However, VSV can effectively replicate in cancer cells due to their common defects in IFN-I signaling (83). Emerging evidence underscores the efficacy of combining VSV with immunotherapeutic strategies, particularly the activation of NK cells. This synergistic approach has been demonstrated to enhance ICD within tumor cells. Moreover, in a murine model bearing 4T1 tumors, this combination therapy yielded superior outcomes compared to individual treatments, extending overall survival and significantly curtailing metastatic burden. These findings illuminate a promising path for advanced cancer therapy strategies (69). 1.3.3.6 Reovirus Reovirus is known as a nonenveloped double-stranded RNA virus and is commonly recognized for its general nonpathogenicity in humans (84). Intriguingly, this virus exhibits a preference for replicating within transformed cells, rendering it a highly appealing candidate for oncolytic therapy (85). Compelling research has revealed that the combination of reovirus with the chemotherapeutic drug doxorubicin can result in a substantial reduction of the primary tumor burden in TNBC in vivo models. Impressively, this combined approach not only led to a notable decrease in the primary tumor size but also exhibited even more significant reductions in metastatic burden compared to individual treatments (70). Similar observations were obtained after administration of reovirus with other topoisomerase inhibitors such as epirubicin and topotecan 21 (71). Combining reovirus with immunotherapeutic strategies has demonstrated remarkable efficacy. In a study conducted by Mostafa et al., the synergy between reovirus and anti-PD-1 antibodies led to a substantial reduction in disease burden and a notable improvement in survival rates in a syngeneic EMT6 murine model of breast cancer. Significantly, this dual treatment regimen also triggered the development of a systemic adaptive anti-tumor immune response, evidenced by the increase in tumor-specific IFN-γ-producing CD8⁺ T cells and conferred immunity against tumor re-challenge (72). Reovirus is currently undergoing extensive evaluation in clinical trials. In a Phase II trial (NCT04445844), it is being tested in combination with retifanlimab, a monoclonal antibody targeting the PD-1, in patients diagnosed with TNBC. Additionally, reovirus has been investigated in combination with Atezolizumab, a monoclonal antibody targeting PD-L1 (NCT04102618). Furthermore, a Phase II clinical study (NCT04215146) has been conducted to assess the safety and efficacy of reovirus in combination with anti-PD-L1 Avelumab and paclitaxel. This trial focuses on patients with HR+/HER2- endocrine-refractory metastatic breast cancer. These studies collectively highlight the extensive research efforts aimed at uncovering the potential of reovirus-based therapies in enhancing treatment outcomes for various subtypes of breast cancer. 1.3.3.7 Measles virus Measles virus is characterized as an enveloped, negative-sense, single-strand RNA virus. It has three distinct cellular receptors: membrane cofactor protein (CD46), signaling lymphocytic activation molecule, and the adherens junction protein Nectin4 (86). Notably, CD46 and adherens junction protein Nectin4 are frequently upregulated in various human tumor cells, including breast cancer cells (87). Recent research has unveiled the potent synergy between MV and the chemotherapeutic agent camptothecin at both low and high dosages. This combination therapy has 22 demonstrated a substantial enhancement in apoptosis within breast cancer cells, surpassing the effects observed with either measles virus or camptothecin treatment in isolation (73). Similarly, the combination of measles virus with the chemotherapy drug paclitaxel has emerged as a highly effective strategy in inducing significantly greater levels of cell death in breast cancer cells (74). 1.3.3.8 Coxsackievirus Coxsackieviruses form a category of non-enveloped, positive-sense, single-stranded RNA viruses that fall under the human Enterovirus species within the Picornaviridae family (88). Initially, Coxsackieviruses were classified into two groups, namely A (CVA) and B (CVB), based on initial observations of the differing tissue damage they induced (89). The exploration of oncolytic CVB began in 1997, with the investigation of the potential of CVB1 in combating human colon cancer. Since then, numerous research teams have embarked on extensive efforts to evaluate the oncolytic properties and safety profiles of CVBs in pre-clinical studies, targeting a variety of tumor forms (90). CVB3, like all members of the Picornaviridae family, is characterized by an icosahedral capsid with a diameter of approximately 30 nm, housing the positive-sense (+) RNA genome (91). The RNA genome of CVB3 is positive-sense (+) and spans approximately 7.5 kilobase pairs in length. This genome consists of a solitary extensive open reading frame, framed by a 5′-untranslated region (5′-UTR) of 742 nucleotides (nt) and a polyadenylated 3‘-UTR measuring approximately 100 nt in length (91). The capsid of CVB3 is composed of twelve pentamers, each constructed from five asymmetric units of the structural proteins VP1 to VP4. Specifically, VP1 to VP3 constitute the viral shell, while VP4 resides at the inner surface of the viral shell, facilitating a connection between the N-termini of the other capsid proteins and the viral RNA. In this capacity, VP4 serves as a crucial stabilizing element during the assembly of the viral capsid pentamers (92). 23 In mice, CVB3 exhibits a distinct pattern of organ susceptibility. The primary target organs for CVB3 in mice are the pancreas and heart. Notably, the pancreas is particularly vulnerable, with CVB3 infections leading to acute pancreatitis characterized by the progressive destruction of the exocrine part of the pancreas. Simultaneously, myocardial infection results in acute and chronic inflammation of the heart, ultimately impairing cardiac contractility and potentially leading to heart failure (93, 94). In contrast, when CVB3 infects humans, it often induces mild, self-limiting illnesses that resemble flu-like symptoms in adults. However, under certain circumstances, including genetic and individual predispositions, more severe outcomes can occur. In human patients, CVB3 infections can result in conditions such as aseptic meningitis, encephalitis, and myocarditis, which are more commonly observed in infants and children (95, 96). The presence of viral receptors on the cell surface plays a pivotal role in determining virus tropism, making it a crucial factor for the successful application of oncolytic CVB3 in cancer treatment. Among these receptors, CAR stands out as the primary one for CVB3 binding and internalization (97). CAR is a transmembrane protein involved in cell adhesion and inflammation and serves as the main receptor for CVB3 in host cells (98). In addition to CAR, CVB3 utilizes the decay accelerating factor (DAF, also known as CD55) as an additional receptor. DAF plays a role in regulating complement activation and cell signaling and functions as a co-receptor, facilitating CVB3 attachment to the host cell surface (99). However, it is important to note that the binding of DAF alone does not suffice to enable viral entry into the cell and subsequent lytic infection (100). Consequently, cancer cells expressing DAF but lacking CAR remain impervious to CVB3, highlighting the significance of CAR as the primary receptor for effective viral therapy. CVB3 stands out as a promising candidate for oncolytic virotherapy due to its impressive oncolytic activity against a variety of cancer types. Notably, CVB3 has demonstrated its potential 24 in selectively targeting and eliminating cancer cells in preclinical studies involving melanoma, breast cancer, colorectal cancer, and other malignancies (101-103). Having said that, preclinical studies have shown that CVB3 can cause cardiotoxicity and pancreatoxicity. One of the significant concerns associated with CVB3 is its potential for cardiotoxicity. This refers to harm to the heart and its functioning. In preclinical studies, CVB3 has been observed to infect and damage heart cells, leading to inflammation and cardiac complications. Efforts are ongoing to understand and mitigate this toxicity to ensure the safety of patients. Another area of concern relates to the pancreas. CVB3 infections can affect the pancreas, potentially leading to pancreatitis or disruptions in pancreatic function. Pancreatic toxicity can have significant implications for overall health and well-being (90, 103). CVB3 exhibits a potent role as an OV in cancer treatment. One of its key attributes is the ability to induce ICD when it infects cancer cells. This process results in the release of DAMPs and PAMPs within the TME. These molecular signals act as alarm bells for the immune system, alerting it not only to the presence of the virus but, critically, to the presence of cancer cells within the tumor (51, 104). What makes this immunogenic response particularly powerful is that it is not localized solely to the immediate tumor site. Instead, it has a systemic effect, meaning that the immune system becomes activated and ready to target and eliminate cancer cells throughout the body. This systemic immunity not only aids in the destruction of primary tumors but also plays a pivotal role in preventing tumor relapse (51). Furthermore, CVB3 possesses the remarkable capacity to transform immunosuppressed "cold" tumors into "hot" tumors. Cold tumors typically lack a robust immune response against cancer cells. CVB3 can change this landscape by infecting immunosuppressive cells within the TME or by converting these cells into pro-inflammatory phenotypes. This transformation is pivotal because it amplifies the immune response against 25 cancer, making the tumor more susceptible to immune-mediated destruction (104). Furthermore, CVB3 stands out as a compelling candidate for virotherapy in cancer treatment, offering additional distinct advantages. Firstly, as an RNA virus, it avoids integrating into the host DNA, reducing genotoxicity concerns associated with other therapies. Secondly, its small RNA genome is easily manipulable, allowing for tailored treatments. Thirdly, CVB3 preferentially targets proliferating cells and relies on oncogenic signaling pathways, enhancing its efficacy against cancer cells. Fourthly, it excels at targeting tumor cells with impaired innate immune responses, particularly those with KRAS mutations. Additionally, while CVB infection can be severe in children, it typically causes mild symptoms in adults. Lastly, CVB2, CVB3, and CVB4 have shown promise in destroying a variety of cancer cell types in large-scale screening studies, suggesting potential for broad-spectrum cancer therapy (90). Numerous endeavors have been undertaken to mitigate the potential toxicity associated with CVB3 in the context of virotherapy. One particularly successful approach involves the integration of target sequences for organ-specific and/or tumor-suppressive miRs into the virus genome. In this refined approach, when miR-modified OVs are internalized by cells containing the specific miRs, the miRs recognize and bind to their designated target sequences strategically embedded within the virus genome. This interaction triggers a cascade of events, leading to the degradation of viral mRNA (105, 106) (Fig. 1-4). 26 Figure 1-4. microRNA-detargeting strategy to improve safety profile of oncolytic virus. Virus genome modification involves the incorporation of targeted microRNA (miR) sequences, which are more prevalent in normal tissues than in cancerous ones. This modification hinders virus replication in normal cells by inducing the degradation of the virus genome upon miR binding to its specific sequence integrated within the virus genome. This precision-guided mechanism represents a remarkable leap in enhancing the safety profile of CVB3-based virotherapy. By harnessing the cell's regulatory machinery through miR-mediated degradation, the virus becomes exquisitely responsive to specific cellular contexts. This means that in healthy tissues or those have abundant miRs, CVB3 remains relatively inert, minimizing collateral damage. Conversely, within the microenvironment of cancerous cells where the specified miRs are absent, CVB3's oncolytic activity is activated. This dynamic approach 27 exemplifies the innovative strides being made in virotherapy to harness the full potential of OVs while safeguarding the health of treated individuals. It has been shown in various study that the miR strategy is effective in reducing the toxicity associated with CVB3 (107, 108). 1.4 Exosome Exosomes (Exo) are small, membrane-bound extracellular vesicles ranging from 30 to 150 nanometers in diameter, secreted by a wide variety of cell types. They play an essential role in intercellular communication by transferring proteins, lipids, and nucleic acids between cells. Exo are generated through the endosomal pathway, which involves the inward budding of the endosomal membrane to create multivesicular bodies (MVBs). When MVBs fuse with the plasma membrane, Exo are released into the extracellular environment (109). This unique biogenesis process gives Exo a specific set of molecular cargos that reflect their cell of origin, making them invaluable in both physiological and pathological contexts. 1.4.1 Exosome biosynthesis The biosynthesis of Exo is a complex and multi-step process centered around the endosomal system within cells. This process can be broken down into several stages: endocytosis, early endosome formation, MVB formation, and Exo release. The process begins with the inward budding of the plasma membrane, which forms early endosomes. This initial stage involves the engulfment of extracellular material and cell membrane proteins into the cell (110). Early endosomes are the primary stage of endocytic vesicles. These endosomes undergo a series of maturation and sorting processes. They can either recycle their contents back to the plasma membrane or progress to late endosomes. As early endosomes mature into late endosomes, inward budding of the endosomal membrane occurs, leading to the creation of intraluminal vesicles (ILVs). This process results in the formation of MVBs or multivesicular endosomes (111). The 28 formation of ILVs within MVBs is primarily mediated by the endosomal sorting complex required for transport (ESCRT) machinery, which comprises several protein complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III). These complexes work together to recognize and sequester cargo molecules into ILVs. Additionally, alternative ESCRT-independent mechanisms involve lipid rafts, tetraspanins (e.g., CD9, CD63), and other proteins that aid in cargo sorting and vesicle formation. MVBs have two potential fates: they can fuse with lysosomes, leading to the degradation of their contents, or they can fuse with the plasma membrane, resulting in the release of ILVs into the extracellular space as Exo (111). This fusion and subsequent exo release are regulated by various Rab GTPases (e.g., Rab27a, Rab27b), Soluble N-ethylmaleimide-sensitive factor activating protein receptor proteins, and other regulatory molecules that facilitate the docking and fusion of MVBs with the cell membrane. During the formation of ILVs, selective incorporation of specific proteins, lipids, and nucleic acids occurs. The cargo of Exo can include membrane proteins (e.g., tetraspanins, integrins), cytosolic proteins (e.g., heat shock proteins, metabolic enzymes), nucleic acids (e.g., mRNAs, miRs, DNA), and lipids (e.g., cholesterol, ceramides). The sorting of cargo into Exo is a highly regulated process influenced by various cellular signals and environmental conditions (110, 112). This intricate biosynthetic pathway ensures that Exo carry a specific set of molecular cargos reflective of their cell of origin, enabling them to play pivotal roles in intercellular communication, physiological processes, and pathological conditions (113). 1.4.2 Exosomes in drug delivery In the field of drug delivery, Exo have attracted significant attention due to their unique properties, making them ideal carriers for therapeutic agents. Their natural biocompatibility and low immunogenicity greatly reduce the risk of adverse immune responses compared to synthetic 29 nanoparticles (114). Additionally, Exo possess surface proteins that enhance their ability to home in on and be taken up by specific recipient cells, thereby improving targeted delivery. This capability is particularly beneficial for cancer therapy, where precise delivery of chemotherapeutic agents to tumor cells while minimizing damage to healthy tissue is crucial (115). There are several methods for loading Exo with therapeutic agents, each with its own advantages and limitations. Passive loading involves incubating Exo with the drug of interest, allowing for spontaneous incorporation. Electroporation uses electrical pulses to temporarily permeabilize the exosomal membrane, facilitating the encapsulation of drugs. Chemical transfection employs transfection reagents to enhance drug loading efficiency. The choice of method depends on the nature of the drug and its intended application (116). The clinical potential of Exo -based drug delivery systems is supported by numerous preclinical and clinical studies. For example, Exo derived from mesenchymal stem cells have shown promise in delivering anti-inflammatory and regenerative agents for treating cardiovascular diseases and tissue injuries (117). In cancer therapy, Exo loaded with doxorubicin, a potent chemotherapeutic drug, have demonstrated enhanced cytotoxicity against tumor cells while reducing systemic toxicity compared to free drug administration. Moreover, Exo engineered to carry specific targeting ligands on their surface can improve the selectivity and efficacy of drug delivery to tumor sites. 1.5 Rationale, Hypothesis and Specific Aims Breast cancer is the most commonly diagnosed malignancy in women worldwide, accounting for approximately 1.7 million new cases annually and roughly 25% of all adenocarcinomas. Conventional treatments such as radiotherapy and chemotherapy fail to significantly improve overall survival of patients with TNBC in light of cancer heterogeneity and rapid development of drug resistance. 30 OVs are a group of genetically engineered viruses with the capability of infecting and killing cancer cells while demonstrating little to no impact on healthy tissues. Recently, a number of OVs, namely poliovirus, Ad, reovirus, HSV, and measles virus, have been applied in various preclinical and clinical studies for the treatment of diverse malignancies. While OVs have shown considerable promise in clinical trials and demonstrated efficacy in some studies, there are several limitations to their use as cancer treatments. One major challenge is the variability in response among different patients and tumor types, which can limit the overall effectiveness of the therapy. Additionally, issues related to the delivery of OVs to the tumor site, such as immune system neutralization and inadequate tumor penetration, can hinder treatment outcomes. Furthermore, there may be potential for off-target effects or unintended damage to healthy tissues, raising concerns about the safety of these therapies.. More importantly, no efficient OV has yet been developed to be able to target and treat breast cancer with high selectivity and low off-target impacts. Therefore, the overall goal of this project is to develop a novel, potent, and safe anti-cancer virotherapy that can be translated into clinical application for breast cancer therapy in the near future. CVB3 is a widespread virus in humans, and infection in adults is usually asymptomatic or causes only mild flu-like symptoms. Recently, it has been demonstrated that CVB3 is a promising anti-tumor virus, capable of infecting and killing different types of malignant cells with minimal off-target effects (118). In prior research, our laboratory successfully engineered a safer variant of CVB3 through a miR-detargeting approach through adding the target sequences of miRs that are overexpressed in normal tissues (miR-1 and miR-216) or downregulated in cancer cells (miR-143 and miR-145) into the viral genome, referred to as miR-CVB3 (107). While miR-CVB3 demonstrated improved safety profiles in immunodeficient mice, a comprehensive assessment of 31 both safety and effectiveness in immunocompetent mice remained pending. Furthermore, despite holding significant potential in crafting cancer therapeutics,one of the main challenges for using OVs is the pre-existing antibodies and rapid clearance by the immune system after systematic administration (119). Exo are a family of phospholipid bilayer-enclosed vesicles that can be secreted by various types of cells, mediating cell-to-cell communication. There is growing evidence proving that modified Exo can be applied for the efficient delivery of therapeutic agents to cancer cells due to their excellent physiochemical stability and biocompatibility. Therefore, for my thesis project, I propose to evaluate the safety and efficacy of miR-CVB3 in breast cancer cells and tumor-bearing mice. I will also develop a safe platform for the systematic administration of miR-CVB3 using Exo. Building upon these discussions, the hypothesis of my thesis is that miR-CVB3, encapsulated into modified extracellular vesicles, is an excellent candidate for development into a novel oncolytic virotherapy for breast cancer. Specific aims : Aim 1: Evaluate the safety and efficacy of miR-CVB3 in different breast cancer cells and in an immunocompetent mouse model of TNBC With this goal in focus, I conducted an evaluation of the effectiveness of the newly developed CVB3 (miR-CVB3) in infecting and eliminating diverse human and mouse breast cancer cells. Additionally, I assessed the safety and efficacy of miR-CVB3 in mice with 4T1-tumor-bearing mice. The results are presented in Chapter 2. Aim 2: Assessment of the therapeutic efficacy and safety of the miR-CVB3 in combination with immunotherapeutic/chemotherapeutic drugs in an immunocompetent mouse model of TNBC With this specific aim, I conducted an assessment of the effectiveness of miR-CVB3 in combination with melittin and CpG sequences, focusing on inducing more robust ICD in vitro. 32 Expanding on this, I also conducted evaluations in vivo to investigate the potential of this combined therapy for tumor reduction and immune system activation in 4T1-tumor bearing mice. The findings are detailed in Chapter 3. Aim 3: Using Exo modified with tumor-targeting agents for the efficient delivery of CVB3 to TNBC and systematic administration of miR-CVB3 in an immunocompetent mouse model of TNBC In this aim, I analyzed the influence of miR-CVB3 on immunological proteins and exosomal cargo. Subsequently, I employed Exo for the effective delivery of miR-CVB3 to breast cancer cells. Additionally, I enhanced the selectivity and efficacy of Exo containing miR-CVB3 by modifying them with AS1411 aptamer and doxorubicin. This modification aims to enhance their specificity and effectiveness against breast cancer in mice bearing 4T1 tumors. The findings are described in Chapter 4. 33 Chapter 2: A new miRNA-modified coxsackievirus B3 inhibits triple-negative breast cancer growth with improved safety profile in immunocompetent mice 2.1 Background Despite significant improvements, the absence of molecular targets among patients suffering from TNBC significantly limits treatment options compared to other subtypes of breast cancer (120). Furthermore, severe off-target effects and multiple drug resistance have been observed for TNBC patients following standard treatments (16, 121). Therefore, there is an urgent need to explore alternative treatments with high efficacy and low side effects. Rationale: In previous research conducted within our laboratory, it was demonstrated that miR-CVB3 exhibits significant promise as an efficient OV, boasting a potent capacity to inhibit lung tumors while maintaining a good safety profile in immunodeficient mice in comparison to the WT-CVB3. The objective of this chapter is to investigate the viability of miR-CVB3 as a novel treatment for breast cancer. The specific aims outlined in this Chapter encompass: (1) assessing the infectivity and cytotoxicity of miR-CVB3 across diverse human and mouse breast cancer cell lines, and (2) evaluating the safety profile of miR-CVB3, alongside its efficacy in impeding tumor growth within immunocompetent mice harboring 4T1 TNBC tumors. 2.2 Materials and methods 2.2.1 Cell lines and culture 4T1 cells [CRL-2539], the mouse triple-negative mammary tumor cell line isolated from Balb/c mice, as well as the HeLa cells (human cervical cancer cells, CCL-2™) were purchased from the American Type Culture Collection (ATCC). MDA-MB-231 and SUM149 breast cancer 34 cell lines were kindly provided by Dr. James Lim (University of British Columbia). MCF-7, T47-D and SK-BR-3 breast cancer cell lines were generous gifts from Dr. Connie Eaves (University of British Columbia). All the cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM; #D6429, Sigma-Aldrich) containing 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO2. 2.2.2 Virus propagation HeLa cells are known to be highly permissive to CVB3, allowing for rapid virus propagation and high-titer virus production. This permissiveness is essential for producing sufficient virus stocks for in vitro and in vivo experiments. HeLa cells were subjected to infection with miR-CVB3 at a multiplicities of infections (MOI) of 10 for a period of 48 hours. Following incubation, the supernatant was collected to isolate the viral particles through ultracentrifugation. The resultant viral stock, reaching a concentration of approximately 5 × 107 plaque-forming unit (PFU)/ml, was stored at a temperature of −80 °C, ensuring its preservation and accessibility for subsequent applications. 2.2.3 Cell viability assay In order to determine the cytotoxic effect of the newly produced miR-CVB3 in different cancer cell lines, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was performed using commercially available kit following the manufacturer's protocol (#G9243, Promega). Briefly, cells (104 cells/well) were seeded into 96-well plates and cultured overnight to reach roughly 80% of confluence. Subsequently, cells were treated with either miR-CVB3 at different MOIs of 0.01, 0.1 and 1 or PBS as the sham negative control. After 1 h incubation, medium containing viral particles was removed and fresh media was added. At different infection time points, 10 μl of MTS solution was added into each well and the 35 plate was incubated at 37° for 3 h. Eventually, the absorbance at 490 nm was measured using a BioTek Synergy H1 microplate reader. 2.2.4 Reverse transcription- quantitative polymerase chain reaction RT-qPCR was employed to determine the level of viral RNA and Ifnb1. For viral RNA measurement, total RNAs were isolated using the Monarch total RNA miniprep kit (T2010, New England Biolabs). This was followed by RT-qPCR with primer pairs targeting CVB3 2A and murine gapdh using the Luna universal one-step RT-qPCR kit (E3005L, New England Biolabs) on a ViiA 7 real-time PCR system. To investigate the response of different breast cancer cells to polyIC (tlrl-pic-5, InvivoGen), cells were transiently transfected with polyIC (1 μg/ml) using Lipofectamine RNAiMAX (13778075, Invitrogen) for 12 h. Then total cellular RNAs were extracted and subjected to RT-qPCR analysis to measure the level of murine Ifnb1 and human IFNB1 as well as murine gapdh and human GAPDH. Primer sequences are depicted in Table 2-1. Table 2-1. The primer pairs used for viral RNA, Human IFNB1 and Murine Ifnb1 Target Forward Reverse CVB3 2A 5′-GCT TTG CAG ACA TCC GTG ATC-3′ 5′-CAA GCT GTG TTC CAC ATA GTC CTT CA-3′ Human IFNB1 5′-CTT GGA TTC CTA CAA AGA AGC AGC-3′ 5′-TCC TCC TTC TGG AAC TGC TGCA-3′ Murine Ifnb1 5′-AAG AGT TAC ACT GCC TTT GCC ATC-3′ 5′-CAC TGT CTG CTG GTG GAG TTC ATC-3′ Human GAPDH 5′-AAT CCC ATC ACC ATC TTC CA-3′ 5′-TGG ACT CCA CGA CGT ACT CA-3′ Murine gapdh 5′-GGC AAA TTC AAC GGC ACA GT-3′ 5′-AGA TGG TGA TGG GCT TCC C-3′ 36 2.2.5 Western blot analysis Various cancer cells (1 × 105 cells/well) were seeded into 12-well plates. When reaching approximately 80% confluence at the next day, cells were infected with WT-CVB3 or miR-CVB3 at different MOIs (0.01, 0.1 and 1). After 1 h incubation, virus-containing medium was removed, and fresh medium was added. After additional 16 h, cells were harvested with protein lysis buffer [25 mM Tris-Cl pH7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% Sodium deoxycholate] and protein concentration was determined via a BCA assay. Cell extracts were subjected to protein separation using 12% SDS-polyacrylamide gels. After transferring, membranes were incubated with primary antibodies, i.e., anti-CVB3 capsid protein VP1 (M47, Mediagnost, Germany) or anti-CAR (16984, Cell Signaling) overnight, followed by probing with horseradish peroxidase-conjugated secondary antibody. After each treatment, membranes were washed three times with PBS containing 0.05% Tween 20 to remove unbound antibodies. Finally, Syngene G:BOX Chemi XRQ gel doc system was used to visualize protein expression. Membranes were also incubated with β-actin antibody (sc-47778, Santa Cruz Biotechnology) to confirm equal loading of samples. 2.2.6 Animal studies All the animal investigations were conducted under the guidelines for the care and use of laboratory animals. All the protocols were approved by the Animal Care Committee at the University of British Columbia (A18-0275). For safety and anti-tumor study, 6-8-week-old female Balb/c mice were inoculated subcutaneously with 2 × 105 of murine 4T1 cells suspended in 100 μL of PBS into the right flank. Once the tumor reached a palpable size (∼20 mm), mice were randomly divided into three groups: sham, WT-CVB3, and miR-CVB3 groups. Mice were treated with PBS (sham group), WT-CVB3, or miR-CVB3 through intratumoral injections. Mouse numbers and viral dosage used for each 37 group are indicated in figure legends. Mice were monitored every other day for signs of clinical symptoms. Body weight and tumor diameter (using caliber) were measured every 2–3 days. Based on our approved protocol, human endpoints were defined as follows: mice lose ≥20% of their initial body weight, ulceration is observed in ≥10% of tumor region, size of tumor reaches ≥1.7 cm diameter or tumor weight exceeds 10% body weight. 2.2.7 Immunohistochemistry Tumor tissues collected were fixed with 4% formaldehyde and then immunostained with anti-CD8 (sc-1177, Santa Cruz Biotechnology), anti-CD4 (sc-19641, Santa Cruz Biotechnology), anti-F4/80 (sc-377009, Santa Cruz Biotechnology), or anti-CVB3 capsid protein VP1 (Mediagnost, Germany) antibodies using the MACH4 Universal HRP-Polymer Detection System (BRI4012H, Biocare Medical) and hematoxylin solution Gill II (GHS232, Sigma-Aldrich) as previously described [28]. Normal mouse IgG1 (sc-3877, Santa Cruz Biotechnology), IgG2A (sc-3878, Santa Cruz Biotechnology), and rabbit IgG (ab172730, Abcam) were used as negative controls. Lastly, Aperio ScanScope AT (Digital slide scanner, Leica Biosystems Inc) and bright-field microscopy were applied to attain images. The staining from at least three separate images for each organ or tumor harvested from at least 3 mice per group was quantified by applying NIH ImageJ (version 1.52p) and presented as relative optical density. 2.2.8 Hematoxylin and Eosin (H&E) staining The mouse organs (heart, lung, pancreas, liver, and spleen) harvested were fixed in 4% formaldehyde and embedded in paraffin. The tissues were then sliced into 5 μm thickness and stained with H&E to visualize and quantify the pathological score of diverse tissues. Sections were evaluated by an experienced pathologist blinded to the specimen status. The damage to the heart and pancreas was assessed based on relative lesion area, cellular vacuolization, calcification, 38 necrosis, and inflammatory infiltration, using the following scale: 0, no or questionable presence; 1, limited focal distribution; 2–3, intermediate severity; and 4–5, coalescent and extensive foci over the entirety of the transversely sectioned tissue. 2.2.9 Statistical analysis Statistical analysis was performed by GraphPad Prism V8.0.1 software and all data are expressed as mean ± standard error of the mean (SEM) (n ≥ 3). The results were analyzed by unpaired Student's t-test or one-way ANOVA followed by Tukey's test to determine differences. The differences between survival rate were evaluated by log-rank test. P-value < 0.05 was considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 2.3 Results 2.3.1 miR-CVB3 shows strong lytic potency towards various breast cancer cells For the initial in vitro study, we utilized different breast cancer cell lines, including mouse 4T1, human MDA-MB-231, SUM149, T47D, SK-RB-3, and MCF-7 cell lines. Cell viability assay revealed that both WT-CVB3 and miR-CVB3 had substantial lytic ability towards all tested cells except for MCF-7 cells, in which only ∼10% decline in cell viability was observed after administration of miR-CVB3 at the highest dosage (Fig 2-1A). To determine whether the oncolytic ability of miR-CVB3 is associated with viral replication, we evaluated viral gene expression and protein synthesis by RT-qPCR and western blotting, respectively. We showed that miR-CVB3 was able to dose-dependently infect and replicate in various breast cancer cells (4T1, MDA-MB-231, SUM149, T47-D and SK-BR-3) even though at a moderately reduced level compared with WT-CVB3 (Fig. 2-1B and C). Interestingly, consistent with the cell viability data, we found that miR-CVB3 failed to show replication efficacy in MCF-7 cells and only minimal expression of VP1 was 39 detected at the highest applied MOI in this study (Fig. 2-1C). Collectively, our results demonstrated that miR-CVB3 efficiently infects and lyses a variety of breast cancer cells, with the exception of MCF-7 cells. 40 41 Figure 2-1. miR-CVB3 potently kills various breast cancer cells through replication. (A) cell viability assay of various breast cancer cell lines, including mouse 4T1, human MDA-MB-231, human SUM149, human T47D, human SK-RB-3, and human MCF-7. Cells were sham-infected or infected with WT- or miR-CVB3-at different MOIs as indicated for 72 h. Cell viability was assessed via MTS assay and presented as mean ± SEM (n = 3–6, single experiment) and results were analyzed by one-way ANOVA followed by Tukey's test to determine differences. *, p < 0.05; ***, p < 0.001 as compared to sham, otherwise specified. (B) Viral RNA copy numbers (mean ± SEM, n = 6) in cells collected from infected 4T1 cells (MOI = 0.1, for 24 h) were assessed by RT-qPCR. The results were analyzed by unpaired Student's t-test. ***, p < 0.001. (C) The levels of viral protein VP1 and β-actin (load control) were examined by western blot analysis in different breast cancer cell lines. Results in this figures represent data from two independent experiments. 42 2.3.2 MCF-7 cells express a low level of coxsackievirus and adenovirus receptor at baseline, but a high level of interferon B1 when exposed to polyinosinic:polycytidylic acid (PolyIC) In order to gain deeper insights into the underlying factors driving the observed insensitivity of MCF-7 cells towards miR-CVB3 our subsequent investigation aimed to pinpoint potential causative elements. The initial focus of our scrutiny centered on the expression status of CAR, a pivotal receptor for the cellular entry of CVB3 and subsequent instigation of lytic infection (118). The examination of CAR expression in various breast cancer cell lines was undertaken as a critical first step. Our analysis yielded intriguing outcomes: the CAR expression levels within MCF-7 cells were found to be relatively low, even bordering on non-detectable levels. This observation substantiates the proposition that the limited expression of CAR could potentially underlie the observed restrained rates of CVB3 infection within MCF-7 cells (Fig. 2-2A). However, it is noteworthy that similar levels of CAR were also evidenced in SK-RB-3 and MDA-MB-231 cells, both of which exhibited a heightened susceptibility to miR-CVB3. This incongruity between CAR expression and cellular susceptibility led us to postulate the involvement of supplementary factors in contributing to the impervious nature of MCF-7 cells towards miR-CVB3. CVB3 is sensitive to IFN-I as part of innate immune response towards viral infection. Tumor cells with impaired IFN-I production are considered susceptible cells to CVB3, while those capable of producing high IFN-I levels are known to be resistant to CVB3 infection (90). To address whether differential response in the context of IFN-I production is a factor responsible for the difference in CVB3 permissiveness, we next examined the IFN-I levels in these cells. RT-43 qPCR results showed that MCF-7 cells expressed the highest IFNB1 after exposure to polyIC, a synthetic double-stranded RNA known to induce IFN-I synthesis (Fig. 2-2B), suggesting that the ability of MCF-7 cells to produce high levels of IFN-I, unlike SK-BR-3 and MDA-MB-231 cells, may contribute to impeded CVB3 replication should the virus internalize into the cells. Taken together, these findings denote that both the low expression of CAR and high potency of MCF-7 in the production of anti-viral IFNB1 are possible factors responsible for the insensitivity of this cell line to miR-CVB3 Future investigation is warranted to determine whether additional factors may also contribute to the resistance of MCF-7 cells to CVB3. 44 Figure 2-2. Human breast cancer MCF-7 cells express low protein level of viral receptor CAR but display the highest Ifnb1 gene expression in response to polyIC treatment. (A) Evaluation of CAR expression in different breast cancer cell lines using western blot analysis, representing from two independent experiments. (B) qPCR results of gene expression of Ifnb1 (mean ± SEM, n = 3) in different breast cancer cell lines after treatment with polyIC (1 μg/ml) for 12 h, normalized to sham-infected cells. The results were analyzed by one-way ANOVA followed by Tukey's test to determine differences. **, p < 0.01 MCF-7 vs other breast cancer cell types. 45 2.3.3 Intratumoral injection of miRNA-CVB3 impedes tumor growth in TNBC-bearing mice with high safety profile After verifying the efficacy of miR-CVB3 in vitro, we then conducted in vivo experiments to investigate the therapeutic efficacy of miR-CVB3 in 4T1-bearing mice. This mouse model was established by subcutaneously injecting 4T1 cells into female Balb/c mice. Once the tumor reached a palpable size (∼20 mm), mice were given intratumorally with PBS (sham), WT-CVB3 (1 × 106 PFU), or miR-CVB3 (1 × 106 PFU). After intratumoral application, we found that both WT-CVB3 and miR-CVB3 were able to hinder tumor growth compared with PBS-treated control group (Fig. 2-3A). At day 18 of the anti-tumor study, the average diameter of the tumor in WT-CVB3 and miR-CVB3 was 30.3 and 39.59 mm, respectively, while tumor size in control group reached 119.37 mm, verifying the high efficacy of WT-CVB3 or miR-CVB3 in tumor suppression. Whilst WT-CVB3-treated group demonstrated the least tumor growth, safety analysis revealed that administration of WT-CVB3 resulted in a significant loss in body weight (Fig. 2-3B). In stark contrast, no significant difference was observed in the body weight of the mice treated with miR-CVB3 compared with mice in control group (Fig. 2-3B). H&E staining revealed similar safety profile for miR-CVB3. Only modest pancreatic pathology (Fig. 2-3C and D) and negligible VP1 staining in the pancreas (not shown) were identified after miR-CVB3 treatment. Notably, a longer survival rate (60% up to day 28 post-treatment) was observed in mice that received miR-CVB3 compared with WT-CVB3 and PBS-treated mice (Fig. 2-3E). These results demonstrated that although WT-CVB3 could suppress tumor growth, its administration led to severe toxicity in which mice were unable to survive longer than the PBS-treated group. However, miR-CVB3 was able to significantly improve survival rate compared with WT-CVB3- and PBS-treated groups, indicating its remarkable efficacy as a safe treatment for breast cancer. 46 47 Figure 2-3. miR-CVB3 limits breast cancer growth in 4T1 tumor-bearing Balb/c mice with high safety profile. Female Balb/c mice at the age of ∼6–8 weeks were subcutaneously injected with 2 × 105 of 4T1 cells into the right flank of the mice. When tumor reached a tangible size, mice were randomly divided into three groups (sham, WT-CVB3, and miR-CVB3). The sham control mice were intratumorally injected with 80 μl of PBS, while mice in WT-CVB3 and miR-CVB3 groups were intratumorally inoculated with 1 × 106 PFU of WT-CVB3 and miR-CVB3, respectively (all groups received one injection). (A) Tumor size was measured every 2–3 days after treatment and presented as mean ± SEM (n = 5 mice in each group). The results were analyzed by one-way ANOVA followed by Tukey's test. *, p < 0.05. (B) Body weight of mice was measured every 2–3 days and expressed as % changes (n = 5 mice in each group). The results were analyzed by one-way ANOVA followed by Tukey's test to determine differences. ***, p < 0.001. (C) H&E staining of various tissues from a different cohort of mice received PBS, WT-CVB3 or miR-CVB3 (n = 5 mice in each group) and harvested at day 18 post-injection. (D) Pathological score of the H&E staining in (C). The results were analyzed by unpaired Student's t-test. **, p < 0.01. (E) Kaplan-Meier plot of survival rate. The differences in survival rate were evaluated by log-rank test. p < 0.05 between miR-CVB3 group and WT-CVB3 or PBS group in survival rate. Scale bar = 100 μM. SC, subcutaneous; IT, intratumoral; BW, body weight. 48 2.3.4 miR-CVB3 promotes immune cell infiltration into the tumor microenvironment and prevents tumor metastasis when given intratumorally Viral quantitation by VP1 immunostaining revealed that WT-CVB3 and miR-CVB3 could effectively replicate in tumor tissues when given by intratumoral injection (Fig. 2-4A and B). To evaluate the effect of WT-CVB3 and miR-CVB3 treatment on the recruitment of immune cells into the tumor site, we stained the tumor tissues for F4/80 (macrophage marker), CD4 (T helper cell marker) and CD8 (cytotoxic T cell marker). As shown in Fig. 2-4C and D, administration of either WT-CVB3 or miR-CVB3 resulted in a significant increase in the number of macrophages and T cells in the TME compared with the PBS control group. Finally, we investigated the possible anti-metastatic effect of WT-CVB3 and miR-CVB3 in TNBC-bearing mice. H&E staining of lung tissues revealed that both WT-CVB3 and miR-CVB3 hindered lung metastasis while evident cancer cell invasion could be found in the lungs of mice from the control PBS group (Fig. 2-4E and F). Together, our data suggest that both WT-CVB3 and miR-CVB3 are able to replicate in and lyse tumor cells, resulting in the recruitment of immune cells into the TME and the hampering of tumor metastasis to other tissues especially lung. 49 50 Figure 2-4. miR-CVB3 replicates in tumor cells, facilitates immune cell infiltration into the tumor microenvironment, and prevents tumor metastasis into the lung. Female Balb/c mice were implanted with 4T1 tumor cells and then treated with PBS, WT-CVB3, or miR-CVB3 . (A-B) IHC staining and quantification of viral capsid protein VP1. (C-D) IHC staining and quantification of immune cell markers, F4/80, CD4, and CD8 in tumors collected at day 18 post-treatment. Relative optical densities for VP1 and immune cell markers are presented as mean ± SEM (n = 3 mice in each group) and shown in (B) and (D), respectively. NIH Image J software was used for all quantification. The results were analyzed by one-way ANOVA followed by Tukey's test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar = 100 μM. (E) Mouse lungs (n = 3 mice in each group) were harvested at day 18 post-treatment for H&E staining (scale bar = 1000 μM). (F) The number of lung nodules was quantified. The results were analyzed by one-way ANOVA followed by Tukey's test. **, p < 0.01. 51 2.4 Discussion Breast cancer is a highly heterogeneous disease characterized by distinct molecular and pathological features that can be caused by a variety of genetic mutations. Growing evidence suggests that breast cancer cells are able to interconvert between different subtypes of tumor cells (122). Therefore, it is imperative to develop treatments that can target and destroy different breast cancer subtypes. Here we selected a number of breast cancer cell lines to evaluate the potency of the newly developed miR-CVB3 against them. We showed that miR-CVB3 is able to target and kill different human breast cancer cell lines, including T47D cells (luminal A), SK-RB-3 cells (HER2-enriched), SUM149 and MDA-MB-231 cells (TNBC), along with murine TNBC cell line (4T1 cells). Our results are in line with a recent study showing that CVB3 can kill diverse breast cancer cell lines (101). Among different cell lines tested, MCF-7 cells (luminal A) are the only insensitive cell line to our fabricated virus, possibly in light of low expression of CAR and high potency to produce IFN-I following virus infection. Consistent with our finding, different studies substantiated that cells with low expression of CAR and high potency of INFB1 production are less sensitive to CVB3 infections (123-125). Although western blot analysis showed low to no expression of the CAR in MCF-7 cells compared to other cell lines, future studies would benefit from further testing and comparing the levels of CAR available in the surface of these cells to facilitate virus attachment. It is recognized that the present study focused solely on the ability of MCF-7 cells to produce IFNB1. Future investigation is also required to compare the response of MCF-7 cells to IFN-I with other breast cancer cell types. The majority of previous studies on oncolytic CVB3 have been conducted in immunocompromised mice, disregarding the effects of immune system on both viruses and cancer 52 cells (90). For example, Tani group reported that intratumoral application of a new recombinant CVB3, established by inserting muscle-specific miR-1 and pancreas-specific miR-217 target sequences into the 3′UTR of the CVB3 genome, markedly suppresses tumor growth with negligible toxicities in nude mice bearing human TNBC cell xenografts (101). Given the recognized importance of intact immune system in cancer therapy as well as in virus-induced immunopathology, here we investigated the safety and anti-tumor activity of miR-CVB3 in a TNBC syngeneic mouse model. Our results showed that intratumoral inoculation of miR-CVB3 significantly restricts tumor growth and metastasis in 4T1-bearing immunocompetent mice compared with PBS-treated control mice. While mice who received WT-CVB3 exhibit significant weight loss and pancreotoxicity, we found that miR-CVB3 merely causes mild pancreotoxicity. It should be noted that, despite being effective in delaying tumor growth and improving survival rate up to 28 days, a single-dose injection of miR-CVB3 was unable to eradicate tumor. The tumor in mice that received miR-CVB3 continued to grow, although at a slower rate, and the mice were euthanized as they reached the human endpoint due to ulceration. Hence, multiple-dose injections might be more efficient in eliminating tumor and further improvement of survival rate. Moreover, we showed that after intratumoral administration, miR-CVB3 effectively replicates within the tumors and recruits immune cells, including macrophages, T helper cells and cytotoxic T cells, to the tumor sites. Although the detailed mechanism by which miR-CVB3 triggers the immune response remains elusive and requires future investigations, it is speculated that lytic viral replication causes the release of PAMPs, DAMPs, TAA, and cytokines into the TME, consequently activating the immune response towards tumor cells. Although the markers used in this study demonstrated increased infiltration of immune cells into the TME following treatment, further investigations can benefit from using a broader range of markers to determine the exact 53 types of immune cells present. For example, distinguishing between M1 and M2 macrophages is crucial as these subsets have distinct roles in tumor progression and immune response (126). Intratumoral versus systemic applications of OVs have their own advantages and restrictions. Here, we demonstrated that intratumoral injection of miR-CVB3 is effective in suppressing tumor growth in 4T1 tumor-bearing mice. However, we found that systemic delivery of miR-CVB3 fails to produce similar benefits (data not shown). The main obstacle for systemic administration of OVs such as miR-CVB3 is the fast recognition and clearance of OVs by the immune system, preventing sufficient delivery to tumor sites (127). Intratumoral injection has been considered as the most frequent route of oncolytic administration in preclinical or clinical trials for various types of tumors, as it guarantees the high accumulation of OVs in the TME; however, it cannot be applied in multifocal or unapproachable tumors, including brain malignancies or pancreatic cancers. Having noted that, the introduction of nanotechnology to the field has provided the opportunity for systemic administration of OVs. Diverse strategies including surface modification of viruses and encapsulation within nanoparticles, have been recently exploited for efficient systemic OV delivery and have shown outstanding potency (128, 129). In this Chapter, I evaluated the safety and efficacy of miR-CVB3 in treating breast cancer in cell culture model and immunocompetent mice. The in vitro data demonstrated that miR-CVB3 effectively infects and lyses a variety of breast cancer cells with different molecular characterizations, except for MCF-7 cells, which are resistant likely due to low expression of CAR and high potency of IFNB1 production. Moreover, the in vivo study revealed that miR-CVB3 is a safe OV with excellent anti-tumor activity towards TNBC. Altogether, these findings support miR-CVB3 as a promising OV candidate to be further characterized in additional in vivo models, such 54 as primary breast cancer mice and non-human primate models, before moving to future clinical application in breast cancer therapy. 55 Chapter 3: Combination of miR-CVB3 with chemo and immunotherapeutic drugs improves breast tumor suppression 3.1 Background Recently, cancer immunotherapy has gained considerable attention for the management of various malignancies. However, the existence of an immunosuppressive TME in some tumors poses a major obstacle and often limits the clinical application of these therapies. There is growing evidence suggesting that applying therapeutic agents with the capability of inducing ICD of tumor cells is an effective strategy to remodel the immunosuppressive TME and enhance therapeutic outcomes (130, 131). Induction of ICD can result in the release of TAAs and DAMPs, followed by commencing anti-tumor immune response (132). Exposure of calreticulin on the surface of tumor cells, together with the release of HMGB1 and secretion of ATP, are considered as the main markers for ICD (133). Exposed calreticulin serves as an 'eat me' signal that improves immunogenicity of the tumors (134), while released ATP acts as a 'find me' signal to enhance immune cell infiltration into the TME (135). Finally, extracellular release of HMGB1 induces inflammation to recruit additional immune cells (136). Release of all these molecules (i.e., DAMPs) along with TAAs can stimulate antigen-presenting cells (macrophages and dendritic cells), leading to activation of cytotoxic T lymphocytes towards cancer cells (137). Hence, combining current cancer immunotherapeutic strategy with ICD inducers can repress tumor growth in a collaborative mechanism. Melittin is the major component of bee venom with 26 amino acid residues (138). As a natural cationic peptide, it possesses numerous biological and pharmacological properties, such as modulating pro-inflammatory response, activating innate and adaptive immunity, and more 56 importantly, stimulating tumor cell cytotoxicity (139, 140). It has been shown that melittin is able to directly kill cancer cells through membrane permeability enhancement and consequent cell death (141, 142). Due to its immunomodulatory and anti-tumor effects, melittin has been employed as a therapeutic agent against various cancers (143, 144). For example, oncolytic Ads carrying the melittin gene showed promising anti-tumor efficacy in tumor-bearing mice (145). Additionally, oligodeoxynucleotides (ODN) containing CpG motifs (CpG-sequence) are well-known agonists for toll-like receptor 9 (TLR9) that can activate host defense mechanisms, including the maturation of antigen presenting cells. Briefly, upon internalization through endocytosis, CpG-sequence are processed in endosomes where TLR9 binds to the CpG motifs, leading to receptor activation (146). This activation triggers Myeloid differentiation primary response 88-dependent signaling pathways, resulting in the activation of transcription factors such as Nuclear Factor kappa-light-chain-enhancer of activated B cells, Interferon regulatory factor 7, and Activator protein 1. These transcription factors drive the production and secretion of various cytokines and chemokines, including IFN-I (e.g., IFN-α and IFN-β), which have antiviral properties and play a crucial role in the activation of both innate and adaptive immunity, pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-12) that enhance the inflammatory response and promote the activation and maturation of immune cells, and chemokines (e.g., CXCL10) that recruit additional immune cells to the site of infection or inflammation (147, 148). Studies have shown that the combination of CpG sequences with OVs can enhance the immune response against cancer cells, as compared to oncolytic therapy alone (149, 150). Rationale: The rationale for the utilization of miR-CVB3, in conjunction with melittin (Mel) and CpG sequences, lies in the ability of Mel and miR-CVB3 to directly target and rupture cancer cells, thereby releasing TAAs into the TME. This approach aims to enhance the efficacy of 57 cancer treatment. Furthermore, these components possess inherent immunostimulatory properties, which, when released together with TAAs, PAMPs, and DAMPs, can potentially reshape the immunosuppressive TME. The ability of Melittin to permeabilize cell membranes can enhance the delivery and spread of miR-CVB3 within the TME. By increasing the permeability of tumor cells, melittin facilitates the entry and replication of the OV, thereby boosting its oncolytic activity. In addition, CpG enhances the body’s immune response against cancer cells. This is particularly beneficial when combined with OVs like miR-CVB3, as it amplifies the anti-tumor immune response initiated by viral oncolysis. The specific aims outlined in this chapter are: (1) evaluating the efficacy of miR-CVB3 in synergy with melittin and CpG sequences to induce potent ICD in vitro, and (2) investigating the potential of this combined therapeutic approach for tumor size reduction and activation of the immune system in a murine model bearing 4T1 tumors. 3.2 Materials and methods 3.2.1 Cell culture The 4T1 cells (CRL-2539, a mouse triple-negative mammary tumor cell line isolated from Balb/c mice), MDA-MB231 cells (HTB-26™, a human triple-negative mammary tumor cell line), RAW 264.7 cells (TIB-71, macrophage-like cell line derived from Balb/c mice), and HeLa cells (CCL-2™, human cervical cancer cells) were purchased from the ATCC. The 4T1 and RAW 264.7 cells were cultured in Roswell Park Memorial Institute (RPMI) containing 10% FBS and 1% antibiotics (streptomycin, 100 μg/mL; penicillin, 100 U/mL). HeLa cells and MDA-MB231 cells were maintained in DMEM containing 10% FBS and 1% antibiotics (streptomycin, 100 μg/mL; penicillin, 100 U/mL). 58 3.2.2 Virus propagation Virus propagation was performed as described in section 2.2.2. 3.2.3 Preparation and characterization of CpGMel A fixed concentration (10 g/ml) of CpG oligodeoxynucleotides (CpG ODNs, 1826, Integrated DNA Technologies) was added to increasing concentrations (0, 3, 6, 12, 25, 40 g/ml) of melittin (>85% in purity, M2272, Sigma-Aldrich) and the mixture was incubated at room temperature for 1 hour. The formation of CpGMel complex was evaluated using gel retardation assay (2.5% agarose gel). Moreover, 2.5% agarose gel was applied to evaluate the probability of binding CpGMel to the surface of miR-CVB3 after incubating them at room temperature for 1 hour and purifying miR-CVB3 using a centrifugal filter. 3.2.4 Cellular uptake of miR-CVB3 and CpGMel To evaluate the internalization/replication of miR-CVB3, 4T1 cells were seeded into the 8-well chamber slides (104 cells per well) and 24-well plates (5 × 104 cells per well). MDA-MB231 cells were also seeded into 24-well plates (5 × 104 cells per well). The following day, cells were exposed to miR-CVB3 (MOI=1), or miR-CVB3+CpGMel (miR-CVB3 at an MOI of 1, melittin at a concentration of 10 g/ml, and CpG ODNs at a dose of 5 g/ml) for 1 hour. Then, the media were removed and replaced with fresh media. For examination of viral internalization/replication by confocal microscopy, after additional 16-hour incubation, cells were washed with PBS. After fixation in 4% paraformaldehyde and permeabilization with 0.1% Triton X-100, cells were blocked and then incubated with VP1 antibody (M47, Mediagnost, Germany) at 4C for overnight. Following additional incubation with Alexa Fluor® 488-conjugated secondary antibody (A11029, Invitrogen) at room temperature for 1 hour, cells were washed with PBS, mounted with 59 fluoroshield with 4, 6-diamidino-2-phenylindole (DAPI; F6057, Sigma-Aldrich), and subjected to Zeiss LSM 880 inverted confocal microscopy for imaging. For measurement of viral entry/replication by western blotting, after additional 16-hour incubation, both 4T1 and MDA-MB231 cells were lysed in buffer (10 mm HEPES pH 7.4, 50 mm Na pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 100 μm Na3VO4, and 0.1% Triton X-100). Western blotting was conducted using VP1 antibody as previously described (107). To evaluate the impact of miR-CVB3 on internalization of CpGMel, CpGMel was prepared using CpG(Cy5) (CpG ODNs, 1826, Integrated DNA Technologies ). Cells were seeded and treated with CpG(Cy5), CpG(Cy5)Mel or miR-CVB3+ CpG(Cy5)Mel (concentration of CpG(Cy5) for all treatments was 5 g/ml) for 5 hours. Confocal microscopy and flow cytometry (Gallios Flow Cytometer) were applied to investigate the uptake of CpG(Cy5). The results of flow cytometry were analyzed with FlowJo version 10 software. 3.2.5 In vitro anti-cancer study 3.2.5.1 Cell viability assay The 4T1 and MDA-MB231 cells were seeded onto a 96-well plate (104 cells per well). The following day, cells were treated with miR-CVB3, CpGMel or miR-CVB3+CpGMel, as described above, for 24 and 48 hours. Subsequently, 10 l of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution (G9243, Promega) was added into the culture, followed by another 3-hour incubation. The absorbance of each solution was measured using a microplate reader (BioTek Synergy H1) at 490 nm. The OD values of untreated cells were set as 100% viability, and the percentage of inhibition was then calculated. 60 3.2.5.2 Apoptosis detection The annexin V- Fluorescein isothiocyanate (FITC) staining was exploited to assess the apoptosis of cells treated with miR-CVB3, CpGMel or miR-CVB3+CpGMel. In brief, after exposure to miR-CVB3, CpGMel or miR-CVB3+CpGMel for 24 hours, 4T1 cells were harvested and resuspended in annexin binding buffer (V13246, Thermofisher Scientific). Subsequently, annexin V-FITC (5 μl) (A13199, Thermofisher Scientific) reagent was introduced to each sample and incubated for 20 mins in the dark. Finally, the stained cells were analyzed using flow cytometry. Data were analyzed with FlowJo version 10 software. 3.2.5.3 Detection of danger-associated molecular patterns Release and cell surface exposure of DAMPs, including ATP, calreticulin, and HMGB1, wereanalyzed after 4T1 cells were exposed to single treatment (miR-CVB3 or CpGMel) or combination therapy (miR-CVB3+CpGMel) for 24 and 48 hours. Specifically, the release of ATP into the supernatant was measured using RealTime-Glo™ extracellular ATP assay kit (GA5010, Promega) according to the manufacturer’s protocol. Briefly, 20 l of 4 RealTime-Glo™ extracellular ATP assay reagent was added into each culture. The luminescence was then measured using a microplate reader (BioTek Synergy H1). HMGB1 release was assessed by western blotting using HMGB1 primary antibody (651401, Biolegend). Briefly, the supernatant after treatment was collected and precipitated by the addition of equal volume of methanol and 0.25 volumes of chloroform. The mixture was vortexed and centrifuged for 10 mins at 20,000g. The upper phase was discarded. Subsequently, 500 l was added into interphase. Afterwards, the mixture was centrifuged for 10 mins at 20,000g. Finally, protein pellet was dried at 55C, resuspended in protein loading buffer, and subjected to western blotting. For calreticulin detection, after treatment, cells were incubated for 60 mins with anti- calreticulin (Alexa Fluor® 647) antibodies (ab196159, 61 Abcam) at 4°C in the dark. Following several washes, flow cytometry was applied to analyze the translocation of calreticulin. Confocal microscopy was also used to visualize calreticulin on the surface of the cells exposed to corresponding treatments. After fixation, the cells were incubated with anti-calreticulin (Alexa Fluor® 647) for 1 hour, followed by DAPI straining. 3.2.5.4 Reverse transcription-quantitative polymerase chain reaction RT-qPCR was conducted to measure the gene level of tnf-α and Il-6 in 4T1 cells treated with CpGMel, miR-CVB3 and miR-CVB3+CpGMel for 8 hours. Primers for the RT-qPCR analysis were synthesized by Integrated DNA Technologies and presented in Table 3-1. Briefly, after incubation, total RNA was isolated using RNeasy Mini Kit (Qiagen, 74104, Qiagen). The qPCR reaction containing 1 μg of RNA was conducted applying the TaqMan™ RNA-to-CT™ 1-Step Kit (4392653, Thermo Fisher Scientific) on a ViiA 7 Real-Time PCR System (Applied Biosystems). The results were normalized to β-actin mRNA. Samples were run in triplicate and analyzed using comparative CT (2−ΔΔCT) method with control samples and presented as relative fold changes. Table 3.1. Designed primers for RT-qPCR Target Forward Reverse Murine tnfα 5’- GTC CCC AAA GGG ATG AGA AGT T-3’ 5’- GTT TGC TAC GAC GTG GGC TAC A -3’ Murine Il6 5’- ACA ACC ACG GCC TTC CCT AC -3’′ 5’- TCT CAT TTC CAC GAT TTC CCA G -3’ Murine β-actin 5’- CAT TGC TGA CAG GAT GCA GAA GG -3’ 5’- TGC TGG AAG GTG GAC AGT GAG G -3’ 3.2.5.5 Macrophage activation To examine the activation of macrophages in vitro, the media of 4T1 cells treated with miR-CVB3, CpGMel or miR-CVB3+CpGMel for 12 hours were transferred to the plates seeded 62 with RAW 264.7 cells, followed by incubation for 24 hours. Subsequently, RAW 264.7 cells were collected and stained with CD80-PE (B340153, Biolegend) and MHC-II-Alexa Fluor® 647 (B346505, Biolegend) antibodies for 30 mins. The macrophage activation was then detected using a flow cytometer. Data were analyzed with FlowJo version 10 software. 3.2.6 In vivo anti-tumor study 3.2.6.1 Animals Six- to eight-week-old female Balb/c (000651, The Jackson Laboratory) mice were used for the in vivo studies. All animal procedures were performed in compliance with strict guidelines for the care and use of laboratory animals and were approved by the Animal Care Committee at the University of British Columbia (A18-0275). The ARRIVE guidelines were used for reporting animal research (151). 3.2.6.2 Therapeutic effects in a murine breast cancer model The 4T1 cells (5 × 105 cells) in 100 μl of cold PBS were subcutaneously injected into the right flank of female Balb/c mice. After about 10 days, once tumor reached a palpable size (~50 mm3), 4T1 tumor-bearing Balb/c mice were randomly divided into 4 groups (n=8 for each group), which were intratumorally treated with PBS, miR-CVB3 (105 PFU/mouse), CpGMel (CpG=50 µg/mouse and melittin=100 µg/mouse) or miR-CVB3+CpGMel, respectively. Treatments were performed twice on days 0 and 5. The length and width of the tumors were measured every three days using a digital caliper, and tumor volumes were calculated using the formula of (volume = length × width2 × 0.52). Furthermore, the tumor suppression rate (TSR) was calculated using the following formula: TSR (%) = [1 − (tumor volume of the treated group)/(tumor volume of the control group)] × 100 (%). According to our approved protocol, humane endpoints were defined as follows: mice losing ≥ 20% of their initial body weight, observation of ulceration in ≥ 10% of 63 the tumor region, the tumor size reaching ≥1.7 cm in diameter, or tumor weight exceeding 10% of body weight. Mice were kept for 40 days to evaluate the survival rate. Mice in each group were euthanized once they reached humane endpoints. Additionally, tumor metastasis into the lungs was assessed at the end of the experiment. Briefly, lung tissues were collected and fixed in 4% paraformaldehyde. Metastatic tumors in the lung were analyzed via H&E staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also performed on tumor tissues (collected at humane endpoints) to assess apoptosis according to the manufacturer’s protocol (G3250, Promega). 3.2.6.3 Safety analysis To assess the safety of each treatment, the body weight of mice in each group was measured every three days until the experimental endpoint. For safety measurement, a different cohort of mice (n=4 for each group) were treated with PBS, miR-CVB3, CpGMel or miR-CVB3+CpGMel as above. At 14 days post-treatment, mice were sacrificed. The heart, liver, spleen, lung, pancreas and kidney were collected and fixed in 4% paraformaldehyde for H&E staining. In addition, facial blood was collected on day 14 for the blood biochemistry analysis for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, lipase and cardiac troponin I levels by Advia 1800 (Advia 1). 3.2.6.4 Immune cell infiltration Two weeks after treatment, mice were sacrificed, tumor samples were paraffin-embedded and then sliced into 5 μm of thicknesses. The sections were deparaffinized, rehydrated, and then stained with anti-CD8 (sc-1177, Santa Cruz Biotechnology), anti-NK1.1 (14-5941-82, eBioscience), and anti-F4/80 (sc-377009, Santa Cruz Biotechnology) antibodies through IHC, as previously described (152), using the MACH4 Universal HRP-Polymer Detection System (BRI4012H, Biocare Medical) and hematoxylin solution Gill II (GHS232, Sigma-Aldrich). Lastly, 64 Aperio ScanScope AT (Digital slide scanner, Leica Biosystems Inc) was applied to attain whole-slide digital images. All the staining images were quantified using NIH ImageJ (version 1.52p) and the results were presented as relative optical density. Moreover, the level of IFN-γ, IL-6 and TNF-α, as well as translocated calreticulin and granzyme B, in tumor tissues were analyzed using immunofluorescence staining. Tumor tissues were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% FBS, and incubated with target antibodies including anti-IFN-γ (505802, Biolegend), anti-IL-6 (504502, Biolegend), anti-TNF-α (506302, Biolegend), and anti- calreticulin antibodies overnight. The following day, tissues were stained with Alexa Fluor® 488-conjugated secondary antibody (A11029, Invitrogen) for 1 hour, followed by mounting with DAPI. Zeiss LSM 880 inverted confocal microscopy was used to visualize proteins. 3.2.6.5 Inhibition of distant metastatic tumor For the distant tumor model, at 4 days after transplanting 4T1 cells into the right flank of mice (primary tumor, n=5), a distant tumor was implanted by subcutaneous injection of 4T1 cells (5 × 105 cells) into the left flank of each mouse. The primary tumors were treated as described before. The length and width of the distant tumors were measured every three days using a digital caliper. 3.2.7 Statistical analysis Statistical analysis was conducted by GraphPad Prism V8.0.1 software and all data are expressed as mean ± standard deviation (SD) (n ≥ 3). The results were analyzed by unpaired Student’s t test or one-way ANOVA followed by Tukey’s test to determine differences. The differences between survival rate were assessed by log-rank test. P-value < 0.05 was considered to be statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). 65 3.3 Results 3.3.1 Characterization of CpGMel and miR-CVB3+CpGMel Gel retardation assay was used to verify the formation of CpGMel and its binding to the surface of miR-CVB3. As shown in Fig. 3-1A, with an increase in the concentration of melittin, more CpG bound to the peptide, resulting in the complex stayed at the top of the gel. Twelve µg/ml of melittin was considered as the optimal concentration that could absorb 10 g/ml of CpG ODNs. This was confirmed by the absence of a band corresponding to free CpG sequence, indicating that maximal binding of CpG to melittin occurred. Moreover, as demonstrated in Fig. 3-1B, no corresponding band for CpGMel was detected after filtration of solution containing CpGMel in the absence of miR-CVB3, while in the presence of miR-CVB3, the CpGMel complex was unable to pass through the filter and the CpGMel band was once again visible, suggesting an interaction between miR-CVB3 and CpGMel. 3.3.2 Viral replication and uptake of CpG The cellular internalization and replication of miR-CVB3 before and after introduction of CpGMel were examined by confocal microscopy (Fig. 3-1C) and western blotting (Fig. 3-1D). Compared with administration of free miR-CVB3, no significant difference was observed in the expression of viral capsid protein VP1 in 4T1 cells and MDA-MB231 cells after applying miR-CVB3+CpGMel, indicating that CpGMel does not appear to interfere with internalization and/or replication of miR-CVB3 in 4T1 cells. Moreover, the uptake of CpG ODNs was visualized and quantified by confocal microscopy (Fig. 3-1D) and flow cytometry (Fig. 3-1F), respectively. Our results showed that addition of melittin improved the internalization of CpG ODNs into the 4T1 cells, while treatment of cells with miR-CVB3+CpGMel further enhanced CpG internalization as compared to melittin alone. Altogether, these results suggest that miR-CVB3 is able to internalize 66 and replicate in 4T1 breast cancer cells in the presence of CpGMel, and that both melittin and miR-CVB3 can enhance cellular uptake of CpG ODNs. 67 68 Figure 3-1. CpGMel does not affect miR-CVB3 internalization and replication in cancer cells, while miR-CVB3 enhances internalization of CpGMel to the cancer cells. (A) Gel retardation assay was performed to verify proper formation of CpGMel. (B) Examination of possible binding of CpGMel to the surface of miR-CVB3. (C) VP1 expression in 4T1 cells after treatment with miR-CVB3 or miR-CVB3+CpGMel for 16 hours using confocal microscopy. The green and blue fluorescences represent VP1 and nucleus, respectively. Scale bar=50 μm (left). The relative fluorescence intensity for each treatment was also quantified using NIH ImageJ and presented as mean ± SD (right). (D) Western blot analysis of VP1 level in 4T1 (left panels) or MDA-MB231 (right panels) cells treated as in (C). Densitometric analysis of VP1 protein levels was conducted applying NIH ImageJ, normalized to β-actin. (E) Assessment of CpG(Cy5) internalization into 4T1 cells after 5-hour treatment of cells with CpG(Cy5), CpG(Cy5)Mel or miR-CVB3+CpG(Cy5)Mel using confocal microscopy. The red and blue fluorescences represent Cy5 and nucleus, respectively. Scale bar=50 μm. (F) Evaluation of CpG(Cy5) internalization into 4T1 cells after 5-hour treatment of cells with CpG(Cy5), CpG(Cy5)Mel or miR-CVB3+CpG(Cy5)Mel using flow cytometry (n=3-5). The concentrations of each agent for cell treatment were as follow: miR-CVB3 at an MOI of 1, melittin at a concentration of 10 µg/ml, and CpG ODNs at a dose of 5 µg/ml. Data in this figure were analyzed by one-way ANOVA followed by Tukey’s test to determine differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). 69 3.3.3 Cell viability and apoptosis induction After characterization of the miR-CVB3+CpGMel, we then sought to determine its effects on cell viability and apoptosis. Firstly, cytotoxicity was quantifiably analyzed by MTS assay, after treatment of 4T1 and MDA-MB231 cells with PBS (sham), miR-CVB3, CpGMel and miR-CVB3+CpGMel for 24 or 48 hours. Our results demonstrated that all three treatments were able to decline cell viability in a time-dependent manner, with miR-CVB+CpGMel group showing the highest cytotoxicity (Fig. 3-2A). Apoptosis is a crucial mechanisms underlying the anti-cancer activity of therapeutic agents. The average percentage of annexin V-staining positive cells (indicative of apoptotic cells) was 40% and 45 % after treatment with CpGMel and miR-CVB3, respectively (Fig. 3-2B). However, combination therapy (miR-CVB3+CpGMel) induced a significantly greater proportion of apoptotic cells (approximately 80 %) in comparison with a single treatment (Fig. 3-2B). These results verify that combination therapy is more efficient to promote apoptosis in cancer cells. 3.3.4 In vitro induction of DAMP release and macrophage maturation It has been proven that ICD is able to generate an anti-tumor immune response in the TME (153). In our next step, the induction of ICD by miR-CVB3+CpGMel and single treatment was investigated by measuring DAMP release (i.e., calreticulin translocation to the surface of the cells, extracellular release of HMGB1 and ATP in treated cancer cells). While incubation with either miR-CVB3 or CpGMel resulted in a notable increase in calreticulin translation, the results obtained from confocal microscopy (Fig. 3-2C) and flow cytometry (Fig. 3-2D) revealed that treatment with miR-CVB3+CpGMel induced significantly increased translocation of calreticulin in 4T1 cells exhibited significantly greater expression of calreticulin compared with either miR-CVB3 or CpGMel alone. Similarly, western blot analysis demonstrated that treatment of cells with 70 miR-CVB3+CpGMel resulted in a greater release of HMGB1 compared to treatment with miR-CVB3 and CpGMel alone (Fig. 3-2E). In addition, measurement of extracellular ATP after 24- and 48-hour incubation of cells with each treatment revealed an increase in ATP level in a time-dependent manner (Fig. 3-2F). Among different treatments, the highest level of ATP in both time points was observed in the cells incubated with miR-CVB3+CpGMel. Collectively, our data indicate that treatment with miR-CVB3+CpGMel can more efficiently induce tumor cell release of DAMPs as compared to miR-CVB3 and CpGMel alone. We next set out to test whether single or combination treatment can stimulate the immune system. We demonstrated that incubation of macrophages with supernatant collected from 4T1 cells treated with either miR-CVB3 or CpGMel induced moderate maturation of macrophages (13.84% and 9.57 %, respectively), as evidenced by the induction of CD80 and MHC-II expression on macrophages (Fig. 3-2G). The cells positive for CD80 and MHC-II were significantly augmented to around 50% once they were cultured with the supernatant of the 4T1 cells treated with miR-CVB3+CpGMel (Fig. 3-2G). Additionally, the level of pro-inflammatory cytokines (Il-6 and Tnf-α) in 4T1 cells was analyzed by RT-qPCR after treating the cells with CpGMel, miR-CVB3 and miR-CVB3+CpGMel. Administration of CpGMel showed no impact on RNA level of IL-6 while significantly increased RNA level of tnf-α was observed upon CpGMel treatment compared with sham-treated cells (Fig. 3-2H). Both miR-CVB3 and CVB3+CpGMel treatments were able to enhance the RNA level of Il-6 and Tnf-α; however, the highest induction of Il-6 and tnf-α was found after incubation of cells with miR-CVB3+CpGMel (Fig. 3-2H). Overall, these findings indicate that miR-CVB3+CpGMel could induce higher DAMP release, greater macrophage maturation, and enhanced pro-inflammatory cytokine production as compared to treatment with individual agent. 71 72 Figure 3-2. miR-CVB3+CpGMel significantly increases apoptosis, DAMPs release and macrophage activation compared with miR-CVB3 and CpGMel. (A) Cell viabilities of 4T1 and MDA-MB231 cells after 24- and 48-hour incubation with CpGMel, miR-CVB3 or miR-CVB3+CpGMel (n=5). (B) 4T1 cells were analyzed for annexin-V after 24-hour incubation with different therapeutic regimens as indicated (n=3-5). (C) 4T1 cells were treated as above, followed by immunostaining of calreticulin. The purple and blue fluorescences represent calreticulin and nucleus, respectively. Scale bars=50 μm. (D) Quantified calreticulin by flow cytometry (n=3-5). (E) HMGB1 level in the supernatant of 4T1 tumor cells after various treatments was measured by western blotting (left) and quantified using NIH ImageJ (right). (F) Measurement of extracellular ATP at 24 and 48 hours after indicated treatments (n=5). The level of extracellular ATP in the different treatment groups was normalized to that of the sham group and presented the differences as %. (G) Evaluation of macrophage maturation (CD80 and MHC-II-positive cells) using flow cytometry after incubation of macrophages with supernatant collected from 4T1 cells treated with PBS (sham), CpGMel, miR-CVB3 and miR-CVB3+CpGMel, and non-treated (n=3). The 73 concentrations of each agent for cell treatment were as follow: miR-CVB3 at an MOI of 1, melittin at a concentration of 10 µg/ml, and CpG ODNs at a dose of 5 µg/ml. (H) RT-qPCR was performed to determine the level of tnf-α and Il-6 RNA in 4T1 cells after 6-hour incubation with various agents (n=3-5). Data in this figure were analyzed by one-way ANOVA followed by Tukey’s test to determine differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). 74 3.3.5 In vivo anti-tumor therapy and safety analysis After verification of the effectiveness in inducing cell death and immune activation in vitro, we then decided to assess the anti-tumor efficiency of the developed treatment in vivo in 4T1 breast tumor-bearing Balb/c mice. The anti-tumor therapeutic schedule is presented in Fig. 3-3A. When the implanted 4T1 tumors reached a volume of around 50 mm3, the mice were randomly separated into 4 groups, and each group received twice intratumoral injections on day 0 and 5. The tumor size and weight in each group are presented in Fig. 3-3B and C. The data showed that both miR-CVB3 and CpGMel treatment were able to significantly delay tumor growth, resulting in an average tumor size of 303.25 mm3 and 329.26 mm3, respectively, at day 21, compared to 745.9 mm3 in the control group. However, the miR-CVB3+CpGMel group exhibited the most outstanding anti-tumor performance, with an average tumor size of 120.75 mm3 at day 21 (Fig. 3-3B). The tumor suppression rate (TSR) was calculated in comparison to the PBS (sham) group. The TSR was 55.8% and 59.4% for the mice that received CpGMel and miR-CVB3, respectively, revealing that treatment with either CpGMel or miR-CVB3 alone efficiently suppressed 4T1 tumor growth (Fig. 3-3D). Notably, the miR-CVB3+CpGMel treatment displayed a more potent inhibition of tumor progression (83.8% TSR) than either treatment alone (Fig. 3-3D). Results of tumor weight confirmed the ability of the miR-CVB3+CpGMel therapy to suppress the growth of the malignant tumor (Fig. 3-3C). Moreover, Fig. 3-3E showed that treatment with miR-CVB3+CpGMel significantly improved the survival rate of 4T1-bearing mice compared with the three other treatment groups. As shown in Fig. 3-3F, no prominent alteration in the body weight was observed following the various treatments. Furthermore, we found that administration of miR-CVB3+CpGMel led to the highest apoptosis rate among all treatment groups, as measured by TUNEL staining of the tumor sections (Fig. 3-3G). Histological analysis was also exploited to 75 assess the safety of the major organs. H&E staining showed that all groups had normal histomorphology with no significant pathological abnormalities detected in the major organs (Fig. 3-3H). Furthermore, blood analyses of various biochemical markers revealed no evidence of cardiac, pancreatic, renal, and hepatic toxicity in any of the treatment groups (Fig. 3-3I). 76 77 Figure 3-3. miR-CVB3+CpGMel significantly inhibits tumor growth in 4T1 breast tumor-bearing Balb/c mice. (A) Diagram illustrating the therapeutic procedures. (B) The tumor size of mice in different groups (n=8 mice/group). (C) Average tumor weights in different groups (n=8 mice/group). (D) Tumor suppression rate after different treatments. (E) Animal survival curves following each treatment (n=8 mice/group). (F) Changes in body weights of mice after various treatment. (G) TUNEL staining of 4T1 tumor (left) and quantification of apoptosis rate by measuring fluorescent intensity signal (right, 3-5 slides/treatment), The green and blue fluorescences represent apoptotic cells and nucleus, respectively. Scale bar=100 μm. (H) H&E staining of different tissues collected at day 14 post-treatment (representative of 3-5 slides/treatment). Scale bar=50 μm. (I) Measurement of biochemical parameters, including creatinine, lipase, ALT, AST, and cardiac troponin I in the blood of 4T1 tumor-bearing mice after 21-day of treatment. Data represent the mean ± SD (n=5). The concentrations of each agent for animal treatment were as follow: miR-CVB3 concentration at 105 PFU/mouse, CpG concentration at 50 µg/mouse and melittin concentration at 100 µg/mouse. Data in this figure were analyzed by one-way ANOVA followed by Tukey’s test to determine differences. The differences in survival rates were evaluated by log-rank test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. IT, intratumoral. 78 3.3.6 In vivo assessment of inflammatory response and immune cell infiltration We next set out to further elucidate the anti-tumor mechanism of the developed treatments. The translocation of calreticulin in the TME was assessed by immunofluorescence (Fig. 3-4A). The tumor sections obtained from mice treated with miR-CVB3+CpGMel presented highest fluorescence intensity compared with sham group and single therapy, verifying the distinguished potency of miR-CVB3+CpGMel to induce ICD. It has been previously proven that TNF-α, IL-6, and IFN-γ play decisive roles in the response of immune cells against cancer cells (154). Immunofluorescence was applied to assess the levels of TNF-α, IL-6 and IFN-γ in tumors. As expected, miR-CVB3+CpGMel was the most effective treatment to boost the level of TNF-α, IL-6, and IFN-γ (Fig. 3-4A). Additionally, miR-CVB3+CpGMel therapy resulted in a significant enhancement in the level of granzyme B in the TME compare to the sham group, suggesting higher cytotoxic effect of T cells (Fig. 3-4A). We also analyzed the infiltration of macrophages, NK cells, and T cells into the TME with the IHC technique using anti-F4/80, NK1.1 and CD8 antibodies. We found that both CpGMel and miR-CVB3 were capable of increasing immune cells including F4/80-positive, NK1.1-positive and CD8-positive cells in the TME, but the highest number of immune cells was observed after miR-CVB3+CpGMel treatment (Fig. 3-4B). These results indicate that the combination treatment modality is the most efficient in stimulating immune cells against cancer cells. 3.3.7 Growth inhibition of distant and metastatic tumor We further investigated the effects of these treatment regimens on distant and pulmonary metastatic tumor. As shown in Fig. 3-4C, intratumoral injection of each treatment directly into primary implanted tumors was able to impede the growth of distant implanted tumors. At the end of the experiment, the average distant tumor sizes of mice that received CpGMel and miR-CVB3 79 were 568.8 and 610.9 mm3, whilst in the group treated with miR-CVB3+CpGMel, the tumor size was 202.2 mm3. Consistently, results on tumor weight substantiated the ability of miR-CVB3+CpGMel to suppress the growth of distant tumors (Fig. 3-4D). In addition, all treatments were able to prevent tumor metastasis into the lung compared with the sham group, as verified by H&E staining followed by counting the number of lung nodules (Fig. 3-4E). These findings support an immunotherapeutic effect of the developed treatment. 80 81 Figure 3-4. miR-CVB3+CpGMel increases immune cell infiltration into the tumor microenvironment and reduces distant tumor growth and tumor metastasis into lungs. (A) Immunostaining of calreticulin, IFN-γ, TNF-α, IL-6 and granzyme B in tumor tissues obtained at 14 days after indicated treatments. The purple, white, green and blue fluorescences represent calreticulin, cytokines, granzyme B and nucleus, respectively (left). Scale bar=50 μm. The relative fluorescence intensity from 3-5 slides/treatment was quantified (right). (B) IHC staining of immune cell markers, F4/80, NK1.1, and CD8 in tumor tissues collected at day 14 post-treatment (top). Scale bar=150 μm. Relative optical densities for immune cell markers are presented (bottom, n=3-5 slides/treatment). Densitometric analysis of protein levels was conducted applying NIH ImageJ. (C and D) Average tumor size (untreated tumor, C) and weight (D) of mice after various treatments (n=5 mice for each group). (E) H&E staining of lungs harvested from mice treated with various formulations at the endpoint (left, n = 5 mice/group, scale bar=500 μm), along with number of nodules in the lungs right). The concentrations of each agent for animal treatment were as follow: miR-CVB3 at 105 PFU/mouse, CpG at 50 µg/mouse and melittin at 100 µg/mouse. Data in this figure were analyzed by one-way ANOVA followed by Tukey’s test to determine differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). 82 3.4 Discussion In recent years, combination therapy has shown promising results for the suppression of various malignancies. Lately, employing OVs with the ability to lyse the cancer cells directly and modulate anti-tumor immunity has recaptured enhanced momentum in cancer treatment. For the current study, we applied a genetically engineered CVB3 through adding the target sequences of miRs that are overexpressed in normal tissues (miR-1 and miR-216) or downregulated in cancer cells (miR-143 and miR-145). It was previously shown that this miR-modified virus has a higher safety profile compared with wild type (WT)-CVB3 while maintaining the oncolytic activity (90). Here we demonstrated that the combination of miR-CVB3 with CpGMel greatly improves cytotoxic effect toward tumor cells both in vitro and in vivo as compared to the use of these agents separately. To prepare the combination treatment, melittin was initially incubated with and bound to the CpG ODNs via electrostatic interaction to form CpGMel complex, followed by addition of miR-CVB3. We found that binding CpG ODNs to melittin prevents the degradation of CpG ODNs by nucleases (data not shown), which is a significant challenge when using CpG ODNs alone for in vivo studies. Moreover, as CpG ODNs bind to melittin via electrostatic interaction, it is expected that alteration in pH, which is typically observed in the TME, leads to the separation of CpG ODNs from melittin without significantly affecting their functions. We confirmed that conjugation of CpGMel to miR-CVB3 has no major impact on viral fitness. Surface modification of OVs has been widely applied for various purposes including improving tumor targeting (155), preventing recognition and elimination by the immune system (156), and increasing therapeutic efficiency (70). Berry et al (70) demonstrated that conjugation of doxorubicin to the surface of reovirus does not influence virus performance, but rather elevates its oncolytic capacity. In this study, we also 83 found that miR-CVB3 improves the cellular uptake of CpGMel. No specific receptors on the surface of the cells were identified for CpG entry. Therefore, it is possible that binding of miR-CVB3 to its receptor facilitates the internalization of CpGMels that are in close contact with miR-CVB3. In this study, we assessed the oncolytic activity and cytotoxicity of miR-CVB3 as single treatment or combining with CpGMel(miR-CVB3+CpGMel). We also investigated possible mechanism of cell death and anti-tumor immunity using in vitro and in vivo models. We found that both miR-CVB3 and melittin have the capability of direct lysis of cancer cells and inducing apoptosis. It is known that miR-CVB3 can replicate and rupture the infected cells (90, 157), and melittin kills the cells by creating pores in the plasma membrane (158). Similar to our findings, different studies validated that the combination of Ovs with other common therapeutic approaches, such as chemotherapy and radiotherapy, could have synergistic effects and enhance cytotoxicity against cancer cells (159, 160). In addition to direct lysis, both Ovs and melittin have been previously shown to elicit anti-tumor immunity (161, 162). By rupturing cancer cells upon OV and melittin treatment, TAAs, DAMPs, and PAMPs are released into the TME, stimulating recruitment and activation of immune cells. Interestingly, we found that administrations of miR-CVB3+CpGMel could cause ICD, as evidenced by the release of DAMPs, such as ATP and HMGB1, and increased translocation of calreticulin. All these DAMPs can be recognized by antigen-presenting cells (macrophages and dendritic cells), followed by T cell recruitment into the tumor (163, 164). It is important to mention that in addition to the release of TAAs and DAMPs, miR-CVB3+CpGMel has two inherent immunostimulatory agents, miR-CVB3 and CpG ODNs, which serve as PAMPs and work together with the released TAAs and DAMPs to initiate a robust immunity toward cancer cells. Our in vitro 84 studies showed strong activation of macrophages after a combination treatment, while in vivo investigation revealed considerable infiltration of macrophages, T cells and NK cells into the TME. Although the markers used in this study demonstrated increased infiltration of immune cells into the TME following treatment, future investigations would benefit from employing a broader range of markers to precisely identify the specific immune cell populations involved. Combination therapy leads to significant rise in the level of proinflammatory cytokines including TNF-α and IL-6 both in vitro and in vivo, which play a crucial role in the induction of immune response. Accumulating evidence suggests that local treatment of established tumors with ICD inducer can result in remission of distant tumors, signifying the establishment of systemic immunity (165). Here we also showed that local administration of miR-CVB3+CpGMel can effectively delay the progression of distant tumors, suggesting a strong capacity of the developed treatment to induce systemic immunity against tumor cells. In this Chapter, the benefit of a combination therapy has been demonstrated using miR-CVB3, melittin, and CpG ODNs. It was shown the miR-CVB3 can improve the internalization of CpGMel into cancer cells, whereas addition of CpGMel does not affect virus performance. The results proved that the combination therapy (miR-CVB3+CpGMel) elicits greater tumor ICD in vitro and in vivo compared to individual treatment (miR-CVB3 and CpGMel). The release of DAMPs as a result of ICD along with miR-CVB3 and CpG ODNs induce increased recruitment of immune cells in the TME and initiate anti-tumor T cell response. Significant tumor suppression was achieved after applying miR-CVB3 and CpGMel as the single treatment; however, additive effect was observed following the administration of miR-CVB3+CpGMel. These findings verify that the combination of cancer immunotherapy, which is based on OV, with a chemotherapeutic agent can be a potential strategy for further clinical applications. 85 Chapter 4: Synergistic viro-chemoimmunotherapy in breast cancer enabled by bioengineered immunostimulatory exosomes and dual-targeted coxsackievirus B3 4.1 Background While exhibiting notable anti-tumor efficacy through intratumoral injection, challenges arise when treating deep-seated and metastatic tumors, diminishing the clinical viability of miR-CVB3. A rationale approach to address this is the systemic delivery. However, the effectiveness of this delivery method is hampered by the presence of neutralizing antibodies and rapid clearance of OVs after intravenous or intraperitoneal injection (166, 167). To tackle these challenges, scientists have endeavored to create innovative carriers for OVs (168). These include cellular carriers like mesenchymal stromal cells and osteosarcoma cells, alongside nanomaterials such as nanogold, graphene oxides, and liposomes (128, 169). As described in Chapter 1, Exo are extracellular nanovesicles surrounded by a lipid bilayer with approximately 30-100 nm in size (170). They hold significant potential as drug carriers (171). These naturally occurring structures are released by all cell types and play a crucial role in intercellular communication by transporting peptides, metabolites, and nucleic acids between both nearby and distant cells (172). Exo can be genetically engineered to enhance their capacity for targeted delivery and efficient uptake (173). Several tumor penetration and targeting agents have been successfully incorporated onto Exo surfaces to enable precise delivery of drugs into the cancer cells (174, 175). Despite demonstrated efficacy of Exo as drug carriers, recent studies indicate that Exo released by cancer cells might carry cargo molecules, such as EGFR, PD-L1, transforming growth factor-β, and other tumor-promoting molecules (176-178). These cargos play 86 a role in promoting immune evasion, triggering epithelial-mesenchymal transition, and ultimately contributing to tumor metastasis (178). The AS1411 aptamer is a non-immunogenic and thermally stable DNA aptamer, comprising 26 guanine-rich nucleotides with a quadruplex structure (179). The AS1411 aptamer can serve not only as a detection tool to identify nucleoin, a cell surface protein that is overexpressed in many cancer cells (180), but also as a targeting agent on the surface of various drug delivery systems (181, 182). In this Chapter, I plan to improve tumor delivery and therapeutic efficacy by encapsulating oncolytic CVB3 within Exo, through modifying Exo with the AS1411 aptamer, and by combining CVB3 with a chemotherapy drug. Rationale: The rationale for this study is that encapsulation of miR-CVB3 inside the Exo (ExomiR-CVB3) can protect the virus from attachment to neutralizing antibodies and clearance by immune system. Moreover, modification of the ExomiR-CVB3 with AS1411 aptamer (ExomiR-CVB3/Apt) and adding doxorubicin in it (ExomiR-CVB3/Dox/Apt) enhance its selectivity and efficacy towards cancer cells. The combination of doxorubicin with miR-CVB3 offers a compelling strategy for cancer therapy by integrating the potent chemotherapeutic effects of doxorubicin with the targeted oncolytic and immune-stimulatory properties of miR-CVB3. This synergistic approach enhances cytotoxicity, stimulates a robust anti-tumor immune response, and reduces the likelihood of resistance, potentially leading to improved treatment outcomes with manageable side effects. The specific aims outlined in this Chapter are: (1) evaluating the impact of miR-CVB3 infection on immune-related proteins and exosomal cargo, (2) comparing the performance of ExomiR-CVB3 with free miR-CVB3; and (3) assessing the the efficacy of ExomiR-CVB3/Dox/Apt in both in vitro and in vivo models. 87 4.2 Materials and methods 4.2.1 Cell culture The 4T1 cells (CRL-2539), a murine triple-negative mammary tumor cell line isolated from Balb/c mice, RAW 264.7 cells (TIB-71), a macrophage-like cell line derived from Balb/c mice, MDA-MB-231 (HTB-26™), SUM149 (CRL-2316), human triple negative mammary tumor cell lines HeLa cells (CCL-2™), human cervical cancer cells and MCF-7 cells (HTB-22™), luminal A mammary tumor cell lines were obtained from the American Type Culture Collection. The 4T1 and RAW 264.7 cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% FBS and 1% antibiotics (100 μg/mL streptomycin, 100 U/mL penicillin). MDA-MB-231, SUM149, and HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% FBS and 1% antibiotics (100 μg/mL streptomycin, 100 U/mL penicillin). 4.2.2 Animals Female Balb/c mice (000651, The Jackson Laboratory), aged between six to eight weeks, were employed for the in vivo investigations. All animal protocols adhered to rigorous standards for the ethical treatment and usage of laboratory animals, approved by the Animal Care Committee at the University of British Columbia (A22-0237). 4.2.3 Virus propagation Virus propagation was performed as described in section 2.2.2. 4.2.4 Isolation of Exo and ExomiR-CVB3 To generate Exo under sham infection (Exosham), 4T1 cells were seeded in T-75 flask. Upon reaching 80% confluency, the old medium was substituted with fresh medium, and the cells were incubated overnight. On the subsequent day, the media was harvested. The collected media underwent centrifugation at 2,000 × g for 30 minutes to remove cell debris and apoptotic bodies. 88 Subsequently, Exo in the supernatant were extracted using a commercially available kit (Thermo Fisher, 4478359). Briefly, the supernatant was mixed thoroughly with Exo isolation reagent at a 1:2 ratio and incubated for 24 hours at 4°C. On the following day, the samples were subjected to centrifugation at 10,000 × g for 1 hour at 4°C. Finally, the supernatant was discarded, and the Exo present in the pellet were resuspended in PBS for subsequent applications. To generate Exo under miR-CVB3 infection (ExomiR-CVB3), 4T1 cells were seeded in T-75 flask. Upon reaching 80% confluency, cells were infected with miR-CVB3 at a multiplicity of infection (MOI) of 1. After overnight infection, the medium was harvested and ExomiR-CVB3 was isolated as described above. To produce miR-CVB3 independently of Exo, cells underwent a 24-hour treatment with GW4869 (10 μM, Selleck Chemicals, S7609), an Exo -release inhibitor. Following this, the cells were exposed to miR-CVB3 infection overnight. The next day, miR-CVB3 present in the media was extracted for subsequent comparison with ExomiR-CVB3. Cryogenic transmission electron microscopy (CryoTEM) was used to visualize the structure of ExomiR-CVB3. Dynamic light scattering (DLS, Malvern Zetasizer) was used to determine size and zeta potential of ExomiR-CVB3. 4.2.5 Generation of ExomiR-CVB3/Dox The encapsulation of Doxorubicin (Dox,15007, Cayman Chemical)) within ExomiR-CVB3 (ExomiR-CVB3/Dox) was achieved through the mixing of Dox (1000 μg/mL) with ExomiR-CVB3 (400 μg/mL) in a triethylamine solution (Sigma-Aldrich, 471283) for overnight. The next day, the mixture was subjected to centrifugation at 10,000 × g for 30 minutes at 4°C to remove residual free Dox. The quantification of encapsulation efficiency (% EE) was undertaken indirectly by evaluating the residual unbound DOX content within the supernatant subsequent to the centrifugation process using the following equation: 89 % EE= (Initial amount of Dox - amount of Dox in supernatant)/Initial amount of Dox × 100. 4.2.6 Generation of ExomiR-CVB3/DoxApt To achieve target-specific delivery, the surface of Exo was covalently modified with the 5′-COOH modified AS1411 aptamer (GGT GGT GGT GGT TGT GGT GGT GGT GG, AS1411 Apt, Bio Basic Inc) employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)- N-hydroxysuccinimide (NHS) coupling. Briefly, Aptamer carboxyl groups were activated by introducing aptamer (10 µM, 50 μl) with EDC (46 mg, 0.3 mmol) and NHS (35 mg, 0.3 mmol) for 1 hour. Subsequently, the mixture was incubated with 1 ml suspension of increasing concentrations of ExomiR-CVB3 (20-80 mg/ml) or ExomiR-CVB3/Dox to generate ExomiR-CVB3/Apt or ExomiR-CVB3/DoxApt, respectively. The stable NHS ester promptly reacted with amino groups on the surface of Exo. Finally, 2.5% (w/v) agarose gel electrophoresis was performed to obtain the optimum concentration of the ExomiR-CVB3 that can absorb all aptamers. 4.2.7 Assessment of the impact of miR-CVB3 on immunological proteins in breast cancer cells and exosomes released by breast cancer cells To evaluate the effect of miR-CVB3 on immunological proteins in breast cancer cells and released Exo cargo, 4T1 cells were seeded in 12-well plates (5 × 105 cells per well). On the subsequent day, cells were subjected to an exposure to miR-CVB3 (MOI=1). After overnight incubation, Exo released in the media were isolated as mentioned above. Then, both cells and isolated Exo were thoroughly washed with PBS and lysed in buffer (10 mm HEPES pH 7.4, 50 mm Na pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 100 μm Na3VO4, and 0.1% Triton X-100). Proteins were subsequently subjected to separation using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated protein bands were then transferred onto a polyvinylidene difluoride membrane. To minimize non-specific binding, the 90 PVDF membranes were blocked using a 5% skimmed milk solution for 1 hour. Following this, the membranes underwent an overnight incubation with the primary antibody at 4 °C. The next day, after washing with TBST three times, membranes were incubated with secondary antibody for 1 hour at room temperature. Finally, chemiluminescent imaging and Image J were conducted to visualize and quantify protein expression, respectively. The antibodies used are as follows: anti-PD-L1 (A1645, ABclonal), anti-B7-H3 (A17216, ABclonal), anti-HSP70 (A4777, ABclonal), anti-calreticulin (A11563, ABclonal), anti-CD63 (A21923, ABclonal), anti-HMGB1 (A19529, ABclonal), anti-CD9 (A19027, ABclonal), anti-Alix (A2215, ABclonal), anti-CD47 (A21904, ABclonal), and anti-VP1 (M47, Mediagnost, Germany). To investigate Exo production following miR-CVB3 infection, T75 flasks were employed to culture 4T1 cells and MDA-MB-231 cells. On the subsequent day, the cells were subjected to either PBS (sham) or miR-CVB3 treatment (MOI=1) for overnight. On the next day, Exo present in the supernatant were harvested following the aforementioned procedure. The obtained Exo from both samples were washed using PBS and subsequently lysed using protein lysis buffer. The resulting proteins then underwent western blot analysis to assess the expression of exosomal marker proteins (Alix and CD9). Moreover, flow cytometry was applied to measure the level of calreticulin, PD-L1, B7-H3 and CD47 on the surface of breast cancer cells after miR-CVB3 infection. Briefly, 4T1 cells were seeded in 12-well plates (5 × 105 cells per well). The next day, cells were infected with miR-CVB3 (MOI=1). After 15 hours, cells were washed with PBS, collected, and incubated with Alexa Fluor® 647-calreticulin antibody (ab196159, Abcam) or PD-L1, B7-H3 and CD47 antibodies at 4 °C in the dark for 1 hour. Following additional 1-hour incubation with Alexa Fluor® 488 antibodies 91 (A11034, Thermo Fisher Scientific), the cells underwent washing steps and were then subjected to flow cytometry analysis. Furthermore, ATP release from cells after miR-CVB3 infection as a DAMP marker was measured. In this regard, 4T1 cells were subjected to either PBS or miR-CVB3 treatment (MOI=1), followed by an overnight incubation. The amount of ATP released into the supernatant was assessed utilizing the RealTime-Glo™ extracellular ATP assay kit (GA5010, Promega), following the manufacturer's instructions. Specifically, 20 μl of the 4 × RealTime-Glo™ extracellular ATP assay reagent was introduced to each culture. Subsequently, luminescence measurements were obtained using a microplate reader (BioTek Synergy H1). 4.2.8 Analysis of the effect of miR-CVB3 on immune-related proteins in the tumor microenvironment and exosomes secreted by tumor cells in mice Female Balb/c mice were subcutaneously injected with 4T1 cells (5 × 105 cells) suspended in 100 μl of cold PBS as previously described (51). After a period of about 10 days, during which the tumors reached a noticeable size of approximately 80 mm3, Balb/c mice harboring 4T1 tumors were randomly allocated into two groups (n=5 for each group). These groups were subsequently subjected to intratumoral interventions of PBS (sham) or miR-CVB3 (at a dosage of 106 PFU per mouse) twice on the day 0 and 5, respectively. Fourteen days following the treatments, the mice were sacrificed, and the tumor tissues were collected and preserved by fixation in 4% paraformaldehyde. The fixed samples were then treated with 0.5% Triton X-100-PBS at 4ºC for 10 minutes. Tissues were blocked with 10% FBS, then incubated overnight at 4ºC with primary rabbit antibodies (PD-L1, B7-H3 and CD47 antibodies). Following PBS washes, sections were stained with secondary Alexa Fluor® 488-conjugated antibody (A11029, Invitrogen) for 30 minutes at room temperature. Eventually, tumor samples were stained with fluoroshield mounting 92 medium containing 4, 6-diamidino-2-phenylindole (DAPI; F6057, Sigma-Aldrich) and subsequently imaged using Zeiss LSM 880 inverted confocal microscopy. In parallel, blood samples (0.5 mL) were collected from the mice and then processed by centrifugation at 3000 rpm for 15 minutes to facilitate the separation of the serum. The Exo present within the serum were isolated utilizing a commercially available kit (4478360, Thermo Fisher). Briefly, the serum underwent centrifugation at 2000 × g for 30 minutes to remove cellular elements and debris. Subsequently, the supernatant was mixed with an Exo isolation reagent (1:5 ratio) and incubated for 30 minutes at 4 °C. Following the incubation, the samples were subjected to centrifugation at 10,000 × g for 10 minutes at room temperature. The resulting pellet, containing the isolated Exo, was re-suspended in PBS. Western blot analysis was performed to compare the level of proteins in the Exo. 4.2.9 MiR-CVB3 and ExomiR-CVB3 comparison 4.2.9.1 Cell internalization Western blot analysis was conducted to evaluate the ability of miR-CVB3 and ExomiR-CVB3 to enter and replicate in cells. For this aim, 4T1 cells and CAR- and DAF-double knockout (KO) HeLa cells generated through CRISPR-Cas 9 knockout system were seeded in 12-well plates (5 × 105 cells per well). The next day, cells were exposed to miR-CVB3 (MOI=1) or ExomiR-CVB3 (MOI=1). After overnight incubation, cells were subjected to western blot analysis for viral protein VP1 detection. 4.2.9.2 Cell viability 4T1 cells, MCF-7 cells and RAW 264.7 cells were seeded in a 96-well plate at 5000 cells per well and cultured for 24 hours. miR-CVB3 (MOI=1) or ExomiR-CVB3 (MOI=1) was introduced into the cells and incubated for 24 or 48 hours. Following the respective incubation 93 times, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution was added to the cell cultures, followed by another 3-hour incubation. A microplate reader (BioTek Synergy H1) was used to measure the absorbance of the solutions at a wavelength of 490 nm. The absorbance of untreated cells served as a baseline, representing 100% cell viability. The percentage of inhibition was then calculated based on this baseline. In a separate experiment, miR-CVB3 and ExomiR-CVB3 were incubated with mouse serum containing CVB3-specific neutralizing antibodies, which was obtained from mice that were immunized with head-deactivated CVB3, at room temperature for 3 hours before being introduced to 4T1 cells. MTS assay was performed to evaluate the cytotoxicity of miR-CVB3 and ExomiR-CVB3 in the presence of these neutralizing antibodies. 4.2.9.3 Stimulation of immune cells To assess the immune-stimulating properties of miR-CVB3 and ExomiR-CVB3, RT-qPCR was employed to investigate the gene expression levels of tnf-α, cxcl10, ccl5, and ifnb1 in RAW 264.7 macrophages following a 2-hour infection with miR-CVB3 and ExomiR-CVB3. For the RT-qPCR analysis, primers were synthesized by Integrated DNA Technologies and are outlined in Table 4-1. Following the incubation, total RNA was isolated utilizing the RNeasy Mini Kit (Qiagen, 74104, Qiagen). The qPCR reaction, which contained 1 μg of RNA, was conducted using the TaqMan™ RNA-to-CT™ 1-Step Kit (4392653, Thermo Fisher Scientific) on a ViiA 7 Real-Time PCR System (Applied Biosystems). The resultant data were normalized to β-actin mRNA levels. Samples were analyzed in triplicate and the comparative CT (2 − ΔΔCT) method was applied using control samples, with the results presented as relative fold changes. 94 Table 4-1. Primers used for the RT-qPCR analysis In addition, following a 12-hour incubation of RAW 264.7 cells with miR-CVB3 and ExomiR-CVB3, the cell surface expression of CD80 and MHCII was assessed via flow cytometry. Briefly, after incubation, RAW 264.7 cells were collected and subjected to staining using CD80-PE (B340153, Biolegend) and MHC-II-Alexa Fluor® 647 (B346505, Biolegend) antibodies for 30 minutes. Subsequently, the activation status of macrophages was determined through flow cytometric analysis. The acquired data were analyzed using FlowJo version 10 software. 4.2.9.4 Antitumor activity A total of 5 × 105 4T1 cells suspended in 100 μl of PBS buffer were subcutaneously injected into the right flank of female Balb/c mice. Upon the tumors reached a size of ~80 mm³, the mice were randomly divided into three groups (n=6 for each group): Sham (PBS), miR-CVB3, and ExomiR-CVB3 groups. On day 0 and 5, mice were intratumorally injected with the same amount of virus (106 PFU per mouse). Following the treatment, tumor dimensions were measured using a digital caliper and calculated using the formula: volume = length × width² × 0.52. The tumor Target Forward Reverse Murine tnf-α 5’- GTC CCC AAA GGG ATG AGA AGT T -3’ 5’- GTT TGC TAC GAC GTG GGC TAC A -3’ Murine cxcl10 5’- GCT GGG ATT CAC CTC AAG AA -3’ 5’- CTT GGG GAC ACC TTT TAG CA -3’ Murine ccl5 5’- GCT TTG CCT ACC TCT CC -3’ 5’- TCG AGT GAC AAA CAC GAC TGC -3’ Murine ifnb1 5′-CTT GGA TTC CTA CAA AGA AGC AGC-3′ 5′-TCC TCC TTC TGG AAC TGC TGCA-3′ Murine β-actin 5′-CAT TGC TGA CAG GAT GCA GAA GG-3′ 5′-TGC TGG AAG GTG GAC AGT GAG G-3′ 95 suppression rate (TSR) was quantified as follows: TSR (%) = [1 - (tumor volume of treated group) / (tumor volume of control group)] × 100%. The mouse body weights were recorded every three days throughout the experiment. In accordance with our protocol, mice were euthanized if any of the following indicators were met: ≥ 20% reduction in body weight, ulceration covering ≥ 10% of the tumor area, tumor diameter ≥ 1.7 cm, or tumor weight exceeding 10% of body weight. Furthermore, to assess treatment effectiveness, survival rates were documented for each group. At the last day of observation, the mice were sacrificed, and tumor tissues were collected to assess apoptosis rates using the TUNEL assay as per the manufacturer’s instructions (G3250, Promega). Moreover, to study the immune stimulatory effect of various treatments, 4T1 tumor-bearing mice were randomly divided into three groups (n=5 for each group) and received the same treatments as described above. At 14 days post-treatment, tumors were harvested and fixed with 4% paraformaldehyde. The subsequent steps involved deparaffinization, rehydration of tumor sections, and staining with antibodies against CD8 (98941, Cell Signaling Technology), and F4/80 (70076, Cell Signaling Technology). IHC was carried out using the MACH4 Universal HRP-Polymer Detection System (BRI4012H, Biocare Medical) and Gill II hematoxylin solution (GHS232, Sigma-Aldrich) as previously documented [28]. Finally, digital images were obtained using the Aperio ScanScope AT (Digital slide scanner, Leica Biosystems Inc), and quantification analysis was executed via NIH ImageJ (version 1.52p), with results presented as relative optical density. Additionally, on day 14, tissues were collected and stained with H&E for pathological assessment. For the immunization study, prior to tumor cell injection, mice were immunized with heat-deactivated CVB3 (PFU=106) three times every 10 days. Following this, immunized-4T1 tumor-bearing Balb/c mice were divided into three groups (n=6 for each group) receiving PBS (sham), 96 miR-CVB3 (PFU=106), or ExomiR-CVB3 (PFU=106) intraperitoneally twice with a 5-day interval between doses. Antitumor activity of each treatment was monitored as described earlier. 4.2.10 Examination of cellular uptake and in vitro cytotoxicity of ExomiR-CVB3/DoxApt The level of nucleolin was evaluated in 4T1, SUM149 and RAW 264.7 cells by western blot analysis with nucleolin antibody (20910, ABclonal). To assess cellular internalization, 4T1 cells were cultured onto 8-well chamber slides at a density of 104 cells per well. On the subsequent day, the cells were exposed to Dox, ExomiR-CVB3/Dox, or ExomiR-CVB3/DoxApt, all at the same concentration of Dox (3 μg/mL). Following 6-hour incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X-100, and then blocked using 3% FBS. Finally, the cells were mounted with DAPI and imaged using Zeiss LSM 880 inverted confocal microscopy. Additionally, the cellular uptake of each formulation in 4T1 cells was quantified using flow cytometry. Briefly, seeded 4T1 cells were exposed to each treatment for 6 hours and the fluorescence signal of Dox in the cells was evaluated by flow cytometry (Gallios Flow Cytometer). Flow cytometry data were analyzed with FlowJo version 10 software. The potential cytotoxic impact of each formulation on 4T1 cells was assessed through the MTS assay. Cells were initially cultured at a density of 5000 cells per well for 24 hours. Subsequently, the cells were exposed to Dox, ExomiR-CVB3/Apt, or ExomiR-CVB3/DoxApt (MOI=1 for all treatments). After 24 hours, cells viability was measured as described above. 4.2.11 Assessment of the performance of ExomiR-CVB3/DoxApt A total of 5 × 105 cells 4T1 cells, suspended in 100 μl of PBS buffer, were subcutaneously implanted into the right flank of female Balb/c mice. Once the tumors reached a volume of 80 mm³, the mice were randomly divided into four groups (n=6 in each group): sham (PBS), ExomiR-CVB3, ExomiR-CVB3/Apt, and ExomiR-CVB3/DoxApt. Mice received two intraperitoneal 97 injections of the above treatments on the day 0 and 5, respectively. The concentration of miR-CVB3 was 105 PFU and concentration of Dox was 3 mg/kg. Antitumor activity of each treatment was evaluated as described earlier. At the end of the experiment, the mice were euthanized, and tumor tissues were collected to assess apoptosis rates using the TUNEL assay. The level of Ki67 in the tumor tissues was also evaluated using immunofluorescence technique with the Ki67 antibody (AB16667, ABclonal). In a separate experiment, 4T1 tumor-bearing mice that received the treatment as above were sacrificed on day 14 (n=5 for each group). Tumor tissues were collected to evaluate the immune cell infiltration in the TME and different organs were obtained for safety analysis. 4.2.12 Statistical analysis Statistical analysis was performed using GraphPad Prism V8.0.1 software. All data are presented as mean ± standard deviation (SD) with a minimum sample size of n ≥ 3. The outcomes were analyzed through unpaired Student’s t-test or one-way ANOVA, followed by Tukey’s test to identify variations. Differences in survival rates were evaluated using the log-rank test. A p-value of less than 0.05 denoted statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001). 4.3 Results 4.3.1 Infection with miR-CVB3 leads to a reduction in immune checkpoints and 'don't eat me' signals, while concurrently boosting 'eat me' and 'find me' signals To study the impact of miR-CVB3 on different immunological proteins, 4T1 cells were infected with miR-CVB3 (MOI=1). Western blot analysis revealed a significant decrease in the levels of PD-L1 and B7-H3, two well-known immune checkpoints, as well as CD47, a “do not eat 98 me” signal (Fig. 4-1A). Moreover, flow cytometry analysis corroborated these findings, showing reduced surface expression of these proteins upon miR-CVB3 treatment (Fig. 4-1B). Although western blot analysis indicated no substantial alterations in the levels of proteins that function as "eat me" or "find me" signals, such as calreticulin, heat shock protein 70 (HSP70), and HMGB1 (Fig. 4-1A), intriguingly, flow cytometry analysis demonstrated the translocation of calreticulin to the surface of cancer cells following infection (Fig. 4-1B). Furthermore, miR-CVB3 infection significantly enhanced the release of ATP, a potent “find me” signal, from cancer cells (Fig 4-1C). Consistently, in vivo studies revealed that infection of 4T1 tumor-bearing mice with miR-CVB3 resulted in decreased levels of PD-L1, B7-H3 and CD47 in the TME (Fig. 4-1D). These results provide compelling evidence of the capability of miR-CVB3 to remodel the landscape of immunological proteins in both cancer cells and the TME. 4.3.2 MiR-CVB3 infection not only amplifies the production and release of Exo from cancer cells but also augments their immunostimulatory potential Exo released from cancer cells often play a role in dampening or evading the immune system (183). These Exo encompass a variety of bioactive molecules, including proteins, nucleic acids, and lipids, which can mediate immunosuppressive effects (184). For instance, they might carry immune checkpoint molecules, like PD-L1, that engage with immune cells, such as T cells, and inhibit their activity (176). We next examined the impact of miR-CVB3 infection on the generation and release of Exo, along with their cargo, from cancer cells. As shown in Fig 4-1E, miR-CVB3 infection significantly enhanced Exo production and release from both MDA-MB-231 and 4T1 cell lines compared with non-infected cancer cells with an equal number of the cells, as indicated by the elevated protein levels of Exo markers CD9 and Alix. The observed increase in Exo production and secretion in infected cells may be attributed to the ability of CVB3 to induce 99 the autophagy process and enhance autophagosome production (185). As shown in Fig 4-1F, Exo released by cancer cells after infection contain miR-CVB3. Characterization of ExomiR-CVB3 using DLS revealed that ExomiR-CVB3 has the average size of 90.53 ± 8.7 and zeta potential of -21.6 ± 4.8. Moreover, western blot analysis revealed that Exo originated from miR-CVB3-infected cells exhibited reduced expression of PD-L1, but higher levels of HSP-70, calreticulin, CD47, and HMGB-1 (Fig. 4-1G). Notably, the detection of VP1 within the Exo released by infected cells serves as tangible evidence of the presence of miR-CVB3 in the exosomal cargo, underscoring the connection between the infection and the exosomal content (Fig. 4-1E, G). Furthermore, we extended our research to an in vivo context, where we examined the protein levels within Exo released by tumor cells following the administration of miR-CVB3 to 4T1 tumor-bearing mice. Strikingly consistent with our in vitro findings, a congruent pattern emerged, highlighting the pivotal role of miR-CVB3 in orchestrating the production of Exo characterized by enriched immunostimulatory cargo (Fig. 4-1H). Together, these findings elucidate the multifaceted impact of miR-CVB3, showcasing its dual role as an OV that induces cancer cell lysis, while simultaneously reshaping the protein landscape within cancer cells and influencing the composition of exosomal cargo. Importantly, this orchestrated transformation aligns favorably with immune system dynamics, suggesting the potential to enhance immune responses. 4.3.3 Exosomes released by cancer cells after miR-CVB3 infection can serve as a potential carrier for miR-CVB3 Currently, the widely accepted viewpoint maintains that pre-existing immunity attenuates the efficacy of OVs upon systematic administration, primarily due to the acceleration of viral clearance (166). Although OVs are unaffected by pre-existing antibodies after intratumoral injection, from a clinical perspective, administration of OVs systematically is more favorable as it 100 not only alleviates patient discomfort but also can be used for tumors that are inaccessible for intratumoral injection (166). 101 102 Figure 4-1. miR-CVB3 infection of breast cancer cells leads to altered expression levels of immunomodulatory proteins in both cell lysates and released exosomes. (A) Western blot (left) and quantitative (right) analysis of different immunomodulatory proteins as indicated in 4T1 cells after sham (PBS, n=3) or overnight infection with miR-CVB3 (MOI=1, n=3). Densitometric analysis of protein levels was conducted applying NIH ImageJ. (B) Flow cytometry analysis of distinct immunomodulatory proteins on the surface of 4T1 cells after sham (n=3-5) or overnight infection with miR-CVB3 (MOI=1, n=3-5). (C) The level of extracellular ATP in the 4T1 cell culture after sham or overnight infection with miR-CVB3 (n=3-5 for each group). (D) Immunofluorescence staining of immunomodulatory proteins (PD-L1, B7-18, and CD47) in the TME of 4T1 tumor-bearing mice treated with PBS (sham, n=3-5) or miR-CVB3 (PFU=106, n=3-5) for 14 days. (E) Western blot (top) and quantitative (bottom) analysis of different Exo markers (Alix and CD9) and VP1 in the Exo isolated from the medium of MDA-MB-231 and 4T1 cells, which were sham-infected (Exosham, n=3) or miR-CVB3-infected (ExomiR-CVB3, MOI=1, n=3) for overnight. Densitometric analysis of protein levels was conducted applying NIH ImageJ. (F) Structural analysis of ExomiR-CVB3 using CryoTEM. (G) Western blot (top left) and quantitative (top right) analysis of different immunomodulatory proteins as indicated in the Exo isolated from the medium of sham-infected (ExoSham, n=3) or miR-CVB3-infected (ExomiR-CVB3, MOI=1, n=3) 4T1 cells for overnight. Schematic illustration of the ExomiR-CVB3 formation after infection of cancer cell with miR-CVB3 (bottom). (H) Western blot (left) and quantitative (right) analysis of different immunomodulatory proteins as indicated in the Exo isolated from the serum of 4T1 tumor-bearing mice treated with PBS (sham, n=5) or miR-CVB3 (PFU=106, n=5). Densitometric analysis of protein levels was conducted applying NIH ImageJ. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by unpaired student’s t test. 103 Building on previous discussions, Exo secreted by cancer cells subsequent to miR-CVB3 infection encapsulate miR-CVB3, enriched with robust immunostimulatory properties that distinguish them from the typical Exo released by cancer cells (Fig. 4-1F). Moreover, these Exo, referred to as ExomiR-CVB3, exhibit increased levels of CD47 that acts as a “do not eat me” signal. By strategically incorporating CD47, ExomiR-CVB3 is expected to gain an extended circulation period post-administration, effectively shielding them from prompt recognition and elimination by immune cells. To corroborate the efficacy of ExomiR-CVB3 in comparison to free miR-CVB3, we conducted a comprehensive array of experiments as below. 4.3.4 ExomiR-CVB3 is able to infect cells that lack the coxsackievirus and adenovirus receptor and the decay-accelerating factor CVB3 predominantly relies on two key receptors, CAR and DAF, to gain access to host cells (91). We have previously shown that CVB3 has low efficacy in targeting and killing cells with little to no CAR receptors (94). Western blot analysis unveiled the successful cellular entry and replication of both ExomiR-CVB3 and free miR-CVB3 within 4T1 cells that are known to express both CAR and DAF receptors (94) (Fig. 4-2A). Moreover, the MTS assay demonstrated a comparable reduction in cell viability with both treatment approaches (Fig. 4-2B). To determine the impact of CAR and DAF receptors on ExomiR-CVB3 entry, we utilized the CAR- and DAF-double KO HeLa cells. We observed that ExomiR-CVB3 induced a significantly higher VP1 expression compared to free miR-CVB3 (Fig. 4-2C), highlighting the infection efficiency of ExomiR-CVB3 in cells expressing low/no levels of CAR and DAF receptors. We conducted additional experiments to assess the cytotoxicity of ExomiR-CVB3 in MCF-7 cells, known for their low CAR expression (94). Our findings indicate that while miR-CVB3 has limited impact on 104 MCF-7 cell viability, ExomiR-CVB3 significantly reduces cell viability. This implies the capacity of ExomiR-CVB3 to effectively infect and disrupt cancer cells characterized by low CAR expression (Fig. 4-2D). Furthermore, MTS assays conducted on RAW 264.7 cells, also characterized by restricted CAR expression, revealed that ExomiR-CVB3 does not induce any toxicity in normal cells. This outcome is attributed to the genome modification of CVB3 using miR modification approach, substantiating the safety profile of ExomiR-CVB3 (Fig. 4-2D). Collectively, these findings emphasize that encapsulation of miR-CVB3 in ExomiR preserves its efficacy in cells expressing CAR and DAF, while concurrently offering the advantage of precisely targeting cancer cells characterized by limited CAR and DAF expression. 4.3.5 ExomiR-CVB3 triggers a more pronounced activation of immune cells in comparison to unbound miR-CVB3 We next investigated the immune-stimulating capabilities of ExomiR-CVB3 by exposing RAW 264.7 murine macrophage cells to ExomiR-CVB3 and free miR-CVB3. Macrophages, which function as antigen-presenting cells, play a crucial role in orchestrating immune responses by releasing a range of pro-inflammatory cytokines. We assessed the expression levels of various proinflammatory genes, including tnf-α, cxcl10, ccl5, and ifnb1, following each treatment. While free miR-CVB3 led to a significant increase in pro-inflammatory gene expression compared to the control group, the most substantial enhancement was observed in cells treated with ExomiR-CVB3 (Fig. 4-2E). Furthermore, we measured the levels of costimulatory molecules, CD80 and MHCII, on the surface RAW 264.7 cells following each treatment using flow cytometry. We showed that ExomiR-CVB3 treatment elevated the percentages of CD80+ and MHCII+ cells by more than 10% and 15%, respectively, compared to miR-CVB3 treatment (Fig. 4-2F). Notably, in addition to miR-CVB3, the Exo released by cancer cells after miR-CVB3 infection contained a diverse array of 105 immunostimulatory molecules, including HSP70 and HMGB1. Importantly, these Exo harbor fewer immune checkpoint molecules, allowing them to synergistically activate immune cells to a higher extent when combined with miR-CVB3, in contrast to the administration of free miR-CVB3. Collectively, these findings underscore the potential of ExomiR-CVB3 as a promising candidate for cancer therapy, presenting remarkable immunostimulatory properties. 106 107 Figure 4-2. ExomiR-CVB3 outperforms free miR-CVB3, efficiently infecting cells with low viral receptor CAR and DAF expression, and stimulating more effective immune cell activations. (A) Western blot (left) and quantitative (right) analysis of VP1 protein in 4T1 cells that were sham-infected (PBS, n=3)) or overnight infected with free miR-CVB3 (MOI=1, n=3) or ExomiR-CVB3 (MOI=1, n=3). Densitometric analysis of protein levels was conducted applying NIH ImageJ. (B) Cell viability of 4T1 cells following 24- or 48-hour sham infection (n=3-5) or infection with miR-CVB3 (MOI=1, n=3-5) or ExomiR-CVB3 (MOI=1, n=3-5). (C) Western blot (top) and quantitative (bottom) analysis of VP1 in CAR- and DAF-double KO Hela cells after sham infection or 36-hour infection with free miR-CVB3 (MOI=1, n=3) or ExomiR-CVB3 (MOI=1, n=3). (D) Cell viability of MCF-7 and RAW264.7 cells following 48-hour sham infection (n=3-5) or infection with miR-CVB3 (MOI=1, n=3-5) or ExomiR-CVB3 (MOI=1, n=3-5). (E) RT-qPCR measurement of the mRNA levels of tnf-α, cxcl10, ccl5 and ifnb1 in RAW 264.7 macrophages treated with PBS (sham), miR-CVB3 (MOI=1) or ExomiR-CVB3 (MOI=1) for 2 hours (n=3-5 for each group). (F) Flow cytometry analysis of the maturation of RAW 264.7 macrophages (CD80 and MHC-II-positive cells) following incubation with PBS (sham), miR-CVB3 (MOI=1), or ExomiR-CVB3 (MOI=1) for overnight (n=3 for each group). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by ANOVA. 108 4.3.6 ExomiR-CVB3 exhibits remarkable antitumor efficacy and effectively mobilizes immune cells to infiltrate the tumor microenvironment Having established the potent cytotoxicity of ExomiR-CVB3 towards cancer cells and its impressive immunostimulatory properties, we proceeded to validate its effectiveness in vivo. To achieve this, we administered treatments to mice bearing 4T1 tumors according to the therapeutic schedule outlined in Fig 4-3A. Results presented in Fig 4-3B and 15C revealed that both ExomiR-CVB3 and miR-CVB3 effectively delayed tumor growth in comparison to sham-treated groups. While the average tumor size in sham group reached 1203.41±231.19 mm3 on day 21 post-treatment, the tumor sizes in miR-CVB3 treated group and ExomiR-CVB3 group were only 699.15±117.88 mm3 and 466.33±51.57 mm3, respectively (Fig. 4-3B). Notably, the highest rate of tumor inhibition was achieved through ExomiR-CVB3 treatment, resulting in a TSR of 62.9±4.2%, whereas the TSR for miR-CVB3 was 41.9±9.7% (Fig. 4-3D). It was also observed that ExomiR-CVB3 significantly prolonged the survival, demonstrating an impressive overall survival rate of 83.3% after 40 days of treatment (Fig. 4-3E). The efficacy was further evidenced by TUNEL staining, which revealed maximal tumor cell apoptosis in mice treated with ExomiR-CVB3 (Fig. 4-3F). Importantly, the body weight of mice across various treatment groups remained relatively stable throughout the treatment period (Fig. 4-3G). Histopathological examination through H&E staining also indicated no discernible pathological changes in major organs (heart, liver, spleen, lung, and kidney) in both ExomiR-CVB3 and miR-CVB3 treatment groups compared to the control group (Fig. 4-3H), suggesting excellent biocompatibility and biosafety. Furthermore, we evaluated the ability of ExomiR-CVB3 and miR-CVB3 to enhance immune cell infiltration within the TME. We observed that ExomiR-CVB3 treatment led to a 109 significant increase in CD8+ immune cells and F4/80 immune cells within tumor tissues compared to miR-CVB3 treatment (Fig. 4-3I). These results collectively highlight that Exo not only successfully deliver miR-CVB3 to tumor cells but also recruit a greater number of immune cells to the TME, ultimately leading to superior antitumor effects. 110 111 Figure 4-3. ExomiR-CVB3 demonstrates enhanced antitumor efficacy compared to free miR-CVB3 and effectively recruits immune cells to infiltrate the tumor microenvironment. (A) Schematic figure depicting the therapeutic protocols. (B) Tumor size in distinct treatment groups (n=6 mice/group). (C) Mean tumor weights across various groups (n=6 mice/group). (D) Tumor suppression rate (TSR%) at 21 days post-treatment in each group (n=6 mice/group). (E) Mouse survival rates after indicated treatment (n= 6 mice/group). (F) TUNEL staining of 4T1 tumors (left, scale bar=50 μm) and quantification of apoptosis rate by measuring fluorescent intensity signal (right, 3–5 slides per treatment). (G) Mouse body weight in each group post-treatment (n=6 mice/group). (H) H&E staining of distinct tissues from female Balb/c mice across various groups obtained at day 14 post-treatment (scale bar=100 µm). (I) IHC staining (top panels, scale bar=100 μm) and quantitative (bottom panels) analysis of immune cell markers (F4/80 and CD8) in tumor tissues collected at day 14 post-treatment (n = 3–5 slides per treatment). Densitometric analysis of protein levels was conducted applying NIH ImageJ. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by ANOVA. 112 4.3.7 Exosomes provide a protective shield for miR-CVB3, mitigating its swift clearance by immune cells following systemic administration Pre-existing immunity is a key challenge for systemic OV use. Existing antibodies from previous infections or vaccinations can rapidly neutralize the virus upon introduction, hindering its ability to reach the targeted tumor site. This scenario can limit the therapeutic effectiveness of the virus, necessitating strategies to bypass or counteract this immune response. To assess the capability of Exo to shield miR-CVB3 from neutralizing antibody binding, ExomiR-CVB3 and miR-CVB3 were incubated with serum containing neutralizing antibodies against CVB3. Subsequently, 4T1 cells were exposed to ExomiR-CVB3 and miR-CVB3. MTS assay demonstrated that in the presence of neutralizing antibodies, miR-CVB3 encapsulated in Exos exhibited a 2-fold increase in cytotoxicity compared to free miR-CVB3 (Fig. 4-4A). This finding implies that neutralizing antibodies can bind to free miR-CVB3, impeding its infectivity. We then extended our investigation to animal models to assess the protective effect of Exo on miR-CVB3. To achieve this, we pre-immunized Balb/c mice with heat-deactivated CVB3 before implanting tumor cells. The schedule of treatments is outlined in Fig 4-4B. The immunized 4T1 tumor-bearing mice were divided into three groups, receiving two intraperitoneal injections of PBS (sham), miR-CVB3, or ExomiR-CVB3. It is expected that Exo will counteract the attachment of neutralizing antibodies to miR-CVB3, consequently enhancing their ability to reach the TME with greater efficiency compared to free miR-CVB3 (Fig. 4-4C). The results, shown in Fig 4-4D and E, indicated minimal changes in tumor size and weight in mice receiving free miR-CVB3 compared to the control groups. In contrast, mice treated with ExomiR-CVB3 exhibited a significantly reduced tumor size and weight compared to the other two groups. Notably, ExomiR-CVB3 achieved a TSR of 45.8±9.7%, while miR-CVB3 treatment resulted in a TSR of only 113 9.3±7.6% (Fig. 4-4F). Moreover, ExomiR-CVB3 contributed to a significant extension in survival rates (Fig. 4-4G). No significant alteration was observed in the body weight following each treatment (Fig. 4-4H). Additionally, tumor tissues from mice treated with ExomiR-CVB3 showed notably higher levels of viral protein VP1 compared to those receiving miR-CVB3 (Fig. 4-4I), suggesting an elevated accumulation and replication of miR-CVB3 within the TME upon encapsulation in Exo. These cumulative results imply that Exo effectively shield miR-CVB3 from immune cells during systemic administration, making them excellent carriers to target cancer cells. 114 115 Figure 4-4. Exosomes provide a protective shield for miR-CVB3, preventing its interaction with neutralizing antibodies and subsequent elimination by immune cells. (A) MTS viability assay of 4T1 cells treated with PBS (sham), miR-CVB3 (MOI=1), or ExomiR-CVB3 (MOI=1) in the presence of mouse plasma containing neutralizing antibodies against CVB3 (n=5 for each group). (B) Schematic illustration depicting the therapeutic protocols. (C) Schematic representation highlighting the advantage of employing ExomiR-CVB3 over miR-CVB3, when neutralizing antibodies are present. (D) Tumor size assessment in distinct groups as indicated (n = 6 mice/group). (E) Tumor weights across various groups (n = 6 mice/group). (F) TSR% at day 18 post-treatment in each group (n = 6 mice/group). (G) Animal survival rate following each treatment (n = 6 mice/group). (H) Mouse body weight following treatment in each group. I) Immunostaining of VP1 (yellow fluorescence, scale bar=50 µm) and quantitative analysis (n=3 per group) in tumor tissues harvested at day 14 post-treatments. Densitometric analysis of protein levels was conducted applying NIH ImageJ. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by ANOVA. 116 4.3.8 Further modification of ExomiR-CVB3 increases its specificity and potency against cancer cells After confirming the potent anticancer and immunostimulatory effects of ExomiR-CVB3, we sought to enhance its therapeutic potential through additional modifications. Our strategy involved the surface modification of ExomiR-CVB3 with the AS1411 aptamer—a tumor-targeting molecule—alongside the incorporation of Dox into the ExomiR-CVB3. The selection of the AS1411 aptamer was based on its specific binding affinity for nucleolin, a protein that is prominently overexpressed on the surfaces of cancer cells as compared to normal cells (Fig. 4-5A). To confirm the successful coupling of the AS1411 aptamer onto ExomiR-CVB3, agarose gel electrophoresis was performed. Fig 4-5B demonstrated that the mobility of the AS1411 aptamer was impeded upon the introduction of ExomiR-CVB3, validating its attachment to the Exo surface (the complex was referred as to ExomiR-CVB3/Apt). Subsequent fluorometric analysis verified the successful loading of Dox, with an entrapment efficiency of approximately 33% (the new complex was designated as ExomiR-CVB3/DoxApt). The schematic illustration of the ExomiR-CVB3/DoxApt complex formation is presented in Fig 4-5C. For the evaluation of cellular uptake, confocal microscopy was employed, involving the treatment of 4T1 cells with Dox, ExomiR-CVB3/Dox, or ExomiR-CVB3/DoxApt. The confocal images revealed an enhanced cellular internalization of Dox when encapsulated within ExomiR-CVB3 (Fig. 4-5D). Significantly, the additional modification of ExomiR-CVB3 with the AS1411 aptamer markedly augmented its uptake by cancer cells (Fig. 4-5D). This finding was further supported by flow cytometry analysis (Fig. 4-5E), which showed a two-fold increase in internalization for ExomiR-CVB3/DoxApt compared to ExomiR-CVB3/Dox. In addition, in vitro evaluation of cytotoxicity revealed a 20% greater reduction in cell viability for ExomiR-CVB3/Apt compared to ExomiR-CVB3 in 4T1 cells 117 (Fig 4-5F). Notably, ExomiR-CVB3/DoxApt exhibited the highest cytotoxicity against 4T1 cells, suggesting the synergistic effect of miR-CVB3 and Dox in cancer cell elimination. Taken together, these findings highlight that the additional modification of ExomiR-CVB3 with the AS1411 aptamer and Dox not only amplifies its cancer cell selectivity but also enhances its therapeutic efficacy. 118 119 Figure 4-5. Further modification of ExomiR-CVB3 with the AS1411 aptamer and Doxorubicin enhances its specificity and cytotoxic potential. (A) Western blot analysis of nucelolin in RAW 264.7 macrophages, 4T1 and SUM149 breast cancer cells. (B) Agarose gel evaluation of the formation of Exo-miR-CVB3/Apt through EDC-NHS coupling. The AS1411 Apt (10 µM) was activated by EDC/NHS and subsequently added to increasing concentrations of Exo-miR-CVB3 as indicated. After a 2-hour incubation at room temperature, agarose gel electrophoresis was performed. (C) Schematic illustration of the assembly process of ExomiR-CVB3/DoxAp. (D) Confocal images of 4T1 cells incubated with different formulations as indicated (n=3 for each group). Scale bars=50 µm. Relative fluorescent intensity was measured using NIH Image J. (E) Flow cytometry results of 4T1 cells treated with different formulations (n=3-5 for each group). (F) MTS viability assay of 4T1 cells treated with different formulations as indicated for 24 hours. *, p<0.05; **, p<0.01; ***, p<0.005; and ****, p<0.001 by ANOVA. 120 4.3.9 ExomiR-CVB3/DoxApt exhibits potent tumor-targeting efficacy in mice bearing breast tumors To evaluate the antitumor efficacy, systemic safety, and immunostimulatory properties of ExomiR-CVB3/DoxApt, we performed an in-depth in vivo study using Balb/c mice bearing 4T1 tumors. When the implanted tumors reached a palpable size, we stratified the Balb/c mice into four distinct groups, with each group receiving two intraperitoneal injections of PBS, ExomiR-CVB3, ExomiR-CVB3/Apt, or ExomiR-CVB3/DoxApt. The experimental workflow is depicted in Fig 4-6A. As shown in Fig 4-6B and 4-6C, tumor growth was variably suppressed across the treatment groups when compared to the PBS control. Specifically, mice administered with ExomiR-CVB3 displayed an average tumor size of 936.2±97.7 mm3 on day 21, whereas the ExomiR-CVB3/Apt-treated group exhibited an average tumor size of 639.1±49.7 mm3 on the same day, indicating that the AS1411 aptamer enhances the accumulation of ExomiR-CVB3 within the TME, consequently yielding enhanced antitumor effects. Significantly, treatment with ExomiR-CVB3/DoxApt resulted in the most pronounced inhibition of tumor growth, as evidenced by an average tumor size of 325.7±102.3mm3 (Fig. 4-6B). It was also observed that TSR% in the ExomiR-CVB3/DoxApt group reached an impressive 75.1±7.7%, markedly surpassing the respective 28.7±7.4% for ExomiR-CVB3 and 51.3±3.7% for ExomiR-CVB3/DoxApt (Fig. 4-6D). Moreover, survival analysis demonstrated a higher survival rate (100% up to 40 days) among mice receiving the ExomiR-CVB3/DoxApt treatment (Fig. 4-6E). Further insights were gained from TUNEL and Ki67 staining, showing that ExomiR-CVB3/DoxApt induced the highest apoptosis rate and significantly inhibited tumor cell proliferation (Fig. 4-6F). Importantly, body weight monitoring throughout the treatment period indicated no notable weight loss within the treated groups (Fig. 4-6G). Treatment safety was validated by histopathological assessment of collected 121 tissues through H&E staining, revealing no significant pathological issues (Fig. 4-6H). This observation suggests the absence of drug leakage and systemic toxicity during treatment. Additionally, we examined the immune responses elicited by each treatment. The immunofluorescence images showed elevated levels of granzyme B in the groups treated with ExomiR-CVB3/DoxApt (Fig. 4-6I). IHC revealed a substantial increase in the population of CD8+ T cells, coupled with an increase in macrophages, following ExomiR-CVB3/DoxApt administration (Fig. 4-6I). Together, these findings indicate the potent anti-tumor effects of ExomiR-CVB3/DoxApt. This treatment not only hinders tumor growth but also stimulates a robust immune response within the in vivo setting, as evidenced by enhanced granzyme B levels and increased infiltration of immune cells. These results collectively validate the potential of ExomiR-CVB3/DoxApt as a promising therapeutic approach. 122 123 Figure 4-6. The systemic administration of ExomiR-CVB3/DoxApt markedly diminishes tumor growth, enhances survival rates, and amplifies the infiltration of immune cells into the tumor microenvironment. (A) Schematic illustration of treatment timeline. (B) Alterations in tumor size in 4T1 tumor-bearing mice after different treatments as indicated (n=6 mice per group). (C) Tumor weight of mice following distinct treatments (n=6 per group). (D) TSR% of each treatment in 4T1-tumor-bearing mice (n=6 per group). (E) Survival curves in mice treated with distinct therapeutic formulations (n=6 per group). (F) Immunostaining of Ki67 (top) and TUNEL (bottom) in tumor tissues isolated from each group (n=3 for each treatment condition). Scale bar=50 µm. (G) Changes in body weight of mice following each treatment (n=6 per group). (H) H&E staining of various organs collected at day 14 post-treatment (n = 3–5 slides per treatment). (I) Immunostaining of granzyme B and IHC images of immune cell markers (F4/80 and CD8) in tumor tissues from different groups harvested at day 14 post-treatment along with quantitative analysis (n=3–5 slides per treatment) using NIH ImageJ. *, p<0.05; **, p<0.01; ***, p<0.005; and ****, p<0.001 by ANOVA. 124 4.4 Discussion The ability of miR-CVB3 to modulate immune-related proteins within both cancer cells and Exo signifies a promising achievement in the field of cancer therapy. This transformation towards a more immunostimulatory profile within the TME holds great promise for amplifying the body's immune response against cancer. By reshaping a previously suppressive microenvironment into one that actively supports the immune system, miR-CVB3 not only directly targets cancer cells but also empowers the body's intrinsic defenses against cancer. Immune checkpoints are pivotal regulatory molecules in maintaining immune system balance. However, in the context of cancer, certain tumors exploit these checkpoints as a means of immune evasion (186). What sets miR-CVB3 apart is its unique ability to not only induce ICD, marked by the upregulation of associated markers but also reduce the expression of immune checkpoint proteins like PD-L1 and B7-H3 in breast cancer cells. This dual effect positions miR-CVB3 as a powerful tool in overcoming the immune evasion tactics employed by cancer. Furthermore, the capacity of miR-CVB3 to alter the cargo of Exo, enhancing their aptitude for activating immune cells, introduces an innovative avenue for therapeutic delivery. Exo released from cancer cells post-miR-CVB3 infection carry both the virus and immunostimulatory cargo, presenting a multifaceted approach to virus-mediated delivery. This dual functionality underscores the diversity of Exo as versatile delivery vehicles in cancer therapy. In the landscape of oncolytic virotherapy, a significant challenge lies in the presence of neutralizing antibodies, especially when considering systemic administration (166). Numerous studies have demonstrated that systemic administration of OVs often proves ineffective due to rapid immune system clearance. Researchers have endeavored to address this challenge by modifying the virus's surface or utilizing nanoparticles (168, 187). However, such modifications 125 may compromise the virus's ability to infect or eliminate cancer cells (188). Notably, this study's observations indicate that Exo can effectively shield miR-CVB3 from neutralizing antibodies and swift immune cell clearance. This revelation indicates the potential of Exo to serve as a protective barrier, ensuring the persistence and effectiveness of therapeutic agents within the TME. Such immune evasion mechanisms are pivotal for achieving sustained and effective treatment outcomes. Finally, the strategic engineering of ExomiR-CVB3 to incorporate the AS1411 aptamer and Doxorubicin (DoxApt) represents a significant advancement. This modification is aimed at enhancing treatment efficacy by selectively targeting breast cancer cells, triggering cell death, and fortifying the presence of immune cells within the TME. Although the markers applied in this study showed enhanced infiltration of immune cells into the TME following treatment, future investigations would benefit from employing a broader range of markers to precisely determine the specific immune cell populations involved. The diverse potential of ExomiR-CVB3/DoxApt offers a comprehensive therapeutic strategy that addresses multiple facets of cancer treatment, ranging from direct tumor cell targeting to bolstering the antitumor immune response In this Chapter , I have shown the potential of miR-CVB3 in reshaping the landscape of immune-related proteins towards a more immunostimulatory profile. Beyond this, I demonstrated that miR-CVB3 exhibits the capacity to modulate the cargo of Exo, thereby enhancing their ability to activate immune cells. Importantly, my observations revealed that the Exo released from cancer cells following miR-CVB3 infection also harbor the virus, presenting a novel avenue for virus-mediated delivery. Moreover, my investigations have demonstrated the capacity of Exo to effectively shield miR-CVB3 from neutralizing antibodies and rapid immune cell clearance, thus positioning them as promising carriers for the systemic delivery of OVs. Expanding on my findings, I have engineered ExomiR-CVB3 to encompass the AS1411 aptamer and Dox, a strategic 126 modification aimed at enhancing therapeutic efficacy. My comprehensive in vitro and in vivo analyses have substantiated the multifaceted potential of ExomiR-CVB3/DoxApt. Beyond its ability to selectively target breast cancer cells and induce cell death, this treatment regime demonstrates a remarkable capability to augment immune cell presence within the TME. Taken together, my findings point to the significant potential of ExomiR-CVB3/DoxApt as one of the leading candidates in breast cancer treatment. The versatility demonstrated by this approach also suggests its potential applicability in diverse cancer types. 127 Chapter 5: Closing Remarks 5.1 Research summary and conclusions Chapter 2: miR-CVB3 can be considered as a potential candidate for breast cancer treatment. The major findings of this chapter are: 1- miR-CVB3 is a potent OV against most human and mouse breast cancer cells. 2- MCF-7 breast cancer cells are resistant to miR-CVB3-1.1 possibly due to low expression of CAR and high potency of Infb1 production. 3- miR-CVB3 is able to decrease tumor volume in 4T1-tumor-bearing mice. 4- Local administration of miR-CVB3 enhances immune cell infiltration into the TME. Chapter 3: Combination of miR-CVB3 with other chemotherapeutic or immunotherapeutic drugs can result in better clinical outcomes. The major findings of this chapter are: 1- While CpG sequence and melittin have no effect on the internalization and replication of miR-CVB3-in 4T1 cells, miR-CVB3 enhances the internalization of CpG into tumor cells. 2- The combination of miR-CVB3 and CpG sequence and melittin reduces cell viability and promotes ICD in breast cancer cells. 3- Compared to individual treatments, the combined application of miR-CVB3 and CpG sequence and melittin exhibits heightened antitumor activity and a greater capacity to stimulate the immune system. Chapter 4: Exo serve as excellent vehicles for the systemic delivery of miR-CVB3. The major findings of this chapter are: 128 1- miR-CVB3 has the potential to transform immunosuppressive cancer cells into immunostimulatory ones through its influence on immune-related proteins and exosomal cargo. 2- The encapsulation of miR-CVB3 within Exo not only shields them from neutralizing antibodies but also facilitates systemic administration, expanding its therapeutic options. 3- The additional modification of miR-CVB3-loaded Exo with AS1411 aptamer and doxorubicin not only enhances their safety profile but also augments their efficacy against cancer cells. 5.2 Conclusion In conclusion, miR-CVB3 emerges as a promising candidate in the realm of cancer therapy. It exhibits the ability to effectively target various cancer cells, demonstrating exceptional safety in mouse models while causing significant tumor shrinkage. The true potential of miR-CVB3 shines when combined with other therapeutic elements, creating a powerful synergy that enhances immune responses and drives ICD within tumors. Additionally, miR-CVB3's role in altering immune-related proteins within cancer cells and Exo contributes to creating a more immune-supportive environment within the TME. his change enhances the antitumor effect and suggests new avenues for immunotherapeutic strategies. Delivering miR-CVB3 via Exo improves both safety and efficacy, providing a versatile and efficient method for therapeutic delivery. Overall, miR-CVB3 offers a promising approach in cancer treatment, highlighting the potential of combination therapies to utilize the immune system while reducing adverse effects. This research paves the way for new possibilities in cancer therapy. 129 5.3 Limitation and future direction 5.3.1 Animal model specificity and diversity While the results obtained in Balb/c mice bearing 4T1 breast tumors are promising, it is imperative to emphasize the need for a comprehensive assessment of the treatment's safety and efficacy across a broader spectrum of animal models. For instance, using genetically engineered mouse models such as MMTV-PyMT mice can provide insights into how the treatment performs in a more genetically relevant setting. Additionally, testing in immunodeficient models like Non-Obese Diabetic Severe Combined Immunodeficiency mice can help understand the role of the immune system in the treatment's efficacy. Furthermore, employing spontaneous tumor models or patient-derived xenografts can offer a closer approximation to human cancer biology. Thus, it becomes imperative to replicate these findings in a range of mouse strains and animal models to ensure the reliability and generalizability of the observed outcomes. 5.3.2 Translational challenges and human complexity Moreover, it is crucial to acknowledge the inherent limitations of using mouse models as proxies for human breast cancer. While these models provide valuable insights, they may not fully recapitulate the complexity of breast cancer in humans, which exhibits diverse molecular subtypes, genetic alterations, and responses to therapy. Therefore, to bridge the translational gap between preclinical studies and clinical applications, it is imperative to expand the research scope to encompass patient-derived xenograft models and other relevant animal systems that better mirror the intricacies of human breast cancer. 5.3.3 Long-term effects and relapse dynamics Another significant aspect that warrants in-depth exploration is the assessment of long-term effects associated with miR-CVB3 treatment. While the short-term outcomes in animal 130 models are promising, understanding the potential for late-stage side effects, tumor recurrence, and the development of treatment resistance over an extended duration is paramount. Such insights are crucial for delineating the sustainability and durability of the treatment, ensuring that it remains effective over the course of a patient's journey. 5.3.4 Immunological considerations and the tumor microenvironment Additionally, this research, while highlighting the immune-enhancing effects of miR-CVB3 in the TME, did not comprehensively explore the multifaceted role of the immune system following treatment administration. To further confirm the immunostimulatory effect of miR-CVB3 administration, it is essential to evaluate the exact subtypes of immune cells present in TME. A comprehensive examination of the immune response within the TME, including the dynamics of immune cell populations, cytokine profiles, and immune checkpoint interactions, is warranted. Understanding how miR-CVB3 modulates these immune components can provide insights into its mechanism of action and guide the development of combination therapies that exploit the full potential of the immune system in combating breast cancer. In summary, while the research conducted on Balb/c mice bearing 4T1 breast tumors is a promising foundation, the journey towards clinical translation and maximizing the impact of miR-CVB3 in breast cancer therapy necessitates a broader and more comprehensive research approach. This includes evaluating diverse animal models, considering the complexities of human breast cancer, assessing long-term treatment effects, and delving deeper into the immunological intricacies of the TME. These endeavors will enhance our understanding and pave the way for more effective and sustainable breast cancer treatments in the future. 131 5.4 Significance Breast cancer is one of the most prevalent and challenging diseases affecting millions of individuals worldwide. Despite substantial progress in understanding and treating it, breast cancer remains a global health concern. Our study offers a novel and innovative approach to tackle this disease, providing hope for improved outcomes and survival rates for patients. The development of miR-CVB3 as a safe and effective treatment option is promising. This innovative approach introduces a new dimension to cancer therapy by selectively targeting cancer cells while sparing healthy tissues. It has the potential to enhance the effectiveness of breast cancer treatment. My study focuses on converting "cold" tumors into "hot" ones by enhancing the immune response within the TME. This immunomodulatory aspect of my research is particularly significant in the context of breast cancer therapy. By leveraging the immune system to fight breast cancer, my approach aligns with the growing field of immunotherapy, offering a promising avenue for patients who may not respond to traditional treatments. Moreover, my exploration of innovative delivery systems, such as utilizing Exo as carriers for miR-CVB3, enhances treatment safety and offers a versatile platform for delivering therapeutic agents such as OV to breast cancer cells. These delivery advancements have the potential to improve drug delivery in breast cancer therapy. The ability of miR-CVB3 to modify proteins within cancer cells and Exo, converting them into immunostimulatory molecules, signifies a fundamental shift in our understanding of the TME. This transformation into a pro-immune environment within the tumor holds the key to enhancing treatment efficacy and long-term outcomes. My research sets the stage for a promising future in breast cancer therapy. The combination of miR-modified viruses, immune enhancement, and innovative delivery systems offers a diverse approach that holds significant potential for reducing 132 tumor growth and improving patient survival. 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Thesis/Dissertation
eng
Pathology and Laboratory Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Luo, Honglin
Development of a safe treatment for breast cancer using an oncolytic Coxsackievirus B3
Text