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Cisplatin partially impedes lung adenocarcinoma-mediated M2 macrophage polarization Conway, Emma 2016

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CISPLATIN PARTIALLY IMPEDES LUNG ADENOCARCINOMA-MEDIATED M2 MACROPHAGE POLARIZATION  by  Emma Conway  BSc Hons, The University of Victoria, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology & Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   January 2016  © Emma Conway, 2016 ii  Abstract Lung cancer is the leading cause of cancer mortality and at 18%, has one of the lowest five-year survival rates of all malignancies.  The majority of patients (>80%) are diagnosed with locally advanced or metastatic disease for which the standard of care is platinum-based doublet chemotherapy.  However, chemotherapy has modest effects on overall survival, highlighting the need for novel and more effective treatments.   Within the past decade, the role of the immune system in tumorigenesis has become increasingly appreciated.  Targeting the immune cells within the tumor microenvironment is a growing field of study that holds exciting therapeutic potential.  Macrophages are a prominent immune cell type in the lung and lung tumors. It is widely accepted that a spectrum of macrophage activation states exists, with the exact phenotype dependent upon the precise composition of signals within the microenvironment.  At opposite ends of this spectrum there exist M1 macrophages which are pro-inflammatory and have antitumor functions, and M2 macrophages which are anti-inflammatory and act in wound healing and thus promote tumorigenesis.   I hypothesized that macrophage differentiation is skewed by lung adenocarcinoma cells to an M2 phenotype and that cisplatin, a commonly prescribed chemotherapeutic, affects macrophage polarity.  I co-cultured human monocytes with human lung adenocarcinoma cells in the absence and presence of physiologically relevant concentrations of cisplatin.  Co-cultured macrophages displayed increased differentiation and an M2 polarity, in part potentially through IL-6 secretion by tumor cells.  Cisplatin impeded macrophage differentiation, with treated macrophages displaying decreased size, granularity, and surface marker expression; however, CD206 expression, an M2 marker, remained elevated, suggesting a role for CD206 in response to treatment.  Additionally, I optimized single cell analysis of clinical specimens in preparation for future projects, specifically ex vivo analysis of the effect of standard first line chemotherapy on macrophage polarity and other immune cells in advanced non-small cell lung cancer.   Collectively, this work has demonstrated that macrophage polarity is affected by lung adenocarcinoma cells and by cisplatin.  Moreover, the optimization of single cell analysis has prepared for the study of the effect of chemotherapy on macrophage polarity over the course of treatment using more physiologically representative specimens.  iii  Preface A version of Chapter 1 has been published as:  [Conway EM], Pikor LA, Kung SHY, Hamilton MJ, Lam S, Lam WL, Bennewith KL (2015). Macrophages, inflammation, and lung cancer.  Am J Respir Crit Care Med. 2015 Nov 19. [Epub ahead of print]. PMID: 26583808 I am the first author of this manuscript.  I researched the topic and presented to the co-authors for input.  I wrote the manuscript and designed the figures to produce a complete draft.  I then asked the co-authors to provide edits and proofreading of the final draft before submission.   iv  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Dedication .....................................................................................................................................xv Chapter 1: Introduction ................................................................................................................1 1.1 Lung cancer ..................................................................................................................... 1 1.2 Inflammation and cancer................................................................................................. 3 1.3 The role of the immune system in tumorigenesis ........................................................... 4 1.4 The immune system in lung cancer ................................................................................ 6 1.5 Macrophages: an immune cell with a spectrum of activation phenotypes ..................... 7 1.6 Macrophages in cancer ................................................................................................. 11 1.7 Tumor initiation – the pathways connecting inflammation and cancer ........................ 11 1.7.1 Extrinsic pathway – tumor microenvironment ......................................................... 12 1.7.2 Intrinsic pathway - genetic ........................................................................................ 13 1.7.3 Interplay between the extrinsic and intrinsic pathways ............................................ 14 1.8 Established tumors – how macrophages promote malignancy ..................................... 18 1.8.1 Cell growth, survival, and motility ........................................................................... 18 v  1.8.2 Immune suppression and evasion ............................................................................. 18 1.9 Tumor associated macrophages and lung cancer prognosis ......................................... 23 1.9.1 Macrophage density and localization in lung cancer ................................................ 23 1.9.2 Macrophage polarization in lung cancer ................................................................... 24 1.10 Targeting the immune system: a novel therapeutic approach for the treatment of lung cancer 25 1.11 Rationale ....................................................................................................................... 26 1.12 Objectives and hypotheses ............................................................................................ 27 1.13 Specific aims and thesis outline .................................................................................... 27 Chapter 2: Co-culture of monocytes with lung AC cell lines increases M2 skewing .............29 2.1 Introduction ................................................................................................................... 29 2.2 Materials and methods .................................................................................................. 31 2.2.1 Cells .......................................................................................................................... 31 2.2.2 Co-culture experiments ............................................................................................. 31 2.2.3 Flow cytometry analysis of macrophage polarity ..................................................... 33 1M2a associated; 2M2b hypothesized; 3M2c associated ....................................................... 34 2.2.4 ELISA analyses ......................................................................................................... 34 2.2.5 qPCR of IL-6 expression .......................................................................................... 35 2.2.6 Statistical analysis ..................................................................................................... 35 2.3 Results ........................................................................................................................... 36 2.3.1 Monocytes passively differentiate in culture ............................................................ 36 2.3.2 Co-culture of monocytes with NSCLC cell lines increases M2 skewing ................. 40 2.3.3 Increased IL-6 signaling in co-cultures with H2228 and H2291 .............................. 48 vi  2.3.4 Co-culture induces IL-6 expression in macrophages ................................................ 50 2.4 Discussion ..................................................................................................................... 53 Chapter 3: Cisplatin impedes macrophage differentiation ......................................................60 3.1 Introduction ................................................................................................................... 60 3.2 Materials and methods .................................................................................................. 61 3.2.1 Cisplatin treated co-cultured experiments ................................................................ 61 3.2.2 Dose response assays ................................................................................................ 62 3.2.3 Cell viability assays .................................................................................................. 62 3.2.4 Detection of apoptosis............................................................................................... 63 3.3 Results ........................................................................................................................... 64 3.3.1 The estimated physiological concentration of cisplatin in tumors impairs proliferation and induces apoptosis in NSCLC cell lines ..................................................... 64 3.3.2 Macrophages treated with cisplatin differ in granularity when cultured alone and when co-cultured ................................................................................................................... 70 3.3.3 Macrophages treated with cisplatin display decreased marker expression ............... 75 3.4 Discussion ..................................................................................................................... 83 Chapter 4: Optimization of single cell analysis for clinical pleural effusion specimens .......88 4.1 Introduction ................................................................................................................... 88 4.2 Materials and methods .................................................................................................. 89 4.2.1 Pleural fluid samples ................................................................................................. 89 4.2.2 Sample processing .................................................................................................... 90 4.2.3 Flow cytometry analysis ........................................................................................... 92 4.3 Results ........................................................................................................................... 94 vii  4.3.1 Centrifugation speed during sample processing can affect cell types for analysis ... 94 4.3.2 Freezing disrupts the antibody binding epitope of CD163 ....................................... 97 4.4 Discussion ................................................................................................................... 100 Chapter 5: Conclusion ...............................................................................................................103 5.1 Summary and future directions ................................................................................... 103 5.1.1 Determine how co-culture of monocytes and NSCLC cell lines affects macrophage differentiation and polarity ................................................................................................. 104 5.1.2 Determine if and how treatment alters macrophage phenotypes and polarity ........ 105 5.1.3 Optimize single cell analysis of surrogate lung cancer specimens ......................... 106 5.2 Conclusions and significance ...................................................................................... 107 References ...................................................................................................................................109  viii  List of Tables  Table 2.1 NSCLC cell lines .......................................................................................................... 32 Table 2.2 Fluorochrome-conjugated cell surface antibody panel used to assess macrophage polarity following culture of monocytes alone or in co-culture with NSCLC cell lines .............. 34 Table 4.1 Fluorochrome-conjugated cell surface antibody panel used to assess macrophage polarity in PF samples ................................................................................................................... 92  ix  List of Figures  Figure 1.1 The spectrum of macrophage phenotypes ..................................................................... 9 Figure 1.2 Pathways connecting inflammation and cancer .......................................................... 16 Figure 1.3 Tumor promoting functions of TAMs in established tumors ...................................... 21 Figure 2.1 Monocytes differentiate into macrophages passively in culture ................................. 37 Figure 2.2 Increasing surface marker expression confirms macrophage differentiation .............. 38 Figure 2.3 Macrophages co-cultured with NSCLC cell lines are larger than macrophages cultured alone .............................................................................................................................................. 39 Figure 2.4 CD80 expression of co-cultured macrophages relative to macrophages cultured alone....................................................................................................................................................... 42 Figure 2.5 Co-cultured macrophages display significantly elevated levels of CD206 ................. 43 Figure 2.6 CD200R expression of co-cultured macrophages relative to macrophages cultured alone .............................................................................................................................................. 44 Figure 2.7 Co-culture with some NSCLC cell lines induces macrophage CD274 expression ..... 45 Figure 2.8 CD210 levels of co-cultured macrophages remain similar to macrophages cultured alone .............................................................................................................................................. 46 Figure 2.9 CD163 levels of co-cultured macrophages remain similar to macrophages cultured alone .............................................................................................................................................. 47 Figure 2.10 IL-6 levels from macrophages cultured alone and in co-culture with NSCLC cell lines ............................................................................................................................................... 49 Figure 2.11 IL-6 levels from macrophages and NSCLC cell lines cultured alone ....................... 51 x  Figure 2.12 qPCR reveals macrophages in co-culture express more IL-6 than when cultured alone .............................................................................................................................................. 52 Figure 3.1 Dose response assays of monocytes and NSCLC cell lines ........................................ 66 Figure 3.2 Cisplatin impairs growth of lung AC cell lines at physiologically relevant doses ...... 67 Figure 3.3 Cisplatin induces apoptosis in lung AC cell lines ....................................................... 68 Figure 3.4 Cisplatin reduces macrophage viability....................................................................... 71 Figure 3.5 Macrophages treated with cisplatin appear smaller and more granular when cultured alone .............................................................................................................................................. 72 Figure 3.6 Macrophages in co-culture appear less differentiated with cisplatin treatment .......... 73 Figure 3.7 Macrophages treated with cisplatin display reduced marker expression .................... 76 Figure 3.8 Macrophage CD206 expression remains elevated with cisplatin treatment ................ 77 Figure 3.9 Macrophage CD274 (PD-L1) expression is decreased with cisplatin treatment relative to untreated counterparts ............................................................................................................... 78 Figure 3.10 Macrophages CD80 expression differs between the four lung AC cell line co-cultures when treated with cisplatin .............................................................................................. 79 Figure 3.11 Macrophage CD200R expression is decreased in the cisplatin treated group ........... 80 Figure 3.12 Macrophage CD210 expression is decreased upon cisplatin treatment .................... 81 Figure 3.13 Macrophage CD163 expression is decreased with cisplatin treatment ..................... 82 Figure 4.1 Flow chart of PF sample processing optimization ...................................................... 91 Figure 4.2 Flow chart of assessment of effect of long term cryopreservation of PF samples ...... 93 Figure 4.3 Centrifugation speed during sample processing can affect cell types for analysis ..... 96 Figure 4.4 CD163 and CD206 binding epitopes may be adversely affected by freezing ............. 98 Figure 4.5 CD163 binding epitope is disrupted by freezing ......................................................... 99 xi  List of Symbols  MΦ  Macrophage xii  List of Abbreviations  AC  Adenocarcinoma  ALK  Anaplastic Lymphoma Kinase BAL  Bronchoalveolar lavage DC  Dendritic Cell EGFR  Epidermal Growth Factor Receptor  FSC  Forward Scatter GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor HLA  Human Leukocyte Antigen IDO  Indoleamine 2,3-Dioxygenase IFN-γ  Interferon Gamma IHC   Immunohistochemistry IL  Interleukin LC  Lung Cancer LCCL  Lung Cancer Cell Line  LPS  Lipopolysaccharide  M-CSF Macrophage Colony-Stimulating Factor MDSC  Myeloid Derived Suppressor Cells MFI  Median Fluorescence Intensity MHC   Major Histocompatibility Complex MMP  Matrix Metalloprotease NK   Natural Killer  xiii  NSCLC Non-Small Cell Lung Cancer  PD-1  Programmed Death-Receptor 1 PD-L1  Programmed Death-Ligand 1  PF  Pleural Fluid PGE  Prostaglandin E SCLC  Small Cell Lung Cancer SSC   Side Scatter SqCC  Squamous Cell Carcinoma  TAM  Tumor Associated Macrophage TCGA  The Cancer Genome Atlas TLR  Toll Like Receptor TME  Tumor Microenvironment Treg  T Regulatory Cell  xiv  Acknowledgements  I would like to acknowledge Dr. Wan Lam and the members of the Wan Lam Lab who provided support, useful insight and discussion, and an enjoyable environment in which to work, think, and laugh.  I thank Dr. LP for being my Yoda and providing coherent answers to my many questions.   I owe particular thanks to my supervisor Dr. Wan Lam and my supervisory committee members Dr. Kevin Bennewith and Dr. Stephen Lam for their mentorship, guidance, and continued support.  xv  Dedication  This thesis is dedicated to my family and friends; in particular to my encouraging friends who inaccurately call me Doctor and Professor Conway, and especially to my parents who have always supported me and just been the best. 1  Chapter 1: Introduction 1.1 Lung cancer Lung cancer (LC) is the leading cause of cancer-related deaths worldwide and accounts for more deaths than breast, prostate, and colon cancer combined1.  The majority of patients      (> 80%) are diagnosed with locally advanced or metastatic disease when surgery, the best curative option, is no longer feasible.  These patients are typically treated with platinum based doublet chemotherapy; however, its effect on survival is modest at best.  Due to the lack of effective treatments for advanced stage lung cancer, in the past decade there has been a significant push towards targeted therapy, guided by the molecular landscape elucidated by next generation sequencing.  Small molecule inhibitors against mutant EGFR and ALK rearrangements (erlotinib or gefitinib and crizotinib, respectively) have been deemed a success, with improved response rates over standard chemotherapy, and have become the standard first line treatment for metastatic lung adenocarcinoma patients harboring these mutations2,3.  Despite these advances, only a fraction of lung tumors (20-25%) harbour these mutations, limiting the application of these targeted therapies. Furthermore, resistance to targeted therapies ultimately develops in all cases, such that the 5-year survival rate has failed to improve significantly over the last 30 years and remains a mere 18%4,5.   Clinically, lung cancer is classified into two broad classifications: small cell lung cancer (SCLC) which accounts for approximately 15% of LCs, and the more prevalent non-small cell lung cancer (NSCLC), which accounts for 85% of all cases.  NSCLC is further subdivided into three histological subtypes; adenocarcinoma (AC), squamous cell carcinoma (SqCC), and large cell carcinoma, which differ in their cells of origin, location within the lung, and growth pattern.  Tobacco smoke exposure is the main etiological factor associated with LC, with smokers having 2  a 14-fold increased risk of developing LC than never smokers6.  While cigarette smoke is associated with all subtypes of LC, it is most strongly associated with SqCC and SCLC, both of which develop in the central airways.  However with the introduction of filtered, lower tar and nicotine containing cigarettes, smokers have tended to inhale deeper than before, leading to the distribution of tobacco and smoke to the periphery of the lungs where ACs arise7.  As a result there has been a dramatic shift in the global trend of LC histology within the past few decades, with a steady decline in SCLC and SqCC, such that AC is now the most prevalent histological subtype of LC8, and the focus of this thesis.   Adenocarcinomas, which account for 40-50% of all LC cases, typically arise in the glandular epithelium of the lung periphery from type II pneumocytes or Clara cells9.  These tumors are characterized by substantial inter- and intratumoral heterogeneity both at the histological and molecular levels.  Within AC, further classification has been proposed by the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society (IASLC/ATS/ERS), based on comprehensive histological subtyping of patients who had pathological stage I AC10.  The proposed classification revealed three overall prognostic groups which included nine histological subtypes, highlighting the histological diversity in lung AC.   Comprehensive molecular profiling of lung AC tumors has further emphasized the heterogeneity in these tumors.  Sequencing of 230 resected lung ACs by The Cancer Genome Atlas (TCGA) Research Network revealed AC to have a high mean somatic mutation rate of 8.87 mutations per megabase of DNA, second only to melanoma, and therefore a highly complex molecular landscape11.  Whole genome sequencing has vastly improved our understanding of the molecular landscape of alterations in AC, identifying recurrent mutations in addition to the well 3  known EGFR and KRAS mutations and EML4-ALK fusions such as TP53, BRAF, ERBB2, STK11, PIK3CA, PTEN, CDKNA, and more recently NF1, RB1, ATM, FGFR4, ERBB4, ARID1A, SMARA4, ASH1L and U2AF1, while simultaneously demonstrating the overwhelming heterogeneity within and between patients12-16.  While targeted therapies have dramatically improved treatment for patients harbouring targetable alterations such as mutant EGFR or genetic translocations involving ALK, RET, or ROS1, roughly half of all ACs lack an identifiable driver oncogene3,17,18 further underscoring the idea that within this histological subtype, tumors arise through diverse and distinct mechanisms, leading to vast heterogeneity11.  In addition to genetic heterogeneity, there are many extrinsic factors that also promote tumorigenesis, such as inflammation.  1.2 Inflammation and cancer Inflammation is a normal and essential process of infection and wound healing, but when it goes unresolved and becomes chronic, the resulting tissue damage can be extensive and disastrous.  Persistent exposure to many factors can cause chronic inflammation including smoking, obesity, and viral infections, and are therefore risk factors for development of diseases such as chronic obstructive pulmonary disease (COPD), diabetes, and cancer19-23.  A link between inflammation and cancer was first established in 1863 by the German pathologist Rudolf Virchow, who noted leukocytes in neoplastic tissues and hypothesized that the ‘lymphoreticular infiltrate’ reflected the origin of cancer at sites of chronic inflammation24.  Although these infiltrates were initially believed to be indicative of immune surveillance and anti-tumor responses, it has recently become apparent that tumor-infiltrating leukocytes can have either tumor-suppressing or tumor-promoting effects, depending on the type of immune cell 4  infiltrate and the nature of the tumor microenvironment24-27.  Chronic inflammatory diseases are associated with increased risk of bladder28, esophagus29, liver30, gallbladder31, stomach32, pancreas33, prostate34, colon35,36, and lung37-39 cancer and mortality associated with cancer is reduced with non-steroidal anti-inflammatory drugs, further emphasizing the contribution of inflammation to cancer40,41.   It is now evident that inflammation is involved in all stages of tumorigenesis, from malignant transformation and tumor initiation, to invasion and metastasis of established tumors.  In fact, it is estimated that underlying infections and inflammatory responses are linked to 15-20% of all cancer related deaths worldwide42.  Tumors that arise at sites of chronic inflammation exhibit ‘smoldering inflammation’25, which is characterized by the presence of infiltrating leukocytes, cytokines, chemokines, growth factors, and matrix-degrading enzymes27.  Among these infiltrating leukocytes are dendritic cells (DCs), T cells, and macrophages, which can have opposing effects on tumorigenesis.   1.3 The role of the immune system in tumorigenesis Within the past decade, the role of the immune system in tumorigenesis has become increasingly appreciated, such that immune evasion is now considered one of the hallmarks of cancer.  Recognition of the immune system’s role in tumorigenesis is illustrated by Hanahan and Weinberg’s Hallmarks of Cancer.  In 2000, they identified six hallmarks, all related to features of cancer cells themselves.  In 2011, they published an updated version of their paper, this time with the addition of four new hallmarks, two of which are related to the now recognized interaction between the tumor and the immune system.  These emerging hallmarks include avoiding immune destruction, in particular by T and B lymphocytes, natural killer (NK) cells, 5  and macrophages, and tumor promoting inflammation wherein inflammation by innate immune cells designed to fight infections and heal wounds instead results in their unintentional support of multiple hallmark capabilities43.   It is now well established that the immune system recognizes transformed cells in order to inhibit the growth of neoplastic tissue, and that both innate and adaptive cell types participate in this immunosurveillance44.  Transformed cells are first detected by NK cells, cells crucial to immunosurveillance, via their encounter with specific ligands on tumor cells, leading to tumor cell destruction with the uptake and processing of debris by macrophages and DCs. As a result macrophages and DCs are activated, secreting inflammatory cytokines and presenting tumor derived antigens to T and B cells.  Activation of T and B cells leads to further production of activating cytokines that promote the expansion of tumor antigen specific T cells and antibodies from plasma cells.  The adaptive immune response results in the elimination of any remaining tumor cells, as well as the generation of memory cells for an expedited response upon future recognition44. The ability of the immune system to keep malignantly transformed cells at bay resides in the capability to recognize antigens specific to tumor cells.  Viral proteins in tumors caused by viruses (e.g. HPV and cervical cancer), mutated proteins, and non-mutated proteins that are aberrantly expressed have been shown to be good tumor antigens promoting an adaptive immune response45.  However, the immune system is clearly unable to eliminate all transformed cells.  Tumor cells are known to escape immunosurveillance, subverting attack by multiple mechanisms including downregulation of tumor antigens and major histocompatibility complex (MHC) Class I molecules45.  Immunosurveillance therefore, is now described as having three possible outcomes: elimination, equilibrium, and escape45.  Where a highly immunogenic tumor in an 6  immunocompetent individual will result in stimulation of the immune system as described above, leading to elimination of tumor cells as they arise, a less immunogenic tumor and/or a less immunocompetent individual can result in incomplete elimination of the transformed cells. Surviving cancer cells can then repopulate, but also re-stimulate the immune system such that malignancy does not progress or resolve, a state described as equilibrium.  Changes in either the tumor which allow it to avoid immunosurveillance or in the immune system that weaken its ability to recognize and respond to tumor cells lead to the final possible outcome - tumor escape45.  While this was originally attributed to changes in the tumor cells themselves (downregulation of tumor antigens, etc.), it is now recognized that tumors are able to influence and co-opt normal regulatory functions of immune cells to aid their progression and dissemination through the release of cytokines and chemokines that attract poorly functioning effector cells as well as immunosuppressive cells that inhibit a potent immune-activating response46-48. 1.4 The immune system in lung cancer While a clear association has been shown between tumor immune responses and clinical outcomes in colorectal and ovarian cancers, the role of immune cells with respect to prognosis in NSCLC is less defined.  CD8+ cytotoxic T cells are thought to have a protective role against tumors, being the main effector cell type in the adaptive arm of the immune system; however, many investigations into their prognostic role in NSCLC have shown no correlation between CD8+ infiltration and survival49-53.  A study analyzing the mRNA ratio of IFN-γ, an effector cytokine secreted by activated and functioning CD8+ T cells, to CD8 in peritumoral areas and within tumor cell nests found that contrary to in the peritumoral areas, there was no significant 7  association between IFN-γ levels and CD8+ T cell count within tumor cell nests, suggesting that intratumoral CD8+ T cells may be inadequately activated53.  However, stromal co-localization of CD8+ and CD4+ T cells has shown an association with improved survival, suggesting that cooperation between these cell populations may allow a more potent anti-tumor response52.  Regulatory T cells (Tregs) are known to suppress the immune response and thought to promote tumor growth. This trend is consistent in NSCLC, with increased Treg counts associated with worse overall and relapse free survival54.  The importance of localization of immune cells has also been shown to be true with respect to tumor associated macrophages (TAMs), where M1 macrophages in tumor cell nests are correlated with good prognosis, while M2 macrophages in the stroma are correlated with poorer outcomes55-57.  Macrophages make up the majority of the immune cell infiltrate58, and are associated with patient prognosis, and therefore the main focus of this thesis. 1.5 Macrophages: an immune cell with a spectrum of activation phenotypes Macrophages are cells of the innate immune system that play critical roles in mounting immunological responses against foreign cells, bacteria, and viruses, but also mediate tissue repair following injury.  Macrophages are extremely plastic cells capable of diverse functions, with their distinct phenotypes contingent on the precise combination of signals present within individual microenvironments59.  It is now widely accepted that a spectrum of macrophage activation states exists, with M1 and M2 phenotypic distinctions representing the extreme ends of this spectrum (Figure 1.1A&B).  M1 macrophages function in a pro-immunogenic (Th1) manner to activate inflammation and eliminate infection, whereas M2 macrophages tune immune 8  responses to an anti-inflammatory (Th2) response, as well as scavenge debris and promote angiogenesis, tissue remodeling and repair59. In healthy tissues, macrophages often express markers associated with both the M1 and M2 phenotypes; therefore, as previously discussed, ‘M1’ and ‘M2’ polarization represent the extreme ends of a continuum of activation states, with the phenotype dependent upon the precise combination of signals in the local microenvironment.  This plasticity is evident in many physiological situations, such as a bacterial infection where macrophages are recruited to the site of infection and activated by TLR agonists and Th1 cytokines (e.g. IFN-γ) to an M1 phenotype to eliminate the invading bacteria and activate an adaptive immune response through production and secretion of pro-inflammatory cytokines and reactive nitrogen and oxygen intermediates60.  Following clearance of the bacteria, resolution of inflammation and tissue repair are initiated, leading to the activation of macrophages with an M2 phenotype60 (Figure 1.1C).  Wound healing generally occurs in a timely manner; however, if it fails to resolve, it can progress inappropriately to a chronic wound, in which inflammation persists, creating an environment ripe for malignant transformation.  In fact, tumors have been described as ‘wounds that do not heal’, reiterating the role of inflammation in disease.     9   Figure 1.1 The spectrum of macrophage phenotypes    10  Figure 1.1 The spectrum of macrophage phenotypes A spectrum of macrophage activation states exists, with M1 (A) and M2 (B) phenotypic distinctions representing the extreme ends of this spectrum. (A) Macrophages are polarized to an M1 (or Th1) phenotype in response to IFN-γ, GM-CSF, and bacterial products (e.g. LPS).  They are characterized by a high production of the pro-inflammatory cytokines IL-12 and IL-23, and with high expression of MHC II molecules and co-activation proteins (CD80 and CD86) are considered excellent antigen-presenting cells that can activate an adaptive immune response and fight foreign insults through the production of toxic intermediates (RNI and ROI).                    (B) Macrophages are polarized to an M2 (or Th2) phenotype in response to M-CSF and anti-inflammatory cytokines and factors.  They produce a variety of anti-inflammatory cytokines, immunosuppressive mediators, and matrix degrading enzymes, and promote angiogenesis, tissue remodeling and repair.  (C) Macrophage polarization is dependent upon the precise combination of signals in the local microenvironment and macrophages alter their phenotypes in response to these changing signals. This plasticity is evident in physiological situations, for example during a bacterial infection where macrophages are recruited to the site are initially activated to an M1 phenotype to eliminate the bacteria and activate an adaptive immune response, and subsequently replaced by M2 functions to resolve inflammation and initiate tissue repair.  Reprinted with permission of the American Thoracic Society. Copyright © 2015 American Thoracic Society. Conway, EM et al. (2015) Macrophages, inflammation, and lung cancer. American Journal of Respiratory and Critical Care Medicine. [Epub ahead of print]. PMID: 26583808 The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.   11  1.6 Macrophages in cancer Macrophages constitute the majority of the inflammatory infiltrate in tumors59.  These tumor associated macrophages (TAMs) mirror the continuum of activation states seen in general physiological situations.  In most cancers, their presence tends to be a negative prognostic indicator as TAMs in established tumors are generally skewed towards the M2 end of the spectrum, promoting tumor survival, progression, and dissemination through cellular processes such as enhanced angiogenesis, epithelial to mesenchymal transition (EMT), and immune suppression61,62.  While M1 infiltration within tumors is less common, it has been observed in colorectal carcinoma, in which the presence of M1 macrophages is associated with better prognosis, even when M2 macrophages outnumber M163,64.  However, there is evidence to suggest that the phenotype of TAMs is dependent upon the stage of tumor development in conjunction with the local tumor microenvironment, and that tumor progression may be the result of the interaction between the extrinsic (microenvironment) and intrinsic (genetic) pathways of tumor development, with one pathway initiating tumorigenesis and driving the other pathway to further promote tumor development25.  1.7 Tumor initiation – the pathways connecting inflammation and cancer Macrophages have significantly different effects on tumorigenesis depending on their phenotype within the tumor microenvironment59.  Although the exact mechanisms through which inflammation promotes cancer are not fully understood, two connected hypotheses have emerged; an intrinsic pathway driven by genetic alterations that lead to neoplasia and inflammation, and an extrinsic pathway driven by inflammatory conditions that increase cancer risk25. 12  1.7.1 Extrinsic pathway – tumor microenvironment Chronic inflammatory conditions increase cancer risk, promote all stages of tumorigenesis from initiation to invasion and metastasis, and are associated with 15-20% of all cancer related deaths29-31,33-39.  Tumors that arise at sites of chronic inflammation exhibit inflammation characterized by the presence of infiltrating leukocytes (dominated by macrophages), cytokines, chemokines, growth factors, and matrix degrading enzymes27.  While M2 macrophages are typically associated with tumor progression, M1 macrophages support initiation of tumorigenesis through the generation of reactive oxygen and nitrogen intermediates (ROI and RNI), which induce DNA damage in proliferating cells65.  As well as causing extensive tissue damage, these reactive intermediates react with superoxide to form peroxynitrite, which inactivates proteins by oxidizing sulfhydryl residues resulting in DNA damage and mutations in the surrounding epithelial cells (Figure 1.2A).  Peroxynitrite also decreases levels of anti-tumorigenic PGI2 and increases levels of pro-tumorigenic PGE265.  The cells of the microenvironment are thus pre-malignantly transformed, predisposing them to neoplastic transformation66.  In response to this tissue damage, inflammatory cytokines are released to recruit cells to initiate repair.  These cytokines further promote tumorigenesis by inhibiting cytochrome P450 and glutathione S-transferase isoenzymes65, leading to an accumulation of DNA damaging agents and subsequently affecting genome integrity and stability.  Pro-survival signals released during the repair process stimulate the proliferation of the premalignant cells, further supporting tumorigenesis66.  Taken together, these observations clearly demonstrate how a microenvironment of chronic inflammation can contribute to tumor initiation.   13  1.7.2 Intrinsic pathway - genetic Genetic alterations that activate oncogenes and inactivate tumor suppressors promote tumorigenesis by deregulating cellular pathways involved in cell proliferation, survival, differentiation, and DNA damage repair – the hallmarks of cancer43.  However, several well-known oncogenes and tumor suppressor genes such as RAS, MYC, VHL, and PTEN have also been implicated in inflammation as they regulate the production of inflammatory mediators (Figure 1.2B).  For example, activated components of the Ras-Raf signaling pathway induce the production of tumor-promoting inflammatory cytokines and chemokines, such as CXCL8 which is required to induce tumor-associated inflammation and neovascularization, thus eliciting a stromal response that further promotes tumor progression67.  Similarly, MYC-dependent cell-autonomous proliferation has been seen to occur via the remodeling of the extracellular microenvironment by inflammatory cells and mediators; in a mouse model of β-cell carcinoma, a MYC-activated transcriptional program lead to increased IL-1β, subsequent angiogenesis, and increased recruitment of mast cells, which are known to be implicated in angiogenesis and tumor growth68,69.  Loss or mutation of the tumor suppressor von Hippel-Lindau (VHL) factor (primarily in renal cancer) results in increased hypoxia induced factor-1alpha (HIF-1α) and subsequently CXCR4, which promotes metastasis through increased cell trafficking via the CXCR4/CXCL12 axis70-72.  Aberrant signaling leading to decreased TGF-β results in increased CXCL5 and CXCL12, which induce myeloid-derived suppressor cells (MDSCs) facilitating metastasis73.  Loss of PTEN can mitigate pro-inflammatory T cell responses as well as induce apoptosis of activated T cells74.  Akbay et al. recently demonstrated that signaling via mutant EGFR in tumor cells in a murine lung tumor model directly upregulates tumor PD-L1 expression, thereby promoting immune escape75.  Taken together, these findings demonstrate 14  that genetic alterations frequently disrupt pathways of innate immunity and inflammation, helping to further promote tumorigenesis through reduced anti-tumor immunity.   While macrophages are largely thought to be involved in tumor initiation and progression through inflammation and the extrinsic pathway, as a result of oncogenic activation, tumor cells recruit monocytic precursors from the blood and promote macrophage differentiation through production of CCL2 and M-CSF, respectively (Figure 1.2C)76-79.  Cytokines such as IL-4, IL-10, and IL-13, which are commonly secreted by tumors as a result of STAT3 and NFκB activation, lead to the differentiation of monocytes into activated M2-like TAMs80,81.  Regardless of whether tumors develop in the presence or absence of chronic inflammation, tumor associated macrophages typically end up promoting tumor progression82,83.   1.7.3 Interplay between the extrinsic and intrinsic pathways Although the intrinsic and extrinsic pathways arise through distinct molecular mechanisms, the two pathways are closely connected and converge through the activation of key transcription factors such as NFκB80, STAT381, and HIF-1α84 in tumor cells (Figure 1.2D).  STAT3 activation leads to increased proliferation and suppression of apoptosis through upregulation of Cyclin D, and Bcl-XL85, respectively, as well as increased capacity for immune evasion by inhibiting DC maturation86,87.   As a tumor grows, a hypoxic environment is generated, particularly at regions furthest from existing vasculature where cells survive with the activation of HIF-1α, further committing them to malignancy88,89.  HIF-1α activation leads to the expression of numerous genes that support neoangiogenesis, migration, and invasion90,91.  Furthermore, necrotic damage to the cells leads to the release of intracellular molecules including danger associated molecular patterns (DAMPs).  The interaction of DAMPs with their receptors that are normally expressed on resident innate immune cells triggers a pro-inflammatory gene 15  expression profile via the activation of NFκB92,93.  NFκB plays a central role in tumorigenesis as it promotes the expression of more than 500 genes involved in essential processes that control functions including proliferation, migration and invasion, suppression of apoptosis, and extracellular matrix remodeling94.  Together, these transcription factors coordinate the production of inflammatory mediators which recruit and activate inflammatory cells and result in the production of additional inflammatory mediators, amplifying the production of an inflammatory microenvironment.  Thus the convergence on the activation of key transcription factors involved in inflammation explains why inflammation is present in most, if not all, solid tumors, irrespective of the developmental trigger.  16    Figure 1.2 Pathways connecting inflammation and cancer  17  Figure 1.2: Pathways connecting inflammation and cancer  (A) The role of M1 macrophages and inflammation in the initiation of solid tumors. M1 macrophages located at sites of chronic inflammation promote tumorigenesis through production of reactive intermediates, tissue damage, and the release of inflammatory cytokines, predisposing the surrounding cells to DNA damage and subsequent neoplastic transformation.  (B) Genetic alterations that activate oncogenes and inactivate tumor suppressors also regulate the production of inflammatory mediators. MYC remodels the microenvironment through production of inflammatory cytokines while loss of the tumor suppressors VHL and PTEN lead to increased HIF-1α and the promotion of metastasis through increased cell trafficking and MDSC recruitment as well as apoptosis of activated T cells. (C) Oncogenic activation in tumor cells leads to the release of chemokines that recruit monocytic precursors from the blood and promote M2 macrophage differentiation and maturation.  (D) The intrinsic and extrinsic pathways converge through activation of NFκB, STAT3, and HIF-1α.  MR: mannose receptor; SRA: scavenger receptor; CCL2: chemokine (C-C) motif ligand 2; DAMPs: danger associated molecular patterns; GST: glutathione S-transferase; P450: cytochrome P450.  Reprinted with permission of the American Thoracic Society. Copyright © 2015 American Thoracic Society. Conway, EM et al. (2015) Macrophages, inflammation, and lung cancer. American Journal of Respiratory and Critical Care Medicine. [Epub ahead of print]. PMID: 26583808 The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.       18  1.8 Established tumors – how macrophages promote malignancy 1.8.1 Cell growth, survival, and motility While M1-like macrophages are thought to predominate at tumor initiation, M2-like macrophages dominate the immune infiltrate of established tumors95.  M2-like TAMs release growth factors (FGF1/2, PDGF, TGF-β, VEGF, TGFα, and IGF) that support tumor cell proliferation, survival and EMT, and have been implicated in contributing to tumor cell invasion and metastasis processes in vivo (Figure 1.3A)96.  In addition to these well characterized growth factors, TAMs also express migration inhibitory factor (MIF), a potent cytokine that has been shown to suppress the activity of p53, exacerbating DNA damage induced by inflammatory cells and limiting immunoediting, thereby increasing immune escape and tumor cell survival (Figure 1.3B)97.  TAMs often accumulate in hypoxic regions of the tumor where they adapt to the low oxygen tension by expressing HIF-1α and promote angiogenesis and support tumor growth through activation of VEGF, IL-8, COX-2, and MMP-9 (Figure 1.3C&D)98,99.  Evidence of TAMs contributing to tumor cell invasion and metastasis has been noted in vivo, with focal areas of basement membrane penetration at the time of malignant transition shown to include high quantities of TAMs100.  Furthermore, there is evidence that tumor cells migrate through these disruptions101,102 which is hypothesized to be due to TAM-derived EGF (Figure 1.3D).  The pro-tumoral functions of M2 macrophages create a microenvironment that supports and promotes angiogenesis, tumor growth and survival, invasion and metastasis, and immune suppression, contributing to tumor progression and tumor aggressiveness. 1.8.2 Immune suppression and evasion With a decreased ability to produce reactive intermediates (RNI and ROI) and inflammatory cytokines (IL-12 and IL-23), as well as reduced antigen presenting capabilities due 19  to the downregulation of MHCII and co-stimulation markers (CD80 and CD86), TAMs have a reduced capacity to promote inflammation.  In addition, through secretion of anti-inflammatory cytokines (interleukin 1 receptor antagonist (IL-1RA), decoy IL-1RII, IL-10), an anti-inflammatory (Th2) environment is generated, preventing the recruitment and activation of Th1 type immune cells to initiate an adaptive immune response with anti-tumoral immunity (Figure 1.3G).  Moreover, TAMs release chemokines and cytokines such as CCL17 and CCL22 that attract T cell subsets (Th2 and regulatory T cells (Tregs)) devoid of cytotoxic functions, further promoting an anti-inflammatory environment (Figure 1.3E)72,103.  Following the recruitment of Th2 cells and expression of the Th2 cytokines IL-4, IL-13, and IL-10, CCL18 expression is induced in TAMs leading to the recruitment of naïve T cells (Figure 1.3F)104,105.  The recruitment of naïve T cells in a microenvironment dominated by M2 macrophages is believed to induce T cell anergy, thereby increasing the numbers of immune cells in the tumor microenvironment devoid of any anti-tumor functional activities.  The expression of IL-10 also promotes the differentiation of monocytes to mature macrophages, blocking their differentiation into DCs, powerful antigen-presenting cells, and downregulating NFκB. In turn, downregulation of NFκB results in downregulation of many pro-inflammatory cytokines, including IL-12 which is involved in the differentiation of naïve T cells, the stimulation of growth and function of T cells, and the production of IFN-γ and TNF-α from T and NK cells.  All together, this results in reduced levels of immune cells with cytotoxic activity while also initiating an autocrine IL-10 signaling loop in activated TAMs, firmly implanting them at the M2 end of the phenotypic spectrum and promoting immune evasion106.   In addition to the reduced number of cytotoxic immune cells within the tumor microenvironment, there is evidence demonstrating TAMs further induce immune suppression 20  through their activation of immune checkpoints (PD-L1, Figure 1.3H) and increased expression of specific metabolic pathways (arginase-1 (Arg-1) and indoleamine-2,3-deoxygenase (IDO), Figure 1.3F).  Engagement of Programmed Death-1 (PD-1) on T cells by PD-L1 on tumor cells and macrophages results in the negative regulation of lymphocyte activation, including the inhibition of proliferation and cytokine production by activated T cells and the induction of T cell exhaustion and apoptosis.  Evidence also suggests that TAMs express PD-L1, directly inducing T cell apoptosis107.  Increased Arg-1 and IDO activity by TAMs leads to a metabolic shift; decreased arginine and tryptophan, amino acids essential for T cell growth108 and activation of CD8+ T cells with anti-tumor functions, respectively.  IDO represents an endogenous tolerogenic pathway that enables tumor immune evasion; IDO-expressing TAMs and importantly, DCs in lymph nodes, can induce systemic tumor tolerance109.  Altogether, the various pro-tumoral functions of M2 macrophages create a microenvironment that supports tumor cell growth and immune evasion/suppression, promoting tumor growth and progression. 21    Figure 1.3 Tumor promoting functions of TAMs in established tumors          22  Figure 1.3 Tumor promoting functions of TAMs in established tumors M2 macrophages create a pro-tumoral microenvironment that contributes to tumor progression and aggressiveness through the secretion of various growth factors and cytokines that promote and support (A) tumor cell proliferation and survival, (B) DNA damage and immune escape, (C) angiogenesis, (D) invasion and metastasis, and (E-H) immune suppression through multiple mechanisms.  Arg-1: arginase-1; IL-1RA: interleukin 1 receptor antagonist; CD210: IL-10 receptor; COX-2: cyclooxygenase-2; VEGF: vascular endothelial growth factor; HIF-1α: hypoxia inducible factor-1 alpha; MIF: migration inhibitory factor; TGF-α: transforming growth factor alpha; PDGF: platelet derived growth factor; FGF1/2: fibroblast growth factor 1 and 2; IGF-1: insulin-like growth factor-1; CCL17: chemokine (C-C) motif ligand 17; CCR4: chemokine (C-C) motif receptor 4.   Reprinted with permission of the American Thoracic Society. Copyright © 2015 American Thoracic Society. Conway, EM et al. (2015) Macrophages, inflammation, and lung cancer. American Journal of Respiratory and Critical Care Medicine. [Epub ahead of print]. PMID: 26583808 The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.           23  1.9 Tumor associated macrophages and lung cancer prognosis Though the presence of leukocytes in neoplastic tissues has been observed for over 150 years, until recently their association with clinical outcomes has been largely unrecognized.  In general, infiltrating leukocytes, particularly macrophages, have been correlated with negative outcomes and poor prognoses in most cancers62.  Encouragingly, studies investigating the association of TAMs with patient prognosis have evolved from looking at TAM density, to the specific anatomical location of TAMs within tumors, to considering the polarization status of TAMs. This recent work highlights the complex roles these cells play in the clinical outcomes of cancer patients.   1.9.1 Macrophage density and localization in lung cancer In the early 2000s, immunohistochemical (IHC) analysis of macrophage infiltration in lung tumors was assessed by several groups.  TAM density was found to be negatively correlated with lung cancer patient survival in a number of studies, and patients with recurrent disease were found to have higher levels of macrophage infiltration in their primary tumors57,110.  Another study revealed a significant association between the extent of infiltration and lung histology, with a high density of TAMs in ACs and low TAM infiltration in SqCCs.  However in this study, no significant association between the extent of infiltration and survival was observed111.  This and other studies, which also failed to observe a significant association between TAM density and prognosis led to a general lack of consensus regarding the prognostic role of TAMs in lung cancer, fuelling research into other aspects of TAMs and cancer biology. Investigation into the micro-anatomical location of TAMs within the lung tumor microenvironment was initiated in the mid-late 2000s in an attempt to clarify and elucidate the prognostic role of TAMs in LC.  Multiple groups found higher numbers of macrophages in 24  tumor islets to be significantly positively correlated with favorable clinical outcomes and longer survival in both surgically resected and advanced stage lung cancers, while high numbers of macrophages in the tumor stroma were negatively correlated with patient outcome112-115.  However, just as with assessment of the extent of TAM infiltration, the prognostic significance of islet or stromal TAMs lacked consensus, with other studies reporting either no association with survival116 or islet TAMs associated with lymph node metastases117.  Despite these contradictory findings, the majority of data suggest that high macrophage densities in tumor islets favor better LC prognosis.   1.9.2 Macrophage polarization in lung cancer Recently, studies investigating the micro-anatomical localization as well as the macrophage polarization status of TAMs have been initiated in order to clarify the prognostic value of TAMs in LC.  IHC double staining of HLA-DR (M1) and CD163 (M2) to assess macrophage densities in tumor islets and stroma in resected NSCLC specimens from 50 patients with an average of 1 year survival and 50 patients with an average of 5 year survival revealed that 70% of TAMs were M2.  However, M1 macrophages were positively associated with patient survival, with the tumor islets and stroma of the long survival group having significantly more M1 macrophages than the short survival group55.  Interestingly, the density of M2 macrophages in tumor islets or stroma did not differ significantly between the long and short survival groups, and was not associated with survival.  Similarly, assessment of M1 and M2 macrophages in surgically resected tumors from patients with extended (92.7 months) and poor (7.7 months) median survival found that M1 and M2 islet densities were greater in the extended survival group relative to the poor survival group, but that M1 density was significantly greater than M2 density.  Patients with an M1 density greater than the median had a 5-year survival rate of 75%, while those below the median 25  had a survival rate of <5%56.  M2 infiltration has also been found to be significantly associated with tumor stage, lymph node metastases83 and poor outcome118, while a higher proportion of circulating M2 cells (CD14+CD204+) correlated with TAM density in the stroma and was indicative of early recurrences119.  Altogether, these studies further demonstrate that TAMs have both pro- and anti-tumoral activity in LC, and that consideration of macrophage counts in tumor islets and stroma in conjunction with assessment of macrophage polarization will be essential in developing consensus on the prognostic significance of TAMs in LC.  1.10 Targeting the immune system: a novel therapeutic approach for the treatment of lung cancer With the recognition of the importance of the immune system in tumorigenesis came the development of immunotherapies aimed at modulating/increasing a patient’s inherent anti-tumor defenses. Lung cancers were historically thought to be non-immunogenic120,121 as patients displayed limited response when treated with non-specific immunotherapies such as the BCG vaccination122 and cytokine therapies, including IL-2 and interferon treatments123.  However, based on the findings from recent immunotherapies targeting immune checkpoints (CTLA-4 and PD-1), it appears that lung cancers are in fact immunogenic, and as a result, a number of cancer immunotherapies are currently in clinical trials as adjuvant or first line therapy for the treatment of LC.  As with most cancer therapies, there is a wide range of responses to immunotherapies.  For example, a Phase II clinical trial of ipilimumab (α-CTLA-4) in combination with chemotherapy (paclitaxel and carboplatin) revealed that squamous lung cancers relative to non-squamous lung cancers had greater clinical benefit in terms of immune-related progression free survival124.  Although the immune response in LC is complex and highly variable between 26  patients, the recent success of immunotherapy trials in NSCLC and other cancers125 has renewed the possibility of the potential efficacy immunotherapies hold in LC, and suggests that combination therapy targeting both malignant cells and the tumor infiltrating inflammatory cells (e.g. macrophages, T cells, etc.), could be effective in eliciting long-lasting adaptive immunity and subsequently disease regression.    1.11 Rationale The tumor microenvironment is recognized as a complex milieu in which tumor and stromal cells engage in crosstalk to promote tumor progression. While the advances in targeted and immunotherapies are encouraging, chemotherapy remains the first line treatment for the majority of patients with NSCLC.  As macrophages make up the majority of the immune infiltrate in tumors, determining how their polarity is affected in response to lung cancer chemotherapies will further our understanding of how these cells contribute to lung cancer progression and response to treatment.  This information could be applied to select patient populations most likely to benefit from certain therapies based on the characteristics of their tumor infiltrating immune profiles.      27  1.12 Objectives and hypotheses The objective of this work was to assess the effect of standard first line chemotherapy on macrophage polarity in the context of advanced lung adenocarcinoma.    This was based on the following hypotheses: 1. Monocytes are skewed by lung adenocarcinoma cells to an M2 phenotype 2. Standard first line chemotherapy affects macrophage polarity toward M2 An additional objective of this work was to optimize single cell analysis of clinical specimens in preparation for future projects, specifically ex vivo analysis of the effect of standard first line chemotherapy on macrophage polarity and other immune cells in advanced NSCLC.  1.13 Specific aims and thesis outline To address the questions of whether 1) monocytes are skewed by lung cancer cell lines to an M2 phenotype and 2) if standard first line chemotherapy affects macrophage polarity; we devised the following specific aims.  Aim 1: Determine how co-culture of monocytes and NSCLC cell lines alters macrophage phenotypes and polarity Chapter 2 describes the effects of culture alone or in combination with lung adenocarcinoma tumor cell lines on macrophage polarity, as assessed by flow cytometry.  Co-culture induced changes in macrophage secreted proteins were analyzed by ELISA based cytokine assays.  We found that M2 skewing was increased by co-culture with lung adenocarcinoma tumor cell lines, with significantly increased expression of CD206 at the end of co-culture with H2228 and H2291.  Interestingly, a corresponding increase in IL-6 signaling was 28  observed in the co-cultures with these two cell lines, suggesting that secretion of IL-6 may be important in M2 skewing.     Aim 2: Determine if and how treatment alters macrophage phenotypes and polarity Chapter 3 describes changes in macrophage polarity following treatment of monocytes cultured alone or in co-culture with tumor cells with clinically relevant doses of cisplatin.  Flow cytometric analysis of macrophage populations revealed that macrophage differentiation was impeded by cisplatin treatment.  Expression of all surface markers assessed was reduced, with the exception of CD206, which remained at levels similar to those of untreated macrophages, suggesting that CD206 may have a role in apoptotic tumor cell clearance.   Aim 3: Optimize single cell analysis of surrogate lung cancer specimens  Chapter 4 describes the effects of sample processing and long-term cryopreservation on cell populations and antibody binding for flow cytometric analysis.  Optimization of these conditions enables banking of surrogate patient samples such as bronchoalveolar lavage (BAL) and pleural fluid, and robust comparison of patient samples collected at different time points. 29  Chapter 2: Co-culture of monocytes with lung AC cell lines increases M2 skewing 2.1 Introduction Macrophages are extremely plastic cells capable of diverse functions, with their phenotypes contingent on the precise combination of signals present within individual microenvironments59.  A spectrum of macrophage activation states exists, with M1 and M2 phenotypic distinctions representing the extreme ends of this spectrum59.  M1, or classically activated macrophages, function in a pro-immunogenic manner to activate inflammation and eliminate infection.  Macrophages are polarized to an M1 (or Th1) phenotype in response to interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and bacterial products (e.g. LPS), and are characterized by a high production of the pro-inflammatory cytokines interleukin (IL)-12 and IL-23126.  These macrophages are considered to have a high capacity to present antigen, with increased expression of MHC class II molecules as well as the co-activation proteins CD80 and CD86, consequently resulting in activation and production of toxic intermediates (RNI and ROI) (Figures 1.1A and 1.2A).  With their bactericidal activity, pro-inflammatory cytokine secretion profile (IL-1, tumor necrosis factor alpha (TNF-α), IL-12, IL-23, chemokine (C-X-C) motif ligand (CXCL)9, CXCL10, and CXCL11126), and immunostimulation activities, M1 macrophages are regarded as having anti-tumor functions.   At the opposite end of the spectrum are M2, or alternatively activated macrophages.  M2 macrophages are polarized in response to IL-4, IL-13, glucocorticoids, IL-10, prostaglandin E (PGE), vitamin D3, macrophage colony-stimulating factor (M-CSF), and immunoglobulin complexes/Toll Like Receptor (TLR) ligands126.  They scavenge debris and promote 30  angiogenesis, tissue remodeling and repair, such that they are regarded as tumor promoting.  Characterized by an IL-10high, IL-12low phenotype, an impaired ability to present antigen (decreased expression of MHCII), and decreased production of toxic intermediates, M2 macrophages tune immune responses to an anti-inflammatory (Th2) response through increased expression of anti-inflammatory cytokines, immunosuppressive mediators, and matrix-degrading enzymes (Figures 1.1B and 1.2B).  These include interleukin 1 receptor antagonist (IL-1RA), decoy IL-1RII, IL-10, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)1/2, platelet derived growth factor (PDGF), IL-8, prostaglandins, matrix metalloproteases (MMPs) , scavenger receptor (SRA), mannose receptor (MR), transforming growth factor beta (TGF-β), chemokine (C-C motif) ligand (CCL)17, CCL18, CCL22, polyamines, and IL-4126.  Importantly, it has been shown that IL-4 induces differentiation of naïve T cells into Th2 cells, initiating a feedback loop in which Th2 cells secrete IL-4 and IL-13, inhibiting the classical activation of macrophages (Figure 1.2B).  M2 macrophages can be further divided into M2a, M2b, and M2c, subsets based on gene expression profiles103.  The subsets can be elicited by different cytokines and immune complexes in vitro and validation of the phenotypic surface markers has been attempted in vivo, but the major challenge remains to define the phenotype-function link in vivo127.  Due to the lack of robust consensus markers for the M2 subtypes, in this work we will not discern between the various M2 subsets.   Despite the significant work described above, and the associations of M1 and M2 phenotypes with prognosis, the precise role TAMs play in lung cancer and how they influence tumor progression, response to treatment, and subsequently patient survival remains unclear.  A basic understanding of the effects of LC tumor cells and their secreted factors have on macrophage differentiation and polarity is therefore required.  In this chapter, human peripheral 31  blood monocytes were cultured alone or in co-culture with human lung adenocarcinoma cell lines in an attempt to decipher how tumor cells influence macrophage polarity.   2.2 Materials and methods 2.2.1 Cells Human CD14+ peripheral blood monocytes were obtained from StemCell Technologies.  All monocytes used were from a non-smoking Caucasian male.  Monocytes were cultured in RPMI 1640 with 10% fetal bovine serum (FBS, Invitrogen).  NSCLC cell lines (H1373, H1819, H2228, and H2291, Table 2.1) were obtained from the American Type Culture Collection and cultured and maintained in RPMI 1640 supplemented with 10% FBS and 0.1% penicillin-streptomycin (Invitrogen), for no more than 10 passages. RPMI 1640 with 10% FBS was used for co-culture experiments.  2.2.2 Co-culture experiments Human CD14+ peripheral blood monocytes were cultured alone or in co-culture with one of four NSCLC cell lines. Co-culture experiments were set up in 24 well plates with monocytes in the bottom compartments and NSCLC cell lines seeded onto cell culture inserts with 3.0 µm pores (VWR).  Monocytes were seeded at 100 000 cells/well, while tumor cell lines were seeded at 6000 cells/insert (H1373, H2228 and H1819) or 10 000 cells/insert (H2291), concentrations optimized for 5 days of growth.  To allow for a tumor cell to macrophage ratio reflective of those observed in vivo, and prevent tumor cells from becoming over confluent, tumor cells were replaced on day 5.  Monocytes/macrophages and supernatants were collected at 4 hours (Day 0), and 1, 3, 5, and 10 days after seeding, and were subsequently assessed by flow cytometry and ELISA assays, respectively.  32  Table 2.1 NSCLC cell lines Cell line Age Gender Ethnicity Tumor subtype Tumor source Smoker Pack-years Stage Morphology Mutation status EGFR KRAS LKB1 TP53 EML4-ALK H1373 56 Male African American Adenocarcinoma Lung Y 30 3A Epithelial WT mutant WT WT negative H1819 55 Female Caucasian Adenocarcinoma Metastasis lymph node Y 80 3 Epithelial WT WT WT WT negative H2228 U Female Unknown Adenocarcinoma Lung primary N 0 U Mesenchymal-like WT WT WT mutant positive H2291 U Male Unknown Adenocarcinoma Metastasis lymph node N 0 U Mesenchymal-like WT mutant WT mutant negative U: unknown; WT: wild type 33  2.2.3 Flow cytometry analysis of macrophage polarity On specified days (outlined in section 2.2.2), monocytes/macrophages were collected for flow cytometry by incubation with 2 mM EDTA in phosphate buffered saline (PBS) for 15 minutes at 4°C with scraping.  Cells were centrifuged at 1000 rpm for 5 minutes and the supernatant removed.  Cells were resuspended in 50 µL FACS buffer (PBS with 10% FBS) and stained with a panel of 8 fluorochrome-conjugated monoclonal antibodies directed at cell surface proteins associated with an M1 or M2 phenotype (Table 2.2) for 45 minutes at 4°C. Cells were washed twice with FACS buffer and centrifugation (at 1000 rpm for 5 minutes) to remove any unbound antibody and resuspended in 1 mL PBS.  Cells were then incubated with eF450 Fixable Viability dye (eBioscience) for 30 minutes at 4°C. Cells were washed by centrifugation as before with FACS buffer.  Cells were resuspended in 300 µL FACS buffer, passed through filter-capped 5 mL flow cytometry tubes (BD Biosciences) and flow cytometry performed using a BD FACSCanto II flow cytometer (BD Bioscience).  Cultured macrophages were used for unstained and viability controls, while UltraComp beads (eBioscience) were used for single stain antibody controls. For single stain controls, 1 test volume of antibody (Table 2.2) was incubated with 1 drop of UltraComp Beads for 20 minutes at 4°C, then washed with 2 mLs FACS buffer and resuspended in 400 µL FACS buffer for flow analysis. 10 000 events were collected for each sample and data were analyzed using FACSDiva version 6.1 software. Live monocytes/macrophages that stained negatively for eF450 viability dye were selected and the median fluorescence intensity (MFI) of each marker was recorded. Results are reported as MFI relative to monocytes cultured alone for each M1 or M2 associated cell surface marker.  34  Table 2.2 Fluorochrome-conjugated cell surface monoclonal antibody panel used to assess macrophage polarity following culture of monocytes alone or in co-culture with NSCLC cell lines Marker Function Conjugated fluorochrome Supplier Clone M1/M2 Volume (µL)/sample  (1 test) CD163 Scavenger receptor FITC BD Pharmingen GHI/61 M23 5 CD210 IL-10 receptor PE BD Pharmingen  1B1.3a M23 20 CD200R  CD200 receptor PerCP-eFluor710 eBioscience OX108 M21 5 CD274  PD-L1 PE-Cy7 eBioscience MIH1 M22,3 5 CD206 Mannose receptor APC BD Pharmingen 19.2 M21,3 20 CD80  Co-stimulatory protein Alexa Fluor 700 BD Pharmingen L307.4 M1 5 CD14 LPS co-receptor V500 BD Horizon M5E2 Monocyte/MΦ 5 Viability  eFluor 450  eBioscience  Viability 1 1M2a associated; 2M2b hypothesized; 3M2c associated 2.2.4 ELISA analyses Supernatants (2.5 ml) were collected from monocytes cultured alone or in co-culture with NSCLC cell lines at the time points specified in section 2.2.2 and stored at -80°C until all time points were collected and could be analyzed together.  ELISA plates (eBioscience) against IL-1β, IL-1RA, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, TNF-α, TGF-β, MCP-1, MMP-9, soluble CD163 were pre-coated overnight with capture antibodies at 4°C, then washed three times with PBS, 0.05% Tween-20 and blocked for 1 hour at room temperature, as per manufacturer’s instructions. 100 µL of supernatant collected from monocytes cultured alone or in co-culture with NSCLC lines were plated in duplicate. After incubation overnight at 4°C, plates were washed three times with PBS, 0.05% Tween-20 and incubated with corresponding detection antibodies at room temperature for one hour. Plates were washed again (3 times), incubated with Avidin conjugated to horseradish peroxidase (eBioscience), washed as before five times, and incubated with TMB substrate solution (eBioscience) for 15 minutes at room temperature. Colour development was stopped with 1M H3PO4 (Sigma-Aldrich), and the plates were quantified by spectrophotometry (EMax plate reader, Molecular Devices) at 450nm with 35  reference to 570nm.  Positive and negative controls were supernatants from in vitro skewed M1 and M2 macrophages and culture media alone, respectively.  2.2.5 qPCR of IL-6 expression Quantitative real-time PCR analysis of IL-6 was performed using the TaqMan Gene Expression Assay Kit (Applied Biosystems) and an Applied Biosystems 7500 Fast Real Time PCR system. RNA from macrophages or NSCLC cells was collected using RNA lysis buffer (Zymo Research). RNA was extracted using Zymo Research RNA MiniPrep kit. 500 ng of sample RNA was converted to cDNA using the TaqMan cDNA Reverse Transcription Kit and 50 ng of cDNA was combined with TaqMan Gene Expression Master Mix without AmpErase Uracil N-glycosylase and IL-6 specific primer (Hs00985639_m1) as per the manufacturer’s instructions (Applied Biosystems). 18S ribosomal RNA (Hs99999901_s1) was used as an endogenous control to normalize cDNA input. RT-qPCR was performed on a reaction volume of 20 µL using default thermal cycling conditions (2 mins at 50°C, 10 mins at 95°C and 1 min at 60°C).  Data were analyzed using 7500 Fast System Software v1.4.  Macrophage IL-6 expression in co-culture was analyzed with reference to macrophage IL-6 expression cultured alone, and a two-fold change between macrophages cultured alone and in co-culture was considered biologically relevant. 2.2.6 Statistical analysis  Median fluorescence intensities (MFIs) for each marker in co-cultured macrophages were normalized to MFI values of macrophages cultured alone.  Comparisons between co-cultured macrophages and macrophages cultured alone were performed using a two-tailed Student’s T test with equal variances in Excel and relative MFIs with a p<0.05 were considered significant.   36  2.3 Results 2.3.1 Monocytes passively differentiate in culture In order to determine the effects of co-culture with NSCLC cell lines on macrophage differentiation and polarity, macrophage size, internal complexity, and cell surface expression of a panel of M1 or M2 associated cell surface proteins were compared between macrophages cultured alone and in combination with lung tumor cell lines over 10 days, as described in section 2.2.2.  Monocytes were in the bottom compartments and NSCLC cell lines were seeded onto cell culture inserts, thereby physically separating the two cell types.  As a result, any observed effects were due to secreted proteins from the tumor cells.  As expected, over the course of the experiment, monocytes cultured alone differentiated into macrophages128.  Flow cytometric analysis revealed a stepwise increase in forward (size) and side (internal granularity) scatter over the course of the experiment, indicative of monocyte differentiation into macrophages129 (Figure 2.1).  This differentiation was further confirmed by the increasing expression of multiple surface markers over the course of the experiment (Figure 2.2).  Macrophages in co-culture with tumor cell lines were more internally complex and were significantly larger than macrophages cultured alone, suggesting increased differentiation (Figure 2.3).       37  Figure 2.1  Figure 2.1 Monocytes differentiate into macrophages passively in culture Monocytes cultured alone were seeded on Day 0, collected 4 hours later, and then on indicated days following seeding. Representative flow plots with forward scattered light (FSC) on x axis and side scattered light (SSC) on y axis. Flow plots show the live monocyte/macrophage population gated in blue. FSC and SSC measured with same voltages on each day so that relative size (FSC) and granularity/internal complexity (SSC) are comparable across collection days.           38  Figure 2.2  Figure 2.2 Increasing surface marker expression confirms macrophage differentiation  Median fluorescence intensities (MFIs) of monocytes/macrophages cultured alone, as measured by flow cytometry.  MFIs of every cell surface marker increase in a stepwise manner over the course of the experiment, indicative of monocyte differentiation into macrophages. Depicted MFIs are representative of 3 biological replicates, as MFIs were recorded with different voltages in independent experiments making calculations of means inappropriate.      050010001500200025000 1 3 5 10Median fluorescence intensity (MFI)DayCD163CD210CD200RCD206CD274CD8039  Figure 2.3  Figure 2.3 Macrophages co-cultured with NSCLC cell lines are larger than macrophages cultured alone Co-cultured macrophages are significantly larger (increased FSC) than macrophages cultured alone (Student’s T-test, p < 0.05) and showed a trend towards greater internally complexity (increased SSC), although this did not reach statistical significance (Student’s T test, p > 0.05).  Median FSC and SSC values of live co-cultured macrophages were normalized to macrophages cultured alone and are reported as mean ± SEM of three independent experiments.      ****00.20.40.60.811.21.4Mφ alone Mφ + H1373 Mφ + 2228 Mφ + 1819 Mφ + 2291Measurement normalized to Mφcultured aloneFSCSSC40  2.3.2 Co-culture of monocytes with NSCLC cell lines increases M2 skewing To determine whether co-culture with lung cancer cell lines is sufficient to induce macrophage skewing, we assessed a panel of established M1 (CD80) and M2 (CD163, CD210, CD200R, CD206, CD274) markers over the course of co-culture.  As expected, co-culture had little effect on M1 skewing, with CD80 levels showing no significant difference between single and the majority of co-cultures.  While CD80 levels remained consistent over the ten day culture period when co-cultured with H1373 and H1819 (Figure 2.4A,C), Day 10 macrophages co-cultured with H2228 and H2291 displayed a trend to slightly increased CD80 expression, reaching statistical significance in H2228 co-culture (Student’s T-test, p < 0.05, Figure 2.4B).  Despite reaching statistical significance, the increase in CD80 expression on Day 10 was less than a two-fold increase relative to macrophages cultured alone, confirming that CD80 expression is not dramatically induced upon co-culture with LCCLs, especially when compared to the upregulation of CD206.  Of the five M2 markers tested, only CD206 displayed significant enrichment over the course of the experiment in the majority of co-cultures relative to monocytes alone (Figure 2.5).  CD206 levels were considerably increased after 24 hours of co-culture and remained appreciably elevated from baseline over the entire course of the experiment, with maximum expression observed on Day 5 (Figure 2.5).  While all four cell lines demonstrated increased CD206 levels in co-culture relative to monocytes alone, this difference was only significant in three cell lines (H1373, H2228, and H2291, Figure 2.5A,B,D), with only macrophages co-cultured with H2228 and H2291 remaining significantly increased at the end of the experiment.   Interestingly, all co-cultured macrophages exhibited significantly lower expression of CD200R, a marker of immune suppression, on Day 3, with H1373 co-cultured macrophages 41  exhibiting significantly lower expression throughout the remainder of the experiment (Figure 2.6A).  H2228 co-cultured macrophages however, displayed increased CD200R expression at the end of co-culture (Figure 2.6B).  Meanwhile, CD274 (PD-L1), a clinically relevant marker of immune suppression, showed increased expression in H2228 and H2291 co-cultures (Figure 2.7B,D), the two co-cultures in which CD206 expression was increased.  H2228 co-cultured macrophages displayed a stepwise increase in CD274 expression with significantly increased expression at Day 10 (Student’s T-test, p < 0.05), while in H2291 co-cultures maximal CD274 expression was achieved on Day 5, with significantly increased expression at Day 10 (Student’s T-test, p < 0.05).  Co-cultured macrophages exhibited CD210 and CD163 levels similar to macrophages cultured alone (Student’s T-test, p > 0.05) (Figures 2.8 and 2.9).  Taken together, although we did not observe unanimous upregulation of M2 markers, it does appear that co-culture of human peripheral blood CD14+ monocytes with the lung adenocarcinoma cell lines H2228 and H2291 promotes M2 skewing, based on cell surface expression of CD206, CD200R (H2228 only), and CD274.           42  Figure 2.4       Figure 2.4 CD80 expression of co-cultured macrophages relative to macrophages cultured alone  Expression of CD80 on co-cultured macrophages relative to macrophages cultured alone.  Monocytes were co-cultured with one of four NSCLC cell lines, H1373 (A), H2228 (B), H1819 (C), or H2291 (D), for 10 days.  Relative MFIs reflect mean ± SEM of three independent experiments.  Macrophages co-cultured with H2228 (B) exhibit significantly higher expression of CD80 at Day 10, while macrophages co-cultured with H2291 displayed the same trend (2-tailed Student’s T-test, p < 0.05).   00.511.522.530 5 10Relative CD80 MFIDays of co-culture(A) H1373*00.511.522.530 5 10Relative CD80 MFIDays of co-culture(B) H222800.511.522.530 5 10Relative CD80 MFIDays of co-culture(C) H181900.511.522.530 5 10Relative CD80 MFIDays of co-culture(D) H229143  Figure 2.5       Figure 2.5 Co-cultured macrophages display significantly elevated levels of CD206 Relative expression of CD206 upon co-culture with NSCLC cell lines.  Macrophages co-cultured with all NSCLC cell lines (A-D) exhibited a sharp increase in CD206 expression relative to monocytes cultured alone although these did not reach significance.  Macrophages co-cultured with H1373 (A), H2228 (B), and H2291 (D) exhibited significantly higher expression of CD206 on Day 5, with those co-cultured with H2228 and H2291 remaining significantly highly expressed for Day 10 (2-tailed Student’s T-test, p <0.05).  Expression is plotted as the MFI for CD206 of co-cultured macrophages relative to monocytes/macrophages cultured alone, for each time point.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p <0.05.  *0246810120 5 10Relative CD206 MFIDays of co-culture(A) H1373**0246810120 5 10Relative CD206 MFIDays of co-culture(B) H22280246810120 5 10Relative CD206 MFIDays of co-culture(C) H1819 **0246810120 5 10Relative CD206 MFIDays of co-culture (D) H229144  Figure 2.6      Figure 2.6 CD200R expression of co-cultured macrophages relative to macrophages cultured alone Relative expression of CD200R upon co-culture with NSCLC cell lines.  Macrophages co-cultured with all NSCLC cell lines (A-D) exhibited significantly lower expression of CD200R on Day 3, with those co-cultured with H1373 (A) exhibiting significantly lower expression of CD200R for the remainder of the co-culture experiment (2-tailed Student’s T-test, p < 0.05).  Macrophages co-cultured with H2228 (B) displayed significantly higher expression of CD200R on Day 10.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.   * **00.511.520 5 10Relative CD200R MFIDays of co-culture (A) H1373**00.511.520 5 10Relative CD200R MFIDays of co-culture(B) H2228*00.511.520 5 10Relative CD200R MFIDays of co-culture(C) H1819*00.511.520 5 10Relative CD200R MFIDays of co-culture(D) H229145  Figure 2.7       Figure 2.7 Co-culture with some NSCLC cell lines induces macrophage CD274 expression   Relative expression of CD274 upon co-culture with NSCLC cell lines.  Macrophages co-cultured with H2228 (B) and H2291 (D) exhibit significantly higher expression of CD274.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.      0123450 5 10Relative CD274 MFIDays of co-culture (A) H1373*0123450 5 10Relative CD274 MFIDays of co-culture (B) H22280123450 5 10Relative CD274 MFIDays of co-culture (C) H1819*0123450 5 10Relative CD274 MFIDays of co-culture (D) H229146  Figure 2.8      Figure 2.8 CD210 levels of co-cultured macrophages remain similar to macrophages cultured alone  Relative expression of CD210 upon co-culture with NSCLC cell lines.  Macrophages co-cultured with H1373 (A) and H2291 (D) exhibited significantly lower expression of CD210 on Day 3, and no significant differences in macrophage expression of CD210 co-cultured with any cell line was observed at the end of co-culture. Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.    *00.20.40.60.811.21.41.60 5 10Relative CD210 MFIDays of co-culture(A) H137300.20.40.60.811.21.41.60 5 10Relative CD210 MFIDays of co-culture(B) H222800.20.40.60.811.21.41.60 5 10Relative CD210 MFIDays of co-culture (C) H1819*00.20.40.60.811.21.41.60 5 10Relative CD210 MFIDays of co-culture (D) H229147  Figure 2.9      Figure 2.9 CD163 levels of co-cultured macrophages remain similar to macrophages cultured alone Relative expression of CD163 upon co-culture with NSCLC cell lines.  Macrophages co-cultured with H1373 (A) and H2228 (B) exhibit significantly lower expression of CD163 on day 3 while H2291 (D) exhibited significantly lower expression on Day 1 (2-tailed Student’s T-test, p < 0.05).  No significant differences in macrophage expression of CD163 co-cultured with any lines at end of co-culture, however H2228 co-cultured macrophages displayed increased CD163 levels.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05. *00.511.522.530 5 10Relative CD163 MFIDays of co-culture(A) H1373*00.511.522.530 5 10Relative CD163 MFIDays of co-culture(B) H222800.511.522.530 5 10Relative CD163 MFIDays of co-culture(C) H1819*00.511.522.530 5 10Relative CD163 MFIDays of co-culture(D) H229148  2.3.3 Increased IL-6 signaling in co-cultures with H2228 and H2291 In an attempt to decipher the cytokines and chemokines associated with in vitro M2 skewing, supernatants collected at the same time as macrophages (Day 0, 1, 3, 5, and 10) were assessed by a panel of ELISAs.  These included IL-1β, IL-1RA, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, TNF-α, TGF-β, MCP-1, MMP-9, soluble CD163, all secreted factors known to be involved in macrophage activation and modularity of polarity59,130,131.  While the majority of examined proteins showed no significant difference between single and co-cultures (data not shown), a significant and stepwise increase of IL-6 in half of the co-cultures (H2228 and H2291) was observed (Figure 2.10).  Interestingly, this increase was observed in the two lines from which co-cultured macrophages displayed significant M2 polarization by CD206, indicating that secretion of IL-6 may be important in M2 skewing and upregulation of CD206.              49  Figure 2.10  Figure 2.10 IL-6 levels from macrophages cultured alone and in co-culture with NSCLC cell lines Cytokine production in supernatants from macrophages cultured alone or in co-culture was assessed by ELISA assays. Supernatants from co-culture with H2228 and H2291 exhibited higher IL-6 concentrations than H1373 and H1819 co-cultures and macrophages cultured alone.  Mean ± SEM of replicate experiments.       -50050100150200250Mφ alone Mφ + H1373 Mφ + H2228 Mφ + H1819 Mφ + H2291Concentration (pg/mL)013510Day50  2.3.4 Co-culture induces IL-6 expression in macrophages In the original co-culture experiments, cell culture inserts with 3.0 µm pores were used so as to allow the movement of cytokines and other small proteins but not cells.  As a result, the ELISA assays were unable to decipher from which cell population the increased IL-6 originated.  ELISA assays of macrophages and tumor cell lines cultured alone indicated that the LCCLs were the primary source of IL-6, as macrophages cultured alone produced little IL-6 (Figure 2.11).  In an attempt to determine whether co-culture induced IL-6 production in macrophages, RNA was extracted from the individual cell populations within the co-cultures, as well as from macrophages and tumor cell lines cultured alone.  qPCR revealed that macrophages in H2228 and H2291 co-cultures expressed increased IL-6; expressing on average 2.09 and 2.14 fold more IL-6 than when cultured alone, respectively (Figure 2.12).  These findings suggest that tumor cell derived factor(s) induce macrophage IL-6 expression when in co-culture and may contribute to the increased M2 skewing upon co-culture.            51  Figure 2.11  Figure 2.11 IL-6 levels from macrophages and NSCLC cell lines cultured alone Cytokine production in supernatants from macrophages and NSCLC cell lines cultured alone was assessed by ELISA assays. Supernatants from H2228 and H2291 cultures exhibited higher IL-6 concentrations than macrophages, H1373, and H1819 cultures.  Mean ± SEM of replicate experiments.        050100150200250300350400450Mφ alone H1373 alone H2228 alone H1819 alone H2291 aloneConcentration (pg/mL)03510Day52  Figure 2.12  Figure 2.12 qPCR reveals macrophages in co-culture express more IL-6 than when cultured alone  Relative expression of IL-6 in co-culture with H2228 and H2291 and macrophages cultured alone. Monocytes were cultured alone and in co-culture with H2228 or H2291 for 10 days and cells collected separately for RNA extraction and RT-qPCR.  18S ribosomal RNA was used as an endogenous control to normalize cDNA input.  Macrophages cultured alone were used as the calibrator to determine the expression of IL-6 by the macrophages in co-culture relative to the macrophages cultured alone.  Macrophages in co-culture with H2228 and H2291 express on average 2.09 and 2.14 fold more IL-6 transcript, respectively. Values reported as mean ± SEM of duplicate experiments.    00.511.522.533.5Mφ cultured alone Mφ in co-culture with H2228 Mφ in co-culture with H2291Relative IL-6 expression 53  2.4 Discussion Macrophages are a key immune cell type in inflammation and cancer.  While their phenotype is dependent upon the specific signals in their local microenvironment, a simplified view of the spectrum of activation states is widely accepted with M1 and M2 phenotypic distinctions representing the two extremes.  Although M1 macrophages are thought to be present at sites of tumor initiation, macrophages typically end up being skewed to an M2 phenotype by most types of advanced cancers.  Here we provided evidence that co-culture of human monocytes with lung AC cell lines results in increased M2 skewing, supporting observations in patient samples where M2 macrophages predominate.  Mantovani and colleagues proposed an M1/M2 macrophage model, in which M1 macrophages were induced by IFN-γ and LPS or TNF, and M2 macrophages were divided further into the subsets M2a, M2b, and M2c to accommodate the phenotypic distinctions between macrophages stimulated with IL-4 and IL-13, immune complexes and certain TLR ligands, and IL-10 and glucocorticoids, respectively103.  M2a macrophages are considered to express CD206, CD204, and Arg-1, promoting Th2 responses through type II inflammation, and participate in the killing and encapsulation of parasites.  On the other hand, M2b or type II macrophages are thought to promote Th2 activation and immunoregulation, and express MHCII and CD86.  Finally, M2c or ‘deactivated’ macrophages express CD206 and CD163, secrete IL-10 and TGF-β, and participate in immunoregulation as well as matrix deposition and tissue remodeling.  Furthermore, in vitro polarization of human macrophages by Ambarus et al. found that IL-4 polarized macrophages upregulated CD206 and CD200R, while IL-10 stimulation induced expression of CD163, CD16, and CD32127.  Key markers associated with these subsets were included in our macrophage panel (CD163, CD206, CD200R), as well as other markers 54  associated with immune suppression, namely CD210 (IL-10 receptor) and CD274 (PD-L1) in an attempt to accurately decipher the macrophage phenotypes induced upon co-culture with tumor cell lines.  We chose to culture monocytes alone in the absence of additional cytokines or growth factors, knowing that monocytes differentiate into macrophages passively in culture over the course of several days.  While others have quickened differentiation with the use of cytokines, we opted not to add M-CSF or GM-CSF to avoid directed polarization, ensuring that any skewing observed was a result of soluble factors secreted by the lung AC cell lines.  Passive differentiation of our cultured macrophages was confirmed by flow cytometry (Figure 2.1-2.2), validating our culture method and agreeing with previous work by Meisel et al.128.  Increased expression of all markers tested was observed over the course of culture, consistent with differentiation.  A substantial stepwise induction of CD163, CD200R, and CD206 expression (Figure 2.2) throughout the culture period was observed, suggesting a trend to M2 skewing in the absence of cytokines.  However, this was not unexpected as it has been noted that the default differentiation pathway of adherent human monocytes on tissue culture plastic is an M2 phenotype, while Chamberlain et al. recently found that murine macrophages following extended culture consistently displayed an M2 phenotype through the upregulation of CD206132.  M2 skewing in culture has also been attributed to the presence of TGF-β in FBS.  ELISA data from media only controls showed the presence of latent TGF-β (data not shown) indicating that the M2 skewing that occurred over the course of culture may have been due to factors within the culture media.  Following co-culture of macrophages with LCCLs, we observed a significant increase in CD206 expression in macrophages co-cultured with tumor cells relative to macrophages cultured 55  alone, trends to increased CD200R, and little to no change in CD163 expression, suggesting a CD206+CD163- polarity.  The co-cultured macrophages thus resemble M2a macrophages as opposed to an M2c phenotype, indicating that IL-4 and/or IL-13 secretion by lung AC cell lines may be responsible for macrophage skewing in vitro.  However, ELISA analysis of individual macrophage and tumor cell cultures as well as co-cultures revealed no IL-4 or IL-13 production in any sample (data not shown), suggesting that the increase in CD206 expression was induced by another mechanism.  Furthermore, although we anticipated increased CD210 expression, due to IL-10 secretion by tumor cells, ELISA assays confirmed no IL-10 was being secreted, providing an explanation as to why this marker failed to be significantly induced upon co-culture.  CD163 has been used as a marker of M2 macrophages in a number of studies, particularly in studies correlating M1/M2 IHC staining with prognosis in lung cancer55,56.  As our hypothesis was that co-culture with advanced lung AC cell lines would skew monocytes to an M2 phenotype, we had expected to observe an increase in CD163 expression.  While in vitro experiments do not accurately represent the complexity of in vivo physiological conditions, it is interesting that an increase in CD163 expression was not observed.  It may be that physical contact is needed between the tumor cell and macrophage in order to initiate its expression.  These results suggest that it is possible that CD206 may be a better marker of M2 macrophages in lung cancer.  While the high levels of CD206 expression suggest that the co-cultured macrophages were skewed to an M2a phenotype, H2228 co-cultures displayed increased expression of CD200R and CD274 (PD-L1), suggesting that these macrophages may have a more immunosuppressive phenotype than the other co-cultured macrophages.  Engagement of CD200R with its ligand CD200 suppresses LPS-induced macrophage cytokine production133, 56  suggesting that these macrophages have been skewed further away from the M1 end of the macrophage activation spectrum.  Recently, macrophages in hepatocellular carcinoma were shown to express PD-L1 supporting tumor progression107, and our observations here suggest this may also be the case in lung AC.  PD-L1 is a clinically relevant marker as monoclonal antibodies have been developed and are in clinical trials for the treatment of NSCLC.  Treatment with these immunotherapies may therefore also target M2 macrophages in the tumor microenvironment as well as the tumor cells.  Interestingly, expression of CD80, a co-stimulatory molecule involved in antigen presentation, activation of an adaptive immune response and subsequently used as an M1 marker, was increased on the macrophages co-cultured with H2228, intriguingly the only cell line harbouring the EML4-ALK gene rearrangement.  Investigation into the similarities and differences of CD80 and CD86, its hitherto seemingly redundant co-stimulatory relative, has suggested that CD80 is probably a more potent ligand for the inhibitory CD152 (CTLA-4) and that CD86 is a more effective ligand for the stimulating CD28134.  It has been suggested that resting and activation states may be differentiated by a change in the balance between CD86:CD28 and CD80:CD152 interactions, where upon activation CD80 is downregulated thereby increasing the number of CD86:CD28 interactions thus leading to immune activation of T cells expressing CD28.  It is therefore plausible that CD80 may be upregulated upon M2 skewing.  This is merely speculative, however, as a limited number of markers were assessed due to technical limitations of the flow cytometer used, and as a result macrophage CD86 surface expression was not examined.  Future experiments with the assessment of a larger panel of markers using a more advanced cytometer may substantiate this proposed mechanism.  It is also possible that the macrophages are displaying a mixed phenotype, as there is a large grey area between the two extreme ends of the macrophage activation spectrum.   57  Based on our ELISA data, the increase in CD206 expression may be in part correlated with increased IL-6 signaling, as IL-6 was observed in the co-cultures with H2228 and H2291, the two cell lines that yielded the ‘most’ M2 skewed macrophages.  Concordantly, IL-6 has been implicated in the alternative activation of macrophages135.  Interestingly, H2228 and H2291 are considered to be more mesenchymal like cell lines whereas H1373 and H1819 are more epithelial in their morphology136,137.  Concomitantly, tumors that have a more mesenchymal morphology are considered more aggressive, in line with the observation that EMT is correlated with aggressiveness and poor prognoses in many cancer types, including lung cancer138,139.  Furthermore, mesenchymal stem cells have been shown to abundantly secrete IL-6 in both mice and humans140,141.  In fact, upon examination of 120 cytokines at both the mRNA and protein levels, it was established that IL-6 is the most highly expressed cytokine in these cells142, supporting our co-culture observations.  Moreover, while H1373 and H1819 are cell lines derived from smokers, H2228 and H2291 were established from lung AC tumors in non-smokers, and therefore would have disparate mutational profiles.  Interestingly, BAL fluid from non-smokers was found to contain significantly more IL-6 than BAL samples from smokers143; however, the samples came from healthy volunteers and asymptomatic smokers with no history of pulmonary disease, and a correlation between ACs from non-smokers and immune cell modulation remains unstudied.  Finally, while all four cell lines were wildtype for EGFR, H1373 and H2291 harboured activating KRAS mutations, and H2228 and H1819 did not.  Interestingly, it has been stated that one of the important functions of Kras activation is to induce expression of IL-6144.  Ancrile et al. found that mRNA and protein levels of IL-6 were greatly increased in cell types genetically transformed to ectopically express RasG12V compared with nontumorigenic cells lacking mutant Ras145.  Conversely, we observed elevated IL-6 secretion in H2228 and 58  H2291 cultures, as opposed to H1373 and H2291 as would be expected based on Ancrile et al.’s findings.  However, the specific residues mutated in KRAS differed between the two studies: Ancrile et al. induced expression of RasG12V, whereas H1373 and H2291 harbour KRAS G12C and G12F mutations, respectively.  Different KRAS mutants in NSCLC have been found to be metabolically diverse146 and to activate different branches of the Ras pathway147, which may result in differential induction of IL-6 and thereby account for the disagreement observed.  It is also possible that different upstream receptor tyrosine kinases or downstream components could be mutated, inappropriately activating the Ras pathway in H2228, leading to IL-6 production.     Interestingly, qPCR revealed that macrophages co-cultured with H2228 and H2291, the two co-cultures in which IL-6 signaling was observed, expressed more IL-6 transcript than when cultured alone, suggesting that IL-6 expressing tumor cell lines induced expression of IL-6 in macrophages.   Further Western blot analysis of protein lysates for IL-6 in each of the cell types would provide a more accurate representation of true IL-6 protein levels in the cell, or incubation with GolgiPlug, which inhibits intracellular protein transport processes resulting in the accumulation of cytokines and protein in the Golgi complex, allowing for detection by flow cytometry.  However, with low cell numbers available for co-culture experiments, qPCR proved to be the most feasible approach.  While taken together these findings support the notion that increased IL-6 signaling from tumor cells (specifically those with a more mesenchymal phenotype) may be important in the M2 skewing of macrophages, further investigation of this trend in a larger panel of NSCLC cell lines is required.   Here we demonstrated that co-culture with lung AC cell lines skews macrophage differentiation to an M2 polarity potentially in part through IL-6 secretion from tumor cells.  As our study was limited to skewing by secreted soluble factors due to the use of cell culture inserts, 59  it is likely that cell-to-cell interactions would result in a further increase of M2 skewing.  However, the physical interaction of macrophages with allogeneic tumor cells may potentially activate phagocytic activity and an M1 phenotype, but was not examined in this scenario.    60  Chapter 3: Cisplatin impedes macrophage differentiation 3.1 Introduction While considerable advances have been made in the treatment of NSCLC within the past decade, particularly for patients whose tumors harbour activating mutations in EGFR or oncogenic fusions involving ALK, RET, or ROS kinases, and more recently through the development of immunotherapies, chemotherapy still remains the most common treatment modality and standard of care for the majority of NSCLC148.  However, current chemotherapy regimens provide a modest survival benefit at best, with resistance ultimately developing in all advanced cases.  A better understanding of the tumor and microenvironmental factors that contribute to or promote resistance are desperately needed in order to better predict patients who are most likely to benefit from specific chemotherapy treatment modalities.   Cisplatin, one of the most commonly prescribed first line chemotherapeutic drugs for NSCLC, is an inorganic platinum agent that forms highly reactive platinum complexes which bind to GC-rich sites in DNA.  Binding induces intra- and inter-strand DNA crosslinks that result in apoptosis and cell growth inhibition149.  As chemotherapy is administered intravenously, cisplatin is delivered to all cells in the vicinity of blood vessels.  Thus, cisplatin reaches tumor cells as well as the other cell types within the tumor microenvironment, including macrophages and recruited monocytes.  As macrophages make up the majority of the immune infiltrate in tumors, determining how their polarity is affected in response to cisplatin will further our understanding of how these cells contribute to lung cancer progression and response to treatment.   Studies in the 1990s found that macrophages were activated by cisplatin treatment resulting in increased antigen presentation150 and a tumoricidal state151.  More recently however, it was found that chemotherapy alters monocyte differentiation to favor an M2 phenotype152.  61  Dijkgraaf et al. investigated the impact of cisplatin on ten different cervical and ovarian cancer cell lines and on their ability to affect the differentiation and function of co-cultured monocytes in vitro.  They found that treatment with cisplatin increased the potency of tumor cell lines to induce IL-10 producing macrophages, which displayed increased levels of activated STAT3 that they deduced was due to tumor-derived IL-6.  They suggested that a chemotherapy-mediated increase in M2 macrophages may create an indirect mechanism for chemoresistance.   We have previously demonstrated (Chapter 2) that co-culture of monocytes with certain NSCLC cell lines favours an M2 phenotype potentially through tumor-derived IL-6.  As this pathway has been shown to be induced through the treatment of cisplatin, in this chapter we sought to determine how cisplatin treatment influences macrophage polarity and whether the co-culture of macrophages and LCCLs with physiologically relevant doses of cisplatin further promotes an M2 phenotype.   3.2 Materials and methods 3.2.1 Cisplatin treated co-cultured experiments Human CD14+ peripheral blood monocytes were cultured alone or in co-culture with lung AC cell lines as in Chapter 2.  Both cell lines and monocytes were treated with 2 µg/mL cisplatin for the duration of the co-culture, a concentration that reflects the estimated physiological concentration of cisplatin attained in tumors152.  Co-culture experiments with cisplatin treatment were performed simultaneously with those previously described in Chapter 2, and treatment induced effects on macrophage polarity were determined by comparing untreated vs. treated co-culture experiments.  62  3.2.2 Dose response assays Dose response assays were performed to determine the IC50 values of monocytes and LCCLs.  Cells were seeded in triplicate in 96 well plates at optimal densities for growth (4000 cells/well for H1373 and H2228, 6000 cells/well for H1819, 7000 cells/well for H2291, and 10 000 cells/well for monocytes).  Cells were subjected to a series of 2-fold dilutions of cisplatin prepared in cell growth media.  The experimental concentrations ranged from 100 µg/mL to 191 pg/mL.  Blank wells contained equal volumes of culture media.  Cells were incubated for 72 hours at 37°C and then treated with 10 µL Alamar Blue cell viability reagent (Invitrogen) according to manufacturer’s instructions.  The reaction product was quantified by measuring absorbance at 570 nm with reference to 600 nm using an EMax plate reader (Molecular Devices).  Dose response curves and IC50 values were generated in Graph Pad v5 using the proportionate response of all twenty drug concentrations.  3.2.3 Cell viability assays MTT assays were performed to assess NSCLC cell viability following cisplatin treatment.  Cells were seeded in triplicate in 96 well plates at optimal densities for growth (3000 cells/well for H1373, H1819, and H2228, and 5000 cells/well for H2291).  Viability was measured over five consecutive days by the addition of 10 µL MTT reagent (every 24 hours) and incubation for an additional four hours, followed by the addition of 100 µL of 20% SDS to solubilize the MTT dye. Plates were quantified by spectrophotometry (EMax Plate Reader, Molecular Devices) at 570 nm with reference to 650 nm. Experiments were performed in triplicate and viability of treated cells (2 µg/mL) compared to their untreated controls.  63  3.2.4 Detection of apoptosis The amount of cell death of treated and untreated tumor cells was assessed by measurement of staining for apoptotic markers propidium iodide (PI) and Annexin V (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen). NSCLC cell lines were seeded in 6 well plates at a density of 100 000 (H1373, H2228, H1819) or 150 000 (H2291) cells/well and were either untreated or treated with 2 µg/mL cisplatin. At 24, 48, 72, and 120 hours post-treatment cells were trypsinized, washed, and processed as per the manufacturer’s instructions. Flow cytometry was performed using a BD FACSCanto II flow cytometer and analyzed with FACSDiva version 6.1 software (BD Biosciences), as described in section 2.2.3. Results are reported as the percent of the population staining positive for PI and/or Annexin V (% apoptosis).             64  3.3 Results 3.3.1 The estimated physiological concentration of cisplatin in tumors impairs proliferation and induces apoptosis in NSCLC cell lines  To assess the effect of cisplatin on monocyte differentiation and macrophage polarity, monocytes were cultured alone or in co-culture with lung AC cell lines in the absence (Chapter 2) or presence of cisplatin (Figures 3.5-3.11). 2 µg/mL of cisplatin was chosen for treatment, as this has been estimated as the physiological concentration attained in tumors, after taking into account the maximum dose of cisplatin given to patients and the poor vascularity of tumors152. To ensure this concentration would be appropriate for our in vitro experiments, dose response curves of cisplatin with monocytes and the four LCCLs were performed. H1373 and H2228 were more sensitive cell lines with IC50 values just above the chosen cisplatin concentration, while the value for monocytes was very close to 2 µg/mL.  H1819 was slightly more resistant with an IC50 value of 5.77 µg/mL, while H2291 was the most resistant line at 18.62 µg/mL (Figure 3.1).  The chosen cisplatin concentration fell below the range of IC50 values, confirming that 2 µg/mL was an appropriate concentration for our experiments.  While macrophages were collected for analysis of M1 and M2 markers by flow cytometry, the effect of cisplatin on LCCLs cells was investigated by assessing viable cell numbers via metabolic activity and assessing apoptosis by MTT assay and Annexin V/PI staining, respectively (Figures 3.2 and 3.3).  Treatment with cisplatin decreased the number of metabolically active cells (Figure 3.2) and induced apoptosis (Figure 3.3) in all cell lines.  H1373 and H2228 cells showed a slight increase in absorbance followed by a decrease in MTT assays (Figure 3.2A and B), consistent with an increase in apoptotic cells as observed by Annexin V/PI staining (Figure 3.3B).  Treated H1819 cells also showed a slight increase in MTT absorbance, but little compared to untreated controls (Figure 3.2C).  Annexin V/PI staining revealed that little apoptosis was occurring due to cisplatin in the 65  first 48 hours (Figure 3.3B); therefore, the lower MTT absorbance in the first two days compared with untreated controls was likely due to decreased cell numbers and/or metabolic activity as opposed to increased cell death.  As cisplatin is known to intercalate DNA during replication, it is likely that the decreased MTT absorbance is a result of reduced proliferation.  Treated H2291 cells appeared to proliferate, with increased MTT absorbance and little cisplatin-induced apoptosis observed by Annexin V/PI staining at 24-72 hours, before crashing rapidly (Figure 3.2D).  As H2291 is a slow growing line, with a doubling time that is substantially longer than the other three cell lines used, and cisplatin targets rapidly proliferating cells, it’s likely that this slower growth rate protected cells from the effects of cisplatin for a little longer relative to the other cell lines.                66  Figure 3.1  -2 0 2 4-0 .20 .00 .20 .40 .60 .8L o g 2  o f c is p la tin  c o n c e n tra tio n  (u g /m L )Response(proportion of control)M o n o c y teH 1 3 73H 2 2 28H 1 8 19H 2 2 91 Cell line IC50 H1373 2.76 H2228 3.73 H1819 5.77 H2291 18.62 Monocyte 2.06  Figure 3.1 Dose response assays of monocytes and NSCLC cell lines Dose response assays were performed to confirm that the chosen cisplatin concentration for co-culture experiments was appropriate.  Monocytes, H1373, and H2228 were the most sensitive with IC50s at 2.06, 2.76, and 3.73 µg/mL, respectively.  H1819 was slightly less sensitive with an IC50 of 5.77 µg/mL, while H2291 was the most resistant with an IC50 of 18.62 µg/mL, likely due to its long doubling time.  Error bars represent the standard error of the mean of replicate experiments.   67  Figure 3.2  Figure 3.2 Cisplatin impairs growth of lung AC cell lines at physiologically relevant doses Cell viability of treated (2 µg/mL) and untreated lung AC cell lines, as measured by MTT assay. Values are reported as mean ± SEM of replicate experiments. Cisplatin impairs metabolic activity, and therefore presumably proliferation in all cell lines relative to untreated controls.        68  Figure 3.3   Figure 3.3 Cisplatin induces apoptosis in lung AC cell lines (A) (B)  69  Figure 3.3 Cisplatin induces apoptosis in lung AC cell lines Cisplatin induces apoptosis in all cell lines relative to untreated controls, as measured by Annexin V/PI staining and flow cytometry.  (A) Representative flow cytometry plots of H2228 showing Annexin V staining on the x-axis and PI staining on the y-axis.  Gates were based on unstained and single stain controls for each cell line.  (B) Cells staining positive for Annexin V only (early apoptotic cells), or both Annexin V and PI (late apoptosis) were quantified by flow cytometry at 24, 48, 72, and 120 hours after cisplatin treatment. Results are reported as the total % apoptosis (Annexin V positive + Annexin V and PI positive) for each cell line and a Pearson’s chi-squared test applied.  Untreated samples are depicted in blue and samples treated with 2 µg/mL cisplatin are shown in red.               70  3.3.2 Macrophages treated with cisplatin differ in granularity when cultured alone and when co-cultured In order to determine the effects of cisplatin on monocyte to macrophage differentiation and macrophage polarity in the context of lung AC cell lines, monocytes were cultured alone or in co-culture with lung AC cell lines either in the presence or absence of cisplatin.  As expected, the number of viable macrophages decreased over the course of the experiment (Figure 3.4).  Following treatment with cisplatin, macrophages cultured alone appeared smaller and more granular based on forward and side scattered light parameters, respectively, than their untreated counterparts (Figure 3.5).  Interestingly, when in co-culture, treated macrophages were again smaller, but now less granular than their untreated counterparts (Figure 3.6), suggesting that co-culture with lung AC cell lines affects macrophage granularity in response to treatment or that the macrophages are less differentiated.              71  Figure 3.4  Figure 3.4 Cisplatin reduces macrophage viability  Cisplatin reduces macrophage viability relative to untreated controls, as measured by eF450 Fixable Viability dye staining and flow cytometry.  Macrophages cultured alone in the presence of cisplatin had significantly decreased viability relative to untreated controls (p < 0.05).  Live macrophages were identified by their exclusion of the viability dye and percentages were normalized to untreated macrophages and reported as mean ± SEM of three independent experiments.  A 2-tailed Student’s T test was performed, * = p < 0.05.        * **00.20.40.60.811.2Day 0 Day 1 Day 3 Day 5 Day 10ViabilityControlTreated72  Figure 3.5  Figure 3.5 Macrophages treated with cisplatin appear smaller and more granular when cultured alone Representative flow cytometry plots of Day 10 untreated macrophages (A) and macrophages treated with 2 µg/mL cisplatin (B) cultured alone.  (C) Macrophages treated with cisplatin had significantly decreased FSC and increased SSC values than untreated macrophages when cultured alone (p < 0.05).  Median FSC and SSC values of the live macrophage population in the cisplatin-treated group were normalized to their untreated counterparts and are reported as mean ± SEM of three independent experiments.  A 2-tailed Student’s T test was performed, * = p < 0.05.       73  Figure 3.6  Figure 3.6 Macrophages in co-culture appear less differentiated with cisplatin treatment  74  Figure 3.6 Macrophages in co-culture appear less differentiated with cisplatin treatment  Representative flow cytometry plots of untreated co-cultured macrophages (A) and co-cultured macrophages treated with 2 µg/mL cisplatin (B), collected on day 10 (macrophages co-cultured with H2228 shown). (C) Co-cultured macrophages treated with cisplatin showed a trend of decreased FSC and SSC values, with significantly decreased SSC values for treated macrophages co-cultured with H1373 and H2228 (2-tailed Student’s T test, p < 0.05).  Median FSC and SSC values of the live macrophage population in co-cultured macrophages treated with cisplatin were normalized to their untreated counterparts and are reported as mean ± SEM of three independent experiments.               75  3.3.3 Macrophages treated with cisplatin display decreased marker expression To assess macrophage polarity following co-culture and treatment, macrophages were collected as described in 3.2.1 and stained with the same panel of cell surface protein markers used previously (Table 2.1) (Figures 3.7-3.13).  Macrophages cultured alone and treated with cisplatin displayed decreased expression of most markers relative to their untreated counterparts (Figure 3.7).  Interestingly, the markers that displayed significantly decreased expression (CD163, CD200R, CD206, and CD274) were those in which greater stepwise increases were observed over the course of macrophage differentiation (see Figure 2.2).  In general, the expression of most markers decreased in treated co-cultured macrophages relative to untreated controls (Figures 3.9-3.13), suggesting that the treated macrophages were less differentiated.  The exception to this trend was CD206, an M2 marker that we previously observed to be induced on macrophages in co-culture (section 2.3.2), which demonstrated the opposite trend, remaining elevated to similar or greater levels than all untreated co-culture controls (Figure 3.8).  Conversely, CD274 (PD-L1), an M2 associated marker of particular clinical interest, displayed significantly decreased expression on macrophages co-cultured with all four cell lines (Figure 3.9). Interestingly, CD80 (a marker of M1 macrophages) showed a trend towards decreasing expression over the course of co-culture with H2228 and H2291 (the two cell lines in which upregulated IL-6 was observed), suggesting less M1 skewing, while a trend towards increased expression and increased M1 skewing at the end of co-culture was observed with the other two cell lines.  However, due to variability between replicate experiments, these trends did not reach statistical significance in any of the cell lines (Figure 3.10).  Nevertheless, these data further emphasize the different effects of our panel of cell lines on macrophage polarity and suggest that the genetic background of tumor cells likely plays an important role in dictating macrophage polarity within tumors.    76  Figure 3.7  Figure 3.7 Macrophages treated with cisplatin display reduced marker expression Treated macrophages display significantly decreased expression of  M2-associated surface markers (Student’s T-test, p < 0.05).  MFIs of live macrophages cultured alone were normalized to their untreated counterparts and are reported as mean ± SEM of three independent experiments.        ****00.20.40.60.811.21.4Control Treated Control Treated Control Treated Control Treated Control Treated Control TreatedCD163 CD210 CD200R CD206 C274 CD80MFIs normalized to untreated Mφ cultured alone77  Figure 3.8       Figure 3.8 Macrophage CD206 expression remains elevated with cisplatin treatment Expression of CD206 on treated co-cultured macrophages relative to untreated co-cultured macrophages.  Monocytes were co-cultured with one of four NSCLC cell lines, H1373 (A), H2228 (B), H1819 (C), or H2291 (D), for 10 days, and treated with 2 µg/mL cisplatin for the duration of the experiment.  Macrophages co-cultured with H1373 (A) and H1819 (C) exhibit CD206 expression at levels greater than their untreated counterparts, while those co-cultured with H2228 (B) and H2291 (D) exhibit similar levels to controls.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.   *00.511.520 5 10Relative CD206 MFIDays of co-culture (A) H137300.511.520 5 10Relative CD206 MFIDays of co-culture (B) H222800.511.520 5 10Relative CD206 MFIDays of co-culture (C) H1819*00.511.520 5 10Relative CD206 MFIDays of co-culture (D) H229178  Figure 3.9      Figure 3.9 Macrophage CD274 (PD-L1) expression is decreased with cisplatin treatment relative to untreated counterparts Expression of CD274 on treated co-cultured macrophages relative to untreated co-cultured macrophages.  Macrophages co-cultured with all lines exhibited significantly lower expression of CD274 by the end of co-culture, as low as 0.4 relative to their untreated counterparts.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.     *00.511.520 5 10Relative CD274 MFIDays of co-culture (A) H1373***00.511.520 5 10Relative CD274 MFIDays of co-culture(B) H2228***00.511.520 5 10Relative CD274 MFIDays of co-culture (C) H1819* *00.511.520 5 10Relative CD274 MFIDays of co-culture (D) H229179  Figure 3.10       Figure 3.10 Macrophages CD80 expression differs between the four lung AC cell line co-cultures when treated with cisplatin Expression of CD80 on treated co-cultured macrophages relative to untreated controls.  Relative MFIs reflect mean ± SEM of three independent experiments.  Macrophages co-cultured with all four cell lines exhibited decreased expression of CD80 early on (2-tailed Student’s T-test, p > 0.05).     *00.511.50 5 10Relative CD80 MFIDays of co-culture(A) H1373*00.511.50 5 10Relative CD80 MFIDays of co-culture (B) H2228**00.511.50 5 10Relative CD80 MFIDays of co-culture (C) H1819* *00.511.50 5 10Relative CD80 MFIDays of co-culture (D) H229180  Figure 3.11       Figure 3.11 Macrophage CD200R expression is decreased in the cisplatin treated group Expression of CD200R on treated co-cultured macrophages relative to untreated co-cultured macrophages.  Macrophages co-cultured with H2228 (B) exhibit significantly lower expression of CD200R at the final collection point.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.      00.20.40.60.811.20 5 10Relative CD200R MFIDays of co-culture (A) H1373**00.20.40.60.811.20 5 10Relative CD200R MFIDays of co-culture(B) H2228*00.20.40.60.811.20 5 10Relative CD200R MFIDays of co-culture(C) H181900.20.40.60.811.20 5 10Relative CD200R MFIDays of co-culture(D) H229181   Figure 3.12       Figure 3.12 Macrophage CD210 expression is decreased upon cisplatin treatment  Expression of CD210 on treated co-cultured macrophages relative to untreated co-cultured macrophages.  Macrophages co-cultured with H1373 (A), H2228 (B), and H2291 (D) exhibit significantly lower expression of CD210 on days 5 and 10 as well as day 3 (B and D), while H1819 (C) exhibited significantly lower expression on day 3 but not at any other collection points.  Relative MFIs reflect mean ± SEM of three independent  experiments, * = p < 0.05.     * *00.20.40.60.811.20 5 10Relative CD210 MFIDays of co-culture (A) H1373* **00.20.40.60.811.20 5 10Relative CD210 MFIDays of co-culture (B) H2228*00.20.40.60.811.20 5 10Relative CD210 MFIDays of co-culture(C) H1819* * *00.20.40.60.811.20 5 10Relative CD210 MFIDays of co-culture(D) H222882  Figure 3.13       Figure 3.13 Macrophage CD163 expression is decreased with cisplatin treatment Expression of CD163 on treated co-cultured macrophages relative to untreated co-cultured macrophages.  Monocytes were co-cultured with H1373 (A), H2228 (B), H1819 (C), or H2291 (D), for 10 days, and treated with 2 µg/mL cisplatin.  Macrophages co-cultured with H1373 (A), H2228 (B), and H1819 (C) exhibit significantly lower expression of CD163 on Day 10 as well as other days, while H2291 (D) exhibited significantly lower expression on days 1 and 3 but not at the final two collection points.  Relative MFIs reflect mean ± SEM of three independent experiments, * = p < 0.05.  * * *00.20.40.60.811.20 5 10Relative CD163 MFIDays of co-culture(A) H1373* * **00.20.40.60.811.20 5 10Relative CD163 MFIDays of co-culture (B) H2228**00.20.40.60.811.20 5 10Relative CD163 MFIDays of co-culture(C) H1819* *00.20.40.60.811.20 5 10Relative CD163 MFIDays of co-culture (D)83  3.4 Discussion Chemoresistance has long been thought to arise as a consequence of intrinsic genetic changes to the tumor that result in increased drug efflux, activation of detoxifying enzymes, diminished sensitivity to apoptosis and decreased drug entry.  However, there is now substantial evidence that suggests chemoresistance can also be modulated by extrinsic factors within the tumor microenvironment including growth factors, cytokines, and infiltrating immune cells.  Macrophages make up the majority of the immune infiltrate in tumors and their presence in tumors is correlated with poor prognosis in multiple cancer types.  Recently, treatment of ovarian and cervical cancer cell lines was shown to induce differentiation to a more M2-like phenotype of macrophages following treatment with platinum based chemotherapeutics.  As platinum based doublet chemotherapy is the standard of care for NSCLC and macrophages are a prominent immune cell type within lung tumors, here we investigated the effect of cisplatin on macrophage differentiation in the context of lung AC, by co-culturing monocytes with lung AC cell lines in the presence of cisplatin.  To assess the effect of cisplatin on lung AC cell lines cell viability and apoptosis assays were performed.  As cell culture inserts containing the LCCLs were replaced on day 5 of co-cultures in an attempt to prevent cells from becoming overconfluent and exiting their exponential growth phase (see section 2.2.2), the final timepoint for viability and apoptosis assays was 5 days/120 hours.  The effect of cisplatin on macrophages was similarly assessed by flow cytometry measurement of a fixable viability dye used to gate live cells in all our previous experiments.  While patients are usually treated with 60 -100 mg/m2, the maximum dose as measured in the blood is 5-6 µg/mL and it has been previously shown that because of tumor bulk and poor vascularity, tumors are typically exposed to concentrations around 2 µg/mL152.  As we 84  wanted to mimic the tumor environment in vivo as closely as possible in our in vitro cultures, using a physiologically relevant concentration of cisplatin was paramount.  However, we first had to ensure that this concentration of cisplatin would only kill a fraction of our tumor cells and macrophages, much like what happens in patient samples.  Dose response assays confirmed that the IC50s for all four lines ranged from 2.76 to 18.62 µg/mL, enabling us to use 2 µg/mL cisplatin, a biologically relevant concentration.  Not surprisingly, following treatment with cisplatin, all cell lines showed stepwise increases in the percent apoptosis (Figure 3.3) and reduced viability (Figure 3.2), compared to untreated controls.  While these trends varied slightly between cell lines, likely because of the different growth characteristics and cisplatin sensitivities, all cell lines displayed phenotypes consistent with chemotherapy induced cell death.   Similar to LCCLs, macrophage viability decreased upon treatment with cisplatin (Figure 3.4).  When cultured alone, viable/live macrophages treated with cisplatin were smaller and more granular/internally complex than their untreated counterparts, suggesting that differentiation was impeded and stress granule formation was induced, respectively, upon sub-lethal doses of cisplatin.  In addition to these morphological changes, we also observed reduced expression of the cell surface markers that were more highly expressed on untreated macrophages (CD163, CD200R, CD206, and CD274).  Cytoplasmic stress granules are specialized regulatory sites of mRNA translation that form under a variety of stress conditions and are known to inhibit translation initiation153, which could explain the decreased expression of cell surface markers we observed.   Alternatively, it has been found that M2-like macrophages are more vulnerable to chemotherapy152.  It is therefore possible that the monocytes were a heterogeneous population, with those more skewed to an M2 phenotype becoming more susceptible to cisplatin induced apoptosis. 85  Interestingly, when macrophages were co-cultured with LCCLs and treated with cisplatin, they again appeared smaller, but this time less granular than untreated controls.  Cell surface marker expression was still largely decreased in these macrophages, suggesting that the formation of stress granules is not solely responsible for reducing protein expression and that impeded differentiation is likely the main factor leading to changes in cell surface expression.  CD206 was the one surface marker that remained elevated to levels similar to that of their untreated counterparts.  CD206 has been implicated on Kupffer cells in a novel ligand-receptor pathway with hepatocyte growth factor beta chain that displayed enhanced ingestion of apoptotic neutrophils by Kupffer cells, potentially providing a new pathway for the enhancement of cell clearance154.  While this was found in Kupffer cells, (i.e. resident liver macrophages), it may be possible that a similar pathway is at play in the M2 skewed macrophages in the co-culture systems containing apoptotic tumor cells and the factors the dying cells secrete.  Conversely, another M2 associated macrophage marker, CD274 (PD-L1), exhibited decreased expression on macrophages in co-culture with all four NSCLC cell lines.  CD274 is of particular clinical interest, with novel immunotherapy agents directed at PD-L1 in advanced stages of development for the treatment of advanced or metastatic NSCLC155.  The PD-1:PD-L1 axis represents a key immunoregulatory checkpoint mechanism, with increased PD-L1 expression by tumor and immune cells associated with immune escape and tumor progression in NSCLC156-158.  The downregulation of PD-L1 by macrophages treated with cisplatin suggests that they are rendered less immunosuppressive by treatment, with the potential to become immune-activating if subsequently stimulated.  Alternatively, it is possible that macrophages expressing PD-L1 were preferentially killed, as M2-like macrophages have been found to be more vulnerable to chemotherapy152.  86  Although marker expression was altered to different extents following cisplatin treatment, on a whole, we found that sub-lethal doses of cisplatin inhibited macrophage differentiation and that the presence of treated tumor cells did not dramatically skew macrophages in either direction.  However, in treated individual macrophage cultures and co-cultures there was a trend to significantly decreased M2 marker expression while the M1 marker CD80 remained relatively stable over the ten day time course, indicating that cisplatin treatment may inhibit M2 skewing and render macrophages more amenable to M1 skewing by IFN-γ with LPS or TNF cytokine therapy.  In fact, in a recent study, Johansson and colleagues engineered TNF-α with a peptide targeting tumor vasculature and were able to accurately deliver low doses of TNF-α into mice with pancreatic neuroendocrine tumors.  They found that vascular remodeling occurred allowing CD8+ T cell infiltration, and that this was in part mediated by TAMs that had been reprogrammed towards a more inflammatory anti-tumoral M1 phenotype159.  Combination therapy involving cisplatin treatment supplemented with an immune activating compound could therefore have the potential to improve treatment response and prognosis.  While further work is required to determine whether or not this synergy is in fact true and leads to a survival benefit, current clinical trials assessing immunotherapies in combination with standard chemotherapies could provide critical insight.   In this study, we looked at the effect of macrophage differentiation in the presence of cisplatin; however, the effect of cisplatin on already differentiated macrophages is an equally important aspect to study.  Dijkgraaf et al. studied an aspect of this with a panel of ovarian and cervical cancer cell lines152.  In their experiments, tumor cell lines were treated with cisplatin for 24 hours and the tumor supernatant (TSN) was then added to monocyte cultures and differentiation assessed after six days.  An increase in the percentage of CD14+CD206+CD163+ 87  M2 macrophages was observed when TSN from treated cancer cells was used compared with TSN from untreated cells, suggesting cisplatin promotes an M2 phenotype.  However, in these experiments, the monocytes/macrophages were not exposed to cisplatin directly, nor were they cultured in the presence of tumor cells as they would be in the tumor microenvironment.  The difficulty in looking at macrophage differentiation in co-culture and subsequently assessing the effect of cisplatin treatment on the same culture, which would provide a better representation of the physiological situation, is feasibility, as attempting to recreate this in vitro becomes very complicated.  An ex vivo analysis of the effect of cisplatin on macrophage polarity would therefore more accurately answer this question, and would be more feasible than attempting to create culture conditions that truly represent physiological conditions.  We therefore thought it best to use clinical pleural effusion specimens to monitor M1/M2 macrophage polarity over the course of chemotherapy treatment.  In order to do this, we first needed to optimize single cell analysis of these specimens as well as determine any technical limitations in working with these samples.  The results of this are described in the next chapter.   88  Chapter 4: Optimization of single cell analysis for clinical pleural effusion specimens 4.1 Introduction  Pleural effusion is the abnormal, excessive collection of fluid in the pleural space, the space between the layers of tissue that line the lungs and the chest cavity.  Both layers of the pleura are covered with mesothelial cells which secrete a small amount of fluid that provide lubrication between the lungs and the chest wall.  A small amount of fluid also continuously seeps out of the blood vessels through the pleura attached to the chest wall into the pleural space, which is absorbed by the pleura attached to the lungs, and then drains into the lymphatic system, thereby returning to the blood.  When pleural effusions develop during malignancy, it is mainly due to increased vascularity resulting in more fluid seeping into the pleural space through the chest wall pleura, as well as tumor cells blocking lymphatic drainage.  Approximately 15% of LC patients have pleural effusion at the time of initial diagnosis, and 50% develop effusions later in the course of disease160.  This is an ominous sign for patients as it is associated with poor prognosis161.  When pleural effusion develops, patients undergo thoracentesis, a procedure in which a needle is inserted into the pleural space and the fluid is drained.  Thoracentesis may be for diagnostic as well as therapeutic purposes, since fluid accumulation can impede lung function, causing shortness of breath and discomfort to patients.  Pleural effusion specimens represent a useful model for studying the immune system involvement in LC.  While it is not equivalent to interrogating the tumor itself, it is generally thought to be more representative of the tumor microenvironment than probing the peripheral blood.  The excess pleural fluid (PF) needs to be drained, and in the vast majority of patients the 89  effusion persists, requiring repeated thoracentesis or the installation of a tunneled catheter (e.g. PleurX catheter) for outpatient drainage.  This allows for time course studies which would not be possible with biopsies like fine needle aspirates sampling the tumor mass.  The PF is normally discarded, making it readily available for research use, providing a suitable model for studying the immune system involvement in LC, specifically during the course of treatment.  As such we sought to optimize single cell analysis of these clinical specimens in preparation for future projects, such as the investigation of macrophage polarity over the course of treatment.  We assessed the effects of sample processing (spin speed) and long-term cryopreservation on cell populations and antibody binding for flow cytometric analysis, as spin speed can reduce cell viability in patient samples and it is unknown whether cryopreservation affects detection of cell surface markers, respectively.  Optimization of these conditions enables banking of surrogate patient samples such as BAL and pleural fluid, and robust comparison of patient samples collected at different time points.   4.2 Materials and methods 4.2.1 Pleural fluid samples Two PF samples were collected by Dr. Stephen Lam at the BC Cancer Agency and were processed immediately following collection.  The first patient (PF1) was a former smoker with newly diagnosed stage IV lung adenocarcinoma with malignant pleural effusion at the time of sample collection.  The tumor has since been classified ALK positive.  The second patient (PF2) was a current smoker with EGFR-positive metastatic stage IV NSCLC.  The pleural fluid was presumed malignant however cytology was negative for malignancy.  The patient had been on gefitinib for four months at the time of sample collection.  90  4.2.2 Sample processing PF was passed through 70 µm nylon cell strainers into 50 mL falcon tubes (BD Biosciences) to remove debris.  Samples were centrifuged at either 1000 rpm, 1500 rpm, or 2000 rpm, for 5 minutes at 4°C, and the supernatant removed.  Red blood cells (RBCs) were depleted by incubation with ammonium chloride (StemCell) for 5 minutes on ice then neutralized and washed with 2 volumes PBS.  The RBC depletion steps were repeated as necessary until the samples were clear.  Centrifugation during washes was performed at the same speeds as the initial centrifugation step.  Cells were counted and aliquoted for flow cytometric analysis.  Cells centrifuged at 1500 rpm were also aliquoted for freezing.  Cells were frozen in 1.0 x 106 cell aliquots in RPMI with 10% FBS and 5 % dimethyl sulfoxide (DMSO) for identical flow cytometric analysis at subsequent time points (2 weeks, 1 month, 2 months, and 3 months).  Figures 4.1 and 4.2 provide flow charts of optimization of sample processing and assessment of effects of long term cryopreservation by flow cytometry, respectively.            91  Figure 4.1  Figure 4.1 Flow chart of PF sample processing optimization  PF was drained from two patients.  Fluid was filtered by passing through 70 µm nylon cell strainers and RBCs lysed by repeated 5 minute incubations with ammonium chloride.  All centrifugation steps were performed at either 1000 rpm, 1500 rpm, or 2000 rpm, and cell populations were compared by flow cytometry with the BD FACSCanto II.     92  4.2.3 Flow cytometry analysis  Cells were stained with a panel of fluorochrome-conjugated monoclonal antibodies directed at cell surface proteins associated with either an M1 or M2 phenotype (Table 4.1) and with eF450 Fixable Viability dye (eBioscience) as described in section 2.2.3.  Following the final wash, cells were resuspended in 100 µL Intracellular (IC) Fixation Buffer (eBioscience) followed by 400 µL FACS buffer and passed through filter-capped flow tubes and flow cytometry was performed using a BD FACSCanto II flow cytometer (BD Biosciences).  Unstained and viability controls were also treated with IC Fixation Buffer before analysis.  Single stain controls were prepared as described in section 2.2.3.  Cell types were determined by differential size and granularity/internal complexity by forward and side scattered light parameters (FSC and SSC), respectively, and cell surface markers.  Macrophage MFIs of fresh and subsequently frozen samples were determined by gating on live myeloid cells based on staining negatively for eF450 viability dye, and their characteristic FSC and SSC values, and then gating on CD45+CD14+ cells.   Table 4.1 Fluorochrome-conjugated cell surface monoclonal antibody panel used to assess macrophage polarity in PF samples Marker Function Conjugated fluorochrome Supplier Clone M1/M2 Volume (µL)/sample  (1 test) CD206 Mannose receptor Alexa Fluor 488 eBioscience 19.2 M2 5 CD200R CD200 receptor PE eBioscience OX108 M2 5 CD45 Leukocyte common antigen PerCP-Cy5.5 eBioscience 2D1 Leukocyte 5 CD14 LPS co-receptor PE-Cy7 eBioscience 61D3 Monocyte/MΦ 5 CD163 Scavenger receptor APC eBioscience GHI/61 M2 5 CD80  Co-stimulatory protein Alexa Fluor 700 BD Pharmingen L307.4 M1 5 HLA-DR MHCII V500 BD Horizon G46-6 Monocyte/MΦ 5 Viability  eFluor 450  eBioscience  Viability 1  93  Figure 4.2  Figure 4.2 Flow chart of assessment of effect of long term cryopreservation of PF samples PF was drained from two patients.  Fluid was filtered by passing through 70 µm nylon cell strainers and RBCs lysed by repeated 5 minute incubations with ammonium chloride.  Specimens were aliquoted and one aliquot was stained with a panel of fluorochrome-conjugated antibodies directed at cell surface markers associated with either the M1 or M2 phenotype and were analyzed by flow cytometry with the BD FACSCanto II.  Additional aliquots were frozen at -80°C for identical flow cytometric analysis upon thawing at the indicated time points.  The MFIs for each marker on live macrophages from thawed aliquots were compared with those assessed from the fresh sample run on the day of specimen collection to identify any effects of freezing on perceived flow cytometric analysis.  94  4.3 Results 4.3.1 Centrifugation speed during sample processing can affect cell types for analysis  As pleural fluid represents a readily available model for studying the immune system involvement in LC, specifically macrophage polarity and phenotypes during the course of treatment, we sought to optimize single cell analysis of these clinical specimens for future projects.  Two PF specimens were collected and processed with centrifugation steps at 1000 rpm, 1500 rpm, or 2000 rpm (Figure 4.3).  The first PF sample contained a population of granulocytes, which were largely lost with centrifugation steps performed at 2000 rpm (Figure 4.3C).  This likely contributed to the decreased number of live cells observed in this sample, with 88.3%, 72.0%, and 60.9% live cells in samples centrifuged at 1000, 1500, and 2000 rpm, respectively (Figure 4.3D).  Figure 4.3E illustrates the effect of the different centrifugation speeds on the ratios of different cell populations present during analysis, with lymphocytes representing a much greater fraction of the populations present when the sample was processed at 2000 rpm.  The results are less striking with the second PF sample, in which granulocytes and monocytes/macrophages make up a small fraction of the cells present across all centrifugation speed samples, and 89.9%, 89.2%, and 86.9% live cells were observed across the three centrifugation speeds (Figure 4.3F-I).  As the second PF sample required one more ammonium chloride incubation than the first PF sample to lyse all the RBCs, it is possible that this additional incubation killed some of the granulocytes and monocytes/macrophages that may have been present in the specimen.  1500 rpm had been the chosen centrifugation speed during preliminary work, and the results of this optimization suggest that this speed is suitable going forward, balancing the desire to include lymphocytes and avoid excessive damage and loss of the larger cell types, however 1000 rpm may be preferable as these results show that lymphocytes were not lost at this speed.  95  Figure 4.3   96  Figure 4.3 Centrifugation speed during sample processing can affect cell types for analysis Flow cytometric analysis of two PF samples processed at three separate centrifugation speeds.  Flow cytometry plots of the first PF sample processed with centrifugation steps at 1000 rpm (A), 1500 rpm (B), and 2000 rpm (C) with a summary of the numbers of different cell types measured from each (D).  Pie charts illustrating the effect of centrifugation on ratios of cell types measured in the first PF (E). (F-I) Flow cytometry plots and a summary of the second PF sample processed in the same manner.  Summaries of population types represent means ± SEM of technical duplicates (D and I).                 97  4.3.2 Freezing disrupts the antibody binding epitope of CD163 In addition to the optimization of processing for fresh pleural effusion specimens, we sought to determine if there are technical limitations in working with frozen samples.  Banking of clinical samples provides a substantial benefit to investigators as it allows interrogation of the most representative samples for a specific question at a later date.  Therefore, we wanted to assess the effect of long term sample cryopreservation on fluorochrome-conjugated antibody binding for flow cytometry analysis.  Aliquots of both PFs processed with all centrifugation steps at 1500 rpm were stained fresh with a panel of fluorochrome-conjugated antibodies for M1/M2 immunophenotyping (Table 4.1) and analyzed by flow cytometry.  Aliquots of each PF were frozen for analysis at a later time point, with MFIs of each marker on live macrophage populations upon thawing compared to those of the macrophage population from the fresh samples (Figures 4.4 and 4.5).  While all antibodies displayed variability across the different timepoints, the majority of markers had comparable MFIs between the fresh and frozen samples.  The exception was CD163 (a marker of M2 macrophages), which showed a substantial reduction in signal at all freezing time points compared to the fresh sample, suggesting that the binding epitope appears to be disrupted by freezing.  In both pleural effusion specimens the MFIs after freezing were significantly lower than those from the fresh samples, suggesting that the antibody was unable to bind its epitope (Figures 4.4 and 4.5).  Interestingly, in the first PF specimen, but not the second, a similar result was seen with CD206 (another marker of M2 macrophages), suggesting that this epitope too may be adversely affected by freezing and therefore unreliable for flow cytometry analysis after cryopreservation (Figure 4.4).    98  Figure 4.4  Figure 4.4 CD163 and CD206 binding epitopes may be adversely affected by freezing MFIs of each marker in the M1/M2 macrophage immunophenotyping flow cytometry panel for the first pleural effusion sample.  MFIs from the sample stained fresh on the day of specimen collection, and after cryopreservation for the indicated periods.  MFIs for each marker were measured for live macrophages and represent means ± SEMs of technical duplicates.  After cryopreservation, the MFIs of CD206 and CD163 were decreased compared to those from staining the fresh sample.  Although the MFIs of other markers varied over the time points, the levels remained similar to those from staining fresh.      PF1 99  Figure 4.5  Figure 4.5 CD163 binding epitope is disrupted by freezing MFIs of each marker in the M1/M2 macrophage immunophenotyping flow cytometry panel for the second pleural effusion sample.  MFIs are from the sample stained fresh on the day of specimen collection, and after cryopreservation for the indicated periods.  MFIs for each marker were measured for live macrophages and represent means ± SEMs of technical duplicates.  Again after cryopreservation, the MFIs of CD163 were greatly decreased compared to that from the freshly stained sample.     PF2 100  4.4 Discussion  Pleural effusion specimens represent a readily available model for studying the immune system involvement in LC, specifically for our interest in macrophage polarity and phenotypes during the course of treatment.  As such we sought to optimize single cell analysis of these clinical specimens so that we could begin banking patient samples for future projects.  PFs were processed with centrifugation steps at 1000 rpm, 1500 rpm, or 2000 rpm.  In the first PF, at 2000 rpm, the majority of granulocytes were no longer present.  Granulocytes are considered to be fragile cells that are easily damaged by improper handling162, explaining their loss at 2000 rpm.  At 1500 rpm, all cell types were present at similar proportions to 1000 rpm, with the exception of granulocytes, which made up more of the sample than at 1000 rpm.  This is interesting as based on FSC values of the granulocyte and lymphocyte population, the two cell types appear very similar in size, but with granulocytes of course being more internally complex based on their SSC values.  Therefore, at 1000 rpm we would have expected to see potentially less lymphocytes and more granulocytes as at lower rpm, smaller less granular cell types may be lost as not enough force was applied to pellet the cells.  While monocytes/macrophages made up less of the 10,000 events at 1500 rpm than at 1000 rpm, we suspect that this is a result of the number of granulocytes as opposed to the monocytes/macrophages being damaged and lost.  In the second PF in which a minimal granulocyte population was observed at all three centrifugation speeds, the numbers of monocytes/macrophages in the three speeds were nearly identical.  1500 rpm had been chosen during preliminary work and the results of this optimization confirm that it is a suitable centrifugation speed for pleural fluid processing, balancing the desire to include lymphocytes while avoiding excessive damage and loss of larger cell types, however 1000 rpm may be preferable as these results show that lymphocytes were not lost at this speed.  101  Clinical specimens are often frozen for long term storage, as this makes analysis more feasible, cost effective, and accurate, minimizing the inherent variability in assaying on different days.  Therefore, prior to generating a large bank of clinical specimens it was essential to assess the effect of long term cryopreservation on cell viability and antibody binding epitopes involved in our flow cytometry M1/M2 immunophenotyping panel.   In the first PF, but not the second, CD206 MFI levels were reduced compared to those measured on fresh macrophages.  It is likely that the epitope that the antibody is specific for was disrupted by freezing, either destroyed or changed in a way that doesn’t allow for recognition, but it is interesting that this wasn’t seen in both PF samples.  Assessment of more samples is necessary to determine how frequently CD206 is affected by freezing and whether this should be taken into account when planning to assess CD206 expression on frozen macrophages.  In both PF specimens, CD163 levels were significantly deteriorated following freezing, suggesting that the antibody epitope is adversely affected by freezing.  Considering that CD206 and CD163 are very commonly used markers to identify M2 macrophage populations, this has profound implications for macrophage phenotyping by flow cytometry.  The antibody clone used for CD163 was eBIOGHI/61.  No other clones are available from the major bioscience companies, namely eBioscience and BD Biosciences, making it impossible to determine wither targeting a different epitope on the CD163 protein would have improved expression following freezing or whether CD163 is for some reason sensitive to freezing.  It is unknown whether any freezing results in this loss of CD163 detection (as it appears it might), or if it is merely a consequence of freezing for a longer period of time (2 weeks or more) as studied here.  Short term freezing, for as long as one or two days, may have the same effect on CD163 detection and thus warrants investigation. 102  Loss of CD163 detection by flow, though troublesome, likely does not concern detection of CD163 by IHC of formalin fixed paraffin embedded (FFPE) tissue samples, a very commonly used technique in macrophage phenotyping and prognosis studies, as tissue samples are not frozen, but fixed with formalin when fresh.  However, samples are sometimes flash frozen before being embedded, and this may adversely affect CD163 detection as the antibody binding epitope may be disrupted.  The results of a similar comparison of fresh FFPE and flash frozen embedded tissue samples could have profound implications on these types of studies.   Concurrently, flash freezing our samples in liquid nitrogen warrants investigation.  Cells in this study were frozen in RPMI-1640 supplemented with 10% FBS and 5% DMSO and stored at -80°C, as this had been the protocol for other human fluid specimens processed in our lab.  Tumor tissue specimens are often flash frozen, suggesting that this method may be more effective in allowing for accurate detection of all cell surface markers in our flow panels.   For the purposes of our studies, CD163 is an important marker and as such, we are exploring more alternative methods of sample storage.  Another method involves fixing the cells with IC Fixation buffer and storing the cells unstained at 4°C until ready to stain many samples in one batch with analysis on the same day.  This is more of a potential short term storage solution, as analysis would take place within weeks as opposed to months.  Anecdotal evidence of increased macrophage autofluorescence exists when describing cells fixed and stored at 4°C.  Assessment of marker loss and autofluorescence will thus need to be carried out, much in the same vein as our assessment of the effect of freezing.   Importantly, the information gleaned from this work can also be applied to the collection of other human fluid specimens such as bronchoalveolar lavage, samples in which macrophages are particularly prevalent, and therefore becoming increasingly valuable in determining the involvement of the immune system in lung cancer.  103  Chapter 5: Conclusion 5.1 Summary and future directions Lung cancer is the leading cause of cancer mortality and at 18%, has one of the lowest five-year survival rates of all malignancies.  This is largely attributed to a late stage of diagnosis and ineffective treatment strategies, which result in the recurrence of more aggressive and rapidly growing tumors.  The majority of patients are diagnosed with locally advanced or metastatic disease for which the standard of care is platinum based doublet chemotherapy.  However, chemotherapy has modest effects on overall survival, highlighting the need for novel and more effective treatments.  Macrophages are a prominent immune cell type in the lungs and lung tumors, and their presence in tumors has been associated with clinical phenotypes including progression and aggressiveness.  It is widely accepted that a spectrum of macrophage activation states exists, with M1 and M2 macrophages exhibiting opposing functions and representing the extremes of this continuum.  The goal of this work was to determine how co-culture of monocytes and NSCLC cell lines alters macrophage differentiation and the effects of cisplatin treatment on this process.  Our analysis revealed that co-cultured macrophages were skewed to an M2 polarity by certain NSCLC cell lines, and that cisplatin treatment impeded differentiation.  The findings from this work, as well as the optimization of single cell analysis of clinical specimens, provide insights into the roles of macrophages following chemotherapeutic treatment and have prepared for the study of the effect of chemotherapy on macrophage polarity over the course of treatment using more physiologically representative specimens.     104  5.1.1 Determine how co-culture of monocytes and NSCLC cell lines affects macrophage differentiation and polarity While significant work has been carried out characterizing in vitro skewed macrophages, and the associations of M1 and M2 phenotypes with prognosis, the precise role macrophages play in lung cancer and how they influence tumor progression, response to treatment, and subsequently patient survival remains unclear.  A basic understanding of the effects of LC tumor cells and their secreted factors have on macrophage differentiation and polarity is thus required.  Therefore, in Chapter 2, we cultured human peripheral blood monocytes alone and in co-culture with human lung AC cell lines in an attempt to decipher how factors secreted from tumor cells influence macrophage polarity.   Our analysis revealed that macrophages co-cultured with LCCLs were bigger than macrophages cultured alone and that they were skewed to an M2 polarity, with macrophages co-cultured with H2228 and H2291 displaying significantly increased expression of two M2 associated markers, CD206 and CD274.  Interestingly, a stepwise increase in IL-6 secretion was observed over the course of co-cultures with H2228 and H2291, suggesting that IL-6 secretion may be important in M2 skewing.  We also found that co-culture with H2228 and H2291 induced IL-6 expression in macrophages compared to when cultured alone.    While the correlation between M2 skewing and IL-6 secretion is an intriguing one, further studies are required to determine whether IL-6 is responsible for this effect.  Repeated co-culture experiments could be carried out, with the addition of tocilizumab, a humanized monoclonal antibody directed against the IL-6 receptor, to the cultures.  Additionally, as our study was limited to skewing by secreted soluble factors due to the use of cell culture inserts, co-culture experiments in which macrophages and LCCLs are in direct contact could provide further 105  insights into macrophage polarity in the context of lung cancer.  While a BD FACSCanto II flow cytometer with eight detection channels was used to analyze macrophage polarity following co-culture in this study, I have since designed a 14 colour flow cytometry panel for use on the more advanced BD FACSAria Fusion.  This panel includes more M1 and M2 associated markers that cover more subtypes and activation states of macrophages to more accurately assess phenotypes, as well as CD45, a pan-leukocyte marker, which would facilitate the identification of macrophages from the tumor cells, and would provide a more in depth assessment of the effects of co-culture on macrophage polarity.   5.1.2 Determine if and how treatment alters macrophage phenotypes and polarity While significant advances have been made in the treatment of NSCLC, with the development of targeted therapies and more recently of immunotherapeutic monoclonal antibodies, chemotherapy remains the most common treatment modality for advanced stage NSCLC, as only a fraction of tumors harbour the activating mutations towards which erlotinib/gefitinib, and crizotinib are directed, and monoclonal antibodies against checkpoint inhibitors have thus far been FDA approved for use only once chemotherapy has failed148,155.  Cisplatin is one of the most commonly prescribed first line chemotherapeutic drugs for treatment of NSCLC.  As macrophages are a prominent cell type in lung tumors, determining how their polarity is affected in response to cisplatin will further our understanding of how these cells contribute to lung cancer progression and response to treatment.  As such, in Chapter 3 we cultured human peripheral blood monocytes alone and in co-culture with human lung AC cell lines with clinically relevant doses of cisplatin to determine the effects of treatment on macrophage polarity.  We found that treatment with cisplatin impeded macrophage differentiation, with treated co-cultured macrophages displaying decreased size, granularity, and 106  surface marker expression, including CD274 (PD-L1), an M2 associated marker of particular clinical interest.  However, CD206 expression, another marker of M2 macrophages, remained elevated, suggesting a role for CD206 in apoptotic tumor cell clearance in response to treatment.  As outlined above, repeated co-cultures with macrophages and LCCLs in direct contact with physiologically relevant doses of cisplatin could provide interesting insights into macrophage functions in response to treatment.  Additionally, co-cultures with combined treatments of cisplatin and tocilizumab or immune activating compounds could provide the first steps towards targeting macrophages in lung cancer.  5.1.3 Optimize single cell analysis of surrogate lung cancer specimens Pleural effusion specimens represent a useful model for studying the immune system involvement in LC. For that reason we sought to optimize single cell analysis of these clinical specimens in preparation for future projects, such as monitoring macrophage polarity over the course of treatment.  Therefore, in Chapter 4, we assessed the effects of sample processing (centrifugation speed) and long term-cryopreservation on cell populations and antibody binding for flow cytometric analysis, as spin speed can reduce cell viability in patient samples and it is unknown whether cryopreservation affects detection of cell surface markers, respectively.  Optimization of these conditions enables banking of surrogate patient samples such as BAL and pleural fluid, and robust comparison of patient samples collected at different time points.   Our analysis revealed that centrifugation speed during sample processing can affect cell types for analysis, with striking differences in the ratios of cell types present during analysis in certain cases.  We also found that cryopreservation of macrophages at -80°C disrupts the antibody binding epitope for CD163, which has profound implications for immunophenotyping of macrophages in banked biofluids.  Accrual of more PF specimens will be critical to assess 107  whether short term freezing has the same effect on CD163 detection, and to examine the effects of alternative preservation methods on antibody detection for immunophenotyping.   The work carried out in Chapter 4 has been in preparation for future projects investigating the changes in immune populations over the course of chemotherapy in pleural effusion specimens.  Pleural effusion specimens represent a readily available surrogate for the study of the immune system involvement in lung cancer and more importantly allow for analysis of a single patient at multiple time points over the course of treatment.  Monitoring a patient’s immune populations over the course of chemotherapy may provide insight into immune profiles that correlate with sensitivity or resistance to chemotherapy.  The information from this project may be applied to select patients based on their immune profiles for whether chemotherapy may better or worsen their prognosis, and whether immunotherapies targeting a certain immune cell type that is correlated with resistance may be combined with chemotherapy to improve lung cancer prognosis which remains one of the lowest of all malignancies. 5.2 Conclusions and significance We hypothesized that 1) monocytes are skewed by lung adenocarcinoma tumor cells to an M2 phenotype and 2) that standard first line chemotherapy affects macrophage polarity toward M2.  Our co-culture experiments revealed that macrophages displayed increased differentiation and an M2 polarity in the presence of lung AC cell line secreted factors, in particular potentially IL-6.  Treatment with cisplatin impeded macrophage differentiation; however, CD206 expression, a marker of M2 macrophages, remained elevated, suggesting a role in response to treatment.  These findings support our hypotheses, with the particularly interesting exception of downregulated macrophage expression of CD274, another M2 marker, upon 108  cisplatin treatment, suggesting that the macrophages are rendered less immunosuppressive by treatment, with the potential to become immune-activating if subsequently stimulated.  Collectively, the findings from this thesis have demonstrated that macrophage polarity is affected by lung adenocarcinoma cells, and provided insight into the mechanisms through which this occurs.  109  References 1 Society, A. C. Cancer Facts and Figures. (2015). 2 Giaccone, G. Epidermal growth factor receptor inhibitors in the treatment of non-small-cell lung cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23, 3235-3242, doi:10.1200/JCO.2005.08.409 (2005). 3 Kwak, E. L. et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. 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