Medicine, Faculty of
DSpace
UBCV
Martin, Lauren Adria
Master of Science - MSc
University of British Columbia
Synovial sarcoma is an aggressive soft-tissue sarcoma that most frequently affects adolescents and young adults and can arise in virtually any area of the body. The biologic behaviour of synovial sarcoma can vary depending on several factors and the 10-year survival rate has been estimated at only 50%. Synovial sarcoma is characterized by a pathognomonic t(X;18)(p11.2;q11.2) translocation which produces a fusion oncogene named SS18-SSX. The cell of origin of synovial sarcoma is currently unknown, although recent research indicates that it may arise from a mesenchymal stem/progenitor cell population. We have generated a unique mouse model of synovial sarcoma where the human SS18-SSX fusion oncogene is conditionally expressed in a population of mesenchymal progenitor cells using a Hic1Cʳᵉᴱᴿᵀ² driver developed by our lab. This model shows robust formation of synovial sarcoma with 100% penetrance and a mean latency period of 16 weeks. The mouse tumours were characterized histologically, using immunofluorescence and hematoxylin and eosin staining, and at the transcriptomic level using single-cell RNA-sequencing. Histologic analysis reveals characteristic cellular morphology and marker expression, while scRNA-seq analyses show that gene expression in these tumours parallels transcript profiles observed in human synovial sarcoma samples. Using this model, we can identify and isolate early neoplastic lesions prior to the development of grossly visible tumours; scRNA-seq of early lesions shows a partially-transformed phenotype and highlights cellular programs involved early in sarcomagenesis. We then generated two modified versions of this model through the addition of stabilized β-catenin (Ctnnb1ᴱˣ³Fᴵ) and knockout of SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (Smarcb1Fᴵ) to assess the effects of activated WNT/ β-catenin signaling and loss of Smarcb1, respectively. Both models show a decreased latency period and more aggressive phenotype and provide additional support for the roles of the WNT/ β-catenin pathway and chromatin remodeling complexes in synovial sarcomagenesis. Overall, these models identify Hic1+ mesenchymal progenitors as a putative cell of origin for synovial sarcoma and provide us with a unique opportunity to address the molecular and cellular mechanisms underlying synovial sarcomagenesis.
GENERATION AND CHARACTERIZATION OF A NOVEL MOUSE MODEL OF SYNOVIAL SARCOMA by Lauren Adria Martin DVM, University of Saskatchewan, 2013 BSc, University of Calgary, conferred 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Cell and Developmental Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2019 © Lauren Adria Martin, 2019 ii Committee Page The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Generation and characterization of a novel mouse model of synovial sarcoma submitted by Lauren Adria Martin in partial fulfillment of the requirements for the degree of Master of Science in Cell and Developmental Biology Examining Committee: Dr. T. Michael Underhill Supervisor Dr. Martin Hirst Supervisory Committee Member Dr. Torsten Nielsen Supervisory Committee Member Dr. Kelly McNagny Additional Examiner Additional Supervisory Committee Members: Dr. Stephen Yip Supervisory Committee Member iii Abstract Synovial sarcoma is an aggressive soft-tissue sarcoma that most frequently affects adolescents and young adults and can arise in virtually any area of the body. The biologic behaviour of synovial sarcoma can vary depending on several factors and the 10-year survival rate has been estimated at only 50%. Synovial sarcoma is characterized by a pathognomonic t(X;18)(p11.2;q11.2) translocation which produces a fusion oncogene named SS18-SSX. The cell of origin of synovial sarcoma is currently unknown, although recent research indicates that it may arise from a mesenchymal stem/progenitor cell population. We have generated a unique mouse model of synovial sarcoma where the human SS18-SSX fusion oncogene is conditionally expressed in a population of mesenchymal progenitor cells using a Hic1CreERT2 driver developed by our lab. This model shows robust formation of synovial sarcoma with 100% penetrance and a mean latency period of 16 weeks. The mouse tumours were characterized histologically, using immunofluorescence and hematoxylin and eosin staining, and at the transcriptomic level using single-cell RNA-sequencing. Histologic analysis reveals characteristic cellular morphology and marker expression, while scRNA-seq analyses show that gene expression in these tumours parallels transcript profiles observed in human synovial sarcoma samples. Using this model, we can identify and isolate early neoplastic lesions prior to the development of grossly visible tumours; scRNA-seq of early lesions shows a partially-transformed phenotype and highlights cellular programs involved early in sarcomagenesis. iv We then generated two modified versions of this model through the addition of stabilized b-catenin (Ctnnb1Ex3Fl) and knockout of SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (Smarcb1Fl) to assess the effects of activated WNT/b-catenin signaling and loss of Smarcb1, respectively. Both models show a decreased latency period and more aggressive phenotype and provide additional support for the roles of the WNT/b-catenin pathway and chromatin remodeling complexes in synovial sarcomagenesis. Overall, these models identify Hic1+ mesenchymal progenitors as a putative cell of origin for synovial sarcoma and provide us with a unique opportunity to address the molecular and cellular mechanisms underlying synovial sarcomagenesis. v Lay Summary Synovial sarcoma (SS) is a type of cancer that primarily affects adolescents and young adults and has an estimated 10-year survival rate of 50%. SS is caused by a unique mutation and we can study SS by creating genetically engineered mouse models that contain this mutation. Using this approach has led to significant advancements in our understanding of this cancer; however, much remains unknown including the cell type, or cell or origin, that gives rise to SS. The goal of this work was to identify a cell of origin for SS and to elucidate the mechanisms underlying the development of SS. We generated a unique mouse model of SS using mesenchymal progenitor cells, a cell type that is found throughout the body and plays a variety of critical roles. This work successfully identified a cell of origin for SS and provides an ideal and powerful model for further study of this cancer. vi Preface The discovery and characterization of Hic1+ MPs was performed in Dr. Underhill’s laboratory. Work on synovial sarcoma included in this thesis, as well as characterization of Hic1+ MPs in the tongue, including literature review, experimental design, data collection, data analysis, and writing was performed by Dr. Lauren Martin under the supervision of Dr. Michael Underhill. Technical support including input on assay design and experimental troubleshooting was received from Dr. Wilder Scott and Martin Arostegui. Protocols including tumour cell isolation and sorting strategies were adapted from established protocols in the Underhill laboratory. Dr. Wilder Scott, Martin Arostegui, Amanda Yang, and Cindy Crichlow all assisted with experiments in various capacities including mouse collection, tissue processing, and image collection. The bone MP scRNA-Seq dataset included in Figure 3.25 was generated by Dr. Wilder Scott. Hematoxylin and Eosin tumour sections were evaluated by Dr. Torsten Nielsen. All animal work was approved by the UBC Animal Care Committee, certificate # A17-0273, A15-0107, and A15-0191. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................. v Preface ........................................................................................................................................... vi Table of Contents ......................................................................................................................... vii List of Tables .................................................................................................................................. x List of Figures ............................................................................................................................... xi List of Symbols ............................................................................................................................ xiii List of Abbreviations .................................................................................................................. xiv Acknowledgements ................................................................................................................... xviii Dedication .................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................ 1 1.1 Overview of synovial sarcoma ........................................................................................ 1 1.1.1 Histologic features and markers of synovial sarcoma ............................................. 2 1.1.2 Molecular mechanisms underlying synovial sarcoma ............................................. 2 1.1.3 WNT/b-catenin signaling in synovial sarcoma ....................................................... 7 1.2 Mouse models of synovial sarcoma ................................................................................ 9 1.3 Cell of origin of synovial sarcoma ................................................................................ 12 1.4 Hic1+ Mesenchymal Progenitors ................................................................................... 14 1.4.1 HIC1 Background .................................................................................................. 14 1.4.2 Form and function of Hic1+ MPs .......................................................................... 15 1.5 Hic1+ MPs as cell of origin for synovial sarcoma ......................................................... 16 viii Chapter 2: Methods ..................................................................................................................... 18 2.1 Mice ............................................................................................................................... 18 2.1.1 Genetics and Breeding ........................................................................................... 18 2.1.2 Husbandry and Genotyping ................................................................................... 22 2.1.3 Tamoxifen Treatment ............................................................................................ 22 2.1.4 EdU Treatment ...................................................................................................... 23 2.1.5 Tissue Collection ................................................................................................... 23 2.2 Immunofluorescent Staining ......................................................................................... 24 2.2.1 Cryoembedding ..................................................................................................... 24 2.2.2 Antibody Staining .................................................................................................. 24 2.2.3 EdU Staining ......................................................................................................... 25 2.2.4 Fluorescent Microscopy ........................................................................................ 25 2.3 Haematoxylin & Eosin Staining .................................................................................... 26 2.3.1 Fixation, Paraffin Embedding, H&E Staining ....................................................... 26 2.3.2 Bright Field Imaging ............................................................................................. 26 2.4 Quantification ................................................................................................................ 26 2.4.1 Vessel Association ................................................................................................. 26 2.4.2 Proliferation ........................................................................................................... 27 2.5 Single-cell RNA-Seq ..................................................................................................... 27 2.5.1 Cell Isolation ......................................................................................................... 27 2.5.2 Fluorescence-Activated Cell Sorting ..................................................................... 29 2.5.3 Sequencing ............................................................................................................ 29 2.6 Statistical Analysis ........................................................................................................ 30 ix Chapter 3: Results ....................................................................................................................... 31 3.1 Effects of conditional expression of SS18-SSX in Hic1+ cells ....................................... 31 3.2 Identification of early and intermediate tumours .......................................................... 41 3.3 Single-cell RNA-Sequencing analysis of mouse tumours ............................................. 44 3.4 Characterization of Hic1+ cells in the tongue ................................................................ 58 3.5 Effects of addition of stabilized b-catenin to Hic1CreERT2; Rosa26hSS2 mouse model .... 64 3.6 Effects of loss of Smarcb1 in Hic1CreERT2; Rosa26hSS2 mouse model ............................ 74 Chapter 4: Discussion .................................................................................................................. 82 4.1 Hic1+ MPs are a cell of origin for synovial sarcoma ..................................................... 82 4.2 Activated WNT/b-catenin signaling potentiates synovial sarcomagenesis ................... 86 4.3 Biallelic loss of Smarcb1 potentiates synovial sarcomagenesis .................................... 87 4.4 Preclinical applications of Hic1CreERT2; Rosa26hSS2 mouse model ................................. 90 4.5 Conclusion ..................................................................................................................... 91 4.5.1 Strengths and limitations ....................................................................................... 91 4.5.2 Future directions .................................................................................................... 92 References .................................................................................................................................... 95 x List of Tables Table 2.1 Genotyping Primers ....................................................................................................... 22 Table 2.2 Primary Antibodies ........................................................................................................ 25 Table 2.3 Secondary Antibodies .................................................................................................... 25 Table 2.4 Antibodies for FACS ..................................................................................................... 28 xi List of Figures Figure 1.1 Schematic of SS18-SSX fusion oncoprotein ................................................................. 3 Figure 1.2 Mouse models of synovial sarcoma ............................................................................. 10 Figure 1.3 HIC1 gene structure ..................................................................................................... 15 Figure 2.1 Overview of Hic1CreERT2; Rosa26hSS2 synovial sarcoma model ................................... 19 Figure 2.2 Overview of synovial sarcoma model with stabilized b-catenin ................................. 20 Figure 2.3 Overview of synovial sarcoma model with loss of Smarcb1 ....................................... 21 Figure 3.1 Kaplan-Meier survival curve of Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 and Hic1CreERT2/CreERT2;Rosa26hSS2/tdTomato animals .................................................................... 31 Figure 3.2 Tumour induction in Hic1+ MPs .................................................................................. 33 Figure 3.3 Histologic analysis of synovial sarcoma tumours from the Hic1CreERT2; Rosa26hSS2 mouse model ...................................................................................................................... 35 Figure 3.4 Immunofluorescent staining of synovial sarcoma tumours from the Hic1CreERT2; Rosa26hSS2 mouse model ................................................................................................... 36 Figure 3.5 Cellular proliferation in mouse tumours ...................................................................... 38 Figure 3.6 Pulmonary lesions in Hic1CreERT2; Rosa26hSS2 mice ..................................................... 40 Figure 3.7 Histologic analysis of tongues from 4 wki Hic1CreERT2; Rosa26hSS2 mice ................... 43 Figure 3.8 Immunofluorescent staining of early, intermediate, and endpoint tongue tumours .... 43 Figure 3.9 FACS strategy for Hic1+ MPs from control tongues ................................................... 45 Figure 3.10 FACS strategy for neoplastic cells at 8 wki and 16 wki ............................................ 46 Figure 3.11 Identification of an intermediate cell population using scRNA-Seq ......................... 48 Figure 3.12 FACS strategy for neoplastic cells at 4 wki ............................................................... 50 xii Figure 3.13 scRNA-Seq analysis of Hic1CreERT2; Rosa26hSS2 Tumours ......................................... 52 Figure 3.14 Identification of 4 wki subpopulations ....................................................................... 55 Figure 3.15 Expression profile of early tumour cells .................................................................... 57 Figure 3.16 Normal histology of the mouse tongue ...................................................................... 59 Figure 3.17 Vessel association of Hic1+ MPs in the tongue ......................................................... 60 Figure 3.18 Proliferation of Hic1+ MPs in the tongue .................................................................. 61 Figure 3.19 scRNA-Seq analysis of control tongue Hic1+ MPs ................................................... 63 Figure 3.20 Kaplan-Meier survival curve of Hic1CreERT2/CreERT2; Rosa26tdTomato/hSS2; Ctnnb1+/Ex3Fl animals ............................................................................................................................... 65 Figure 3.21 Tumour induction in Hic1+ MPs with stabilized b-catenin ....................................... 66 Figure 3.22 Histologic analysis of synovial sarcoma tumours with stabilized b-catenin ............. 67 Figure 3.23 Immunofluorescent analysis of synovial sarcoma tumours from the Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1+/Ex3Fl mouse model ................................................................. 69 Figure 3.24 FACS strategy for neoplastic cells from b-catenin-stabilized tumours ..................... 71 Figure 3.25 scRNA-Seq analysis of Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ Tumours ....... 73 Figure 3.26 Synovial sarcoma model with loss of Smarcb1 ......................................................... 76 Figure 3.27 Histologic analysis of Smarcb1-deficient synovial sarcoma tumours ....................... 77 Figure 3.28 Immunofluorescent analysis of Smarcb1-deficient synovial sarcoma tumours ......... 78 Figure 3.29 Pulmonary lesions in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl mice ............ 79 Figure 3.30 Histologic analysis of pulmonary lesions in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl mice ............................................................................................................... 80 xiii List of Symbols a - alpha b - beta µm - micrometre xiv List of Abbreviations Alpl: alkaline phosphatase AF700: alexa fluor 700 APC: allophycocyanin ATAC-Seq: assay for transposase-accessible chromatin using sequencing Axin2: axis inhibition protein 2 BAF: Brg/Brm-associated complexes Bmp3: bone morphogenetic protein 3 BSA: bovine serum albumin Cd34: cluster of differentiation 34 cDNA: complementary DNA ChIP: chromatin immunoprecipitation CpG: cytosine – guanine CRISPR/Cas9: clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 Ctnnb1: b-catenin EDTA: ethylenediaminetetraacetic acid EdU: 5-ethynyl-2’-deoxyuridine EGR1: early growth response-1 Epha4: erythropoietin-producing hepatocellular A4 Ex3Fl: floxed exon 3 FACS: fluorescence-activated cell sorting FAP: fibro/adipogenic progenitor xv FFPE: formalin-fixed paraffin-embedded Fl: floxed Fzd1: frizzled 1 Fzd10: frizzled 10 Gbx2: gastrulation brain homeobox 2 GFP, EGFP: green fluorescent protein, enhanced green fluorescent protein H&E: hematoxylin and eosin H3K4me3: histone H3 lysine 4 trimethylation H3K27me3: histone H3 lysine 27 trimethylation HDAC: histone deacetylases Hic1: hypermethylated in cancer 1 hSS2: Rosa26SS18-SSX-EGFP allele IF: immunofluorescent IP: intraperitoneal KDM2B: lysine demethylase 2B MET: mesenchymal-epithelial transition MP: mesenchymal progenitor MPNST: malignant peripheral nerve sheath tumour MSC/hMSC: mesenchymal stem cells, hPSC-derived human mesenchymal stem cells MUC1: mucin 1 Myf5: myogenic factor 5 NCC/hNCC: neural crest cells, hPSC-derived human neural crest cells Ostn: osteocrin xvi PBS: phosphate-buffered saline PCR, RT-PCR: polymerase chain reaction, reverse transcription polymerase chain reaction Pdgfra: platelet-derived growth factor a Pdgfrb: platelet-derived growth factor b PE-Cy7: phycoerythrin-Cy7 PFA: paraformaldehyde PI: propidium iodide PRC1/2: polycomb repressive complex 1/2 PSC/hPSC: pluripotent stem cell, human pluripotent stem cell Rgs5: regulator of G protein signaling 5 Sca1: stem cell antigen 1 scRNA-Seq: single-cell RNA sequencing shRNA: short hairpin RNA Smarcb1: SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily B, member 1 SNH: SYT N-terminal homology domain Sox2: SRY-box 2 SS: synovial sarcoma SS18: synovial sarcoma translocation, chromosome 18 SSX: synovial sarcoma X SSXRD: SSX repression domain TAM: tamoxifen TCF/LEF: T-cell factor/lymphoid-enhancing binding factor xvii tdTomato: tandem dimer Tomato Thy1: thymocyte differentiation antigen 1 TLE1: transducin-like enhancer of split wki: weeks post injection WNT5a: WNT family member 5a Zic5: zinc finger protein of the cerebellum 5 xviii Acknowledgements I would like to thank my supervisor, Dr. Michael Underhill, for welcoming me into the lab and for his guidance and support throughout my time in grad school. I would also like to thank my supervisory committee members, Drs. Martin Hirst, Torsten Nielsen, and Stephen Yip for their valuable input and willingness to help whenever it was needed. Special thanks to Torsten who was always available to review slides, discuss the project, and provide feedback. The head of the CELL program, Dr. Cal Roskelley, was tremendously helpful when I was at a crossroads and I am very appreciative of his help. I am grateful for the members of the Underhill lab, both past and present, for their hard work, help, and friendship. In no particular order I would like to thank: Martin Arostegui, Dr. Wilder Scott, Cindy Crichlow, Dr. Kim Wiegand, Dr. Kathleen Martin, Amanda Yang, Will Bastedo, Atefeh Samani, Dr. Lesley Hill, Tara Stach, Ryan Vander Werff, Bernie Zhao, Germain Ho, and Dan Tausan. As always, I am especially grateful for my partner, Conor, my family, and my wonderful friends. Thank you for your unwavering support. Lastly, I appreciate the financial support from the Canadian Institutes of Health Research, the Terry Fox Research Foundation, and the UBC Faculty of Medicine. xix Dedication Dedicated to the people affected by synovial sarcoma and to the animals who were sacrificed for this work. 1 Chapter 1: Introduction 1.1 Overview of synovial sarcoma Synovial sarcoma (SS) is a malignant soft-tissue sarcoma that primarily affects adolescents and young adults and can arise in virtually any area of the body. The biologic behaviour of SS can vary depending on a variety of factors including histologic grade, tumour size, and age at diagnosis, and the 5- and 10-year survival rates have been estimated at approximately 60% and 50%, respectively1,2. SS typically arise in the deep soft tissues of the extremities, often near large joints3, and the second most common locations have been variably reported as the head and neck regions3 and the trunk2. Treatment for synovial sarcoma includes wide surgical resection, occasionally resulting in limb amputation, combined with radiotherapy and chemotherapy. Despite aggressive treatment, local recurrence and metastasis occur in approximately 50% of patients, with metastatic disease often occurring after 5 years4. Synovial sarcoma is a translocation-associated sarcoma characterized by a t(X;18)(p11.2;q11.2) translocation which produces one of three fusion oncogenes: SS18-SSX1, SS18-SSX2, or less commonly SS18-SSX4 5-9. The eight C-terminal amino acids (aa) of SS18 are replaced by 78 C-terminal amino acids of SSX1, 2, or 4, resulting in a fusion protein containing the majority of the SS18 protein (379 aa) and 78 aa from SSX6,7. The SS18-SSX fusion oncoprotein is sufficient to drive the development of SS, although the exact mechanisms underlying SS18-SSX-mediated sarcomagenesis are incompletely understood. In addition, the cell of origin of SS is unknown – it does not arise from synovium as the name suggests – although recent research suggests that it may arise from a mesenchymal stem/progenitor cell population10,11. 2 1.1.1 Histologic features and markers of synovial sarcoma There are two main histologic subtypes of SS: monophasic and biphasic. The monophasic subtype is characterized by tightly-packed spindle cells with uniform nuclei and often contains a hemangiopericytic vascular pattern12. The biphasic subtype consists of both a spindle cell component, as in the monophasic subtype, and an epithelial component containing epithelial cells forming gland-like structures. In addition to the two main subtypes, there are several other less common subtypes including monophasic epithelial (made up entirely of epithelial cells), poorly differentiated, myxoid, and ossifying3. The variable histologic appearance of SS can make it difficult to distinguish from other tumour types, both mesenchymal and epithelial in origin, and antibody staining for a range of markers is commonly used to aid in the diagnosis of SS. Epithelial markers including cytokeratins and epithelial membrane antigen (EMA1 or MUC1 in the mouse) are expressed in over 80% of SS tumours, even in tumours of the monophasic spindle cell subtype13. Bcl-2, CD99, and S-100 are also commonly expressed in SS, while CD34 is typically negative3. Transducin-like enhancer of split (TLE1) has more recently been recognized as a marker of SS, with nuclear expression in over 90% of cases14,15. Along with immunohistochemical staining, detection of the SS18-SSX fusion oncogene using reverse-transcription PCR, cytogenetics, or fluorescence in situ hybridization is used for the definitive diagnosis of SS3. 1.1.2 Molecular mechanisms underlying synovial sarcoma While the SS18-SSX fusion oncoprotein is the known driver of synovial sarcomagenesis, it is unclear exactly how this protein causes neoplastic transformation in cells carrying the oncogene. 3 Early studies of SS18-SSX demonstrated that the fusion oncoprotein is localized to the nucleus and is involved in transcriptional regulation, yet neither the SS18 nor the SSX component of the fusion contain a DNA-binding domain16-18. The SS18 protein contains two domains: an N-terminal domain termed SNH, involved in protein-protein interaction, and a C-terminal domain termed QPGY (glutamine, proline, glycine, and tyrosine rich) that acts as a transcriptional activator, both of which are present in the fusion (Figure 1.1)16,19. The SSX portion of the fusion contains a C-terminal repressive domain (SSXRD) which functions as a transcriptional repressor17. The SS18-SSX fusion oncoprotein therefore has both activating and repressive capabilities, leading to overall aberrant gene expression. Figure 1.1 Schematic of SS18-SSX fusion oncoprotein. The SS18-SSX fusion oncoprotein contains the majority of the SS18 protein including the SNH domain, responsible for interactions with BAF complexes, and the QPGY domain, both considered activating domains, as well as the 78 C-terminal amino acids of the SSX protein containing the C-terminal repressive domain (SSXRD) responsible for interactions with PRC complexes. 4 Early studies also demonstrated that SS18 and SS18-SSX interact with BRM, a component of the mammalian SWI/SNF (mSWI/SNF) or BAF (Brg/Brm-associated complexes) complexes19-21, and that the presence of SS18-SSX induces changes in histone modifications and DNA methylation patterns, suggesting an epigenetic mechanism underlying synovial sarcomagenesis22. The native SS18 protein was later found to be a core subunit of the BAF complexes, and the presence of the SS18-SSX fusion was shown to disrupt BAF complexes leading to altered transcription genome-wide, further supporting an epigenetic basis for the development of synovial sarcoma23,24. BAF complexes are ATP-dependent chromatin remodelers which enable transcription factors to access their target genes through nucleosome remodeling25. BAF complexes have been identified as important tumour suppressors, and mutations in BAF complex subunits occur in approximately 20% of human cancers26. One of the main roles of BAF complexes is to oppose Polycomb Repressive Complexes (PRC1/2). PRC complexes are histone modifiers that facilitate transcriptional repression through the placement of specific histone marks such as H3K27 trimethylation (H3K27me3), and they are also responsible for the maintenance of repression of “bivalently marked” promoters – promoters which have both H3K27me3 and H3K4me3 marks and are poised for expression upon the loss of H3K27me327-29. The H3K27me3 mark is placed by enhancer of zeste homolog 2 (EZH2), a SET-domain methyltransferase and the catalytic subunit of PRC227, which is closely associated with histone deacetylases (HDAC), another group of epigenetic modifiers30. As with BAF complexes, mutations in PRC complex subunits have been implicated in a wide variety of human cancers27, thus providing a rationale for targeted treatment of certain malignancies with EZH2 inhibitors and HDAC inhibitors. In addition to its 5 interaction with BAF complexes, SS18-SSX has been shown to associate with PRC complexes, suggesting an additional and opposing mechanism through which the fusion protein can cause epigenetic dysregulation31,32. One proposed mechanism underlying the epigenetic dysregulation in SS is that SS18-SSX acts as a bridge between activating transcription factor 2 (ATF2) and TLE1, two proteins which do not normally interact32. Su et al. (2012) found that by interacting with ATF2, SS18-SSX is targeted to promoters of ATF2 target genes, which leads to recruitment of PRC2 complexes, placement of the H3K27me3 repressive histone mark, and repression of target genes. These findings support previous work which showed that the transcription factor early growth response-1 (EGR1), an ATF2 target gene, is downregulated in synovial sarcoma through recruitment of SS18-SSX to the EGR1 promoter33. In addition, treatment with an HDAC inhibitor was found to cause derepression of ATF2 target genes, including EGR1, and apoptosis of SS cells, further supporting a role for PRC2 in the pathogenesis of SS32-34. Altogether, these findings suggest a mechanism whereby SS18-SSX drives synovial sarcomagenesis through epigenetic silencing of tumour suppressor genes, mediated at least in part by PRC complexes. Another proposed mechanism underlying the epigenetic dysregulation in SS focuses on the role of SS18-SSX in the BAF chromatin remodeling complex. Kadoch and Crabtree (2013) showed that the SS18-SSX fusion oncoprotein integrates into BAF complexes in the place of native SS18, leading to altered composition and targeting of the complex. They found that the integration of SS18-SSX into the BAF complex results in the ejection of BAF complex subunit SMARCB1 (BAF47) and targeting of the BAF complex to the Sox2 locus leading to loss of 6 H3K27me3 and subsequent Sox2 expression, thus proposing a mechanism where the SS18-SSX-mediated disruption of BAF complexes drives synovial sarcomagenesis23. The same group later found that SS18-SSX retargets BAF complexes to broad PRC2-occupied sites throughout the genome, causing loss of H3K27me3 and activation of bivalently marked genes24. They found both gains and losses of BAF complex occupancy sites, suggesting that this retargeting may exert oncogenic effects both through the gain of expression of oncogenes and the loss of expression of tumour suppressors. A third proposed epigenetic mechanism for synovial sarcomagenesis hypothesizes that SS18-SSX drives the development of SS not through PRC2 but through association with a non-canonical PRC complex, PRC1.135. PRC1.1 contains the histone demethylase KDM2B as a core subunit36-38 and represses gene expression in a non-PRC2-dependent manner39. Banito et al. (2018) identified KDM2B as an epigenetic dependency in SS cell lines and found that KDM2B/PRC1.1 interacts with SS18-SSX and recruits SS18-SSX-containing BAF complexes to CpG-rich promoters, leading to increased expression of target genes. These findings propose yet another mechanism through which SS18-SSX may drive the development of SS through interaction with both BAF complexes and PRC complexes leading to aberrant gene activation. Interestingly, both Banito et al. (2018) and McBride et al. (2018) found that the retargeted SS18-SSX-containing BAF complexes activated genes associated with neural development and embryogenesis. These findings support previous gene-expression profiling studies which have shown that SS has a neurodevelopmental signature which is unique among soft-tissue sarcomas40-44, and may reflect the cell of origin of this neoplasm. 7 While the exact molecular mechanisms driving SS remain unknown, there is clear evidence for epigenetic dysregulation through interactions with both activating BAF complexes and repressive PRC complexes. The SS18-SSX fusion oncogene appears to cause widespread transcriptional dysregulation, with both gene activation and repression, leading to a proliferative phenotype and a unique neurodevelopmental expression pattern. 1.1.3 WNT/b-catenin signaling in synovial sarcoma Activation of the WNT/b-catenin signaling pathway is a common feature of SS. In addition to its role in embryogenesis and development, activation and dysregulation of the WNT/b-catenin pathway is frequently implicated in the development, maintenance, and metastasis of various cancers45. Early studies of SS found accumulation of nuclear b-catenin in up to 50% of SS tumour samples and identified nuclear b-catenin staining as a negative prognostic indicator46-49. Several expression profiling studies also confirmed the overexpression of WNT pathway components in SS40,43,50 and SS18-SSX has been shown to recruit b-catenin to the nucleus and activate WNT/b-catenin signaling51,52, suggesting that activation of this pathway likely plays an important role in the pathogenesis of SS and may represent an attractive target for treatment. Indeed, xenograft studies using an antibody against Frizzled 10 (FZD10), a cell-surface component of the WNT signaling pathway, found significantly decreased SS tumour growth in treated animals53, and several small molecule inhibitors of WNT/b-catenin signaling decreased tumour size and caused proliferative arrest in xenograft models and in vitro, respectively52,54. In addition, constitutive WNT/b-catenin signaling was found to be necessary for tumour formation 8 in a conditional mouse model of SS54, and addition of stabilized b-catenin to other mouse models enhanced tumour development and caused highly invasive phenotypes55,56. While WNT/b-catenin pathway activation appears to play an important role in SS, the underlying mechanisms are unclear. SS18-SSX has been shown to activate WNT signaling in a ligand-independent manner, both by forming a transcriptionally active complex with b-catenin51 or by directly binding WNT transcription factors (TCF/LEF transcription factors) without interacting with b-catenin57. SS18-SSX may also function in a ligand-dependent manner, by upregulating expression of WNT pathway target genes and creating an autocrine WNT/b-catenin loop54. Mutations in WNT pathway components including adenomatous polyposis coli (APC) and b-catenin (CTNNB1) are common causes of WNT pathway activation in cancer45, however mutations in these genes are only found in 4-8% of SS tumours47,58-60, and overall SS has a low rate of secondary mutations24,61-63, indicating that there are other mechanisms responsible for WNT/b-catenin activation in this neoplasm. 9 1.2 Mouse models of synovial sarcoma Animal models, particularly mouse models, are a valuable tool in cancer research which allow the in vivo study of cancer initiation, progression, and metastasis as well as pre-clinical evaluation of potential therapeutics. The first mouse model of SS, developed by Mario Cappechi’s group in 2007, utilized an inducible Cre-Lox system to conditionally express SS18-SSX2 in myogenic factor 5-positive (Myf5+) committed myoblasts (Figure 1.2A), based on the hypothesis that SS may arise from myogenic cells64. They generated a conditional allele termed hSS2 wherein a human SS18-SSX2 construct along with a GFP fluorescent marker were knocked in to the Rosa26 locus and then used a Myf5-Cre driver to drive expression of SS18-SSX2. This model produced tumours with 100% penetrance, the majority of which were found in skeletal muscle tissues, and all tumours were histologically, immunohistochemically, and transcriptionally consistent with human SS tumours. Both biphasic and monophasic murine tumours were generated and tumours expressed markers of SS including cytokeratins and Bcl-2. 10 Figure 1.2 Mouse models of synovial sarcoma. A. Expression of hSS1 or hSS2 in Myf5+ cells leads to formation of SS tumours. B. Genetic loss of b-catenin prevents tumour formation in the Myf5-Cre/hSS2 model. C. Conditional expression of hSS2 with concomitant stabilization of b-catenin leads to periosteal SS tumour formation. D. Expression of hSS2 in Prx1+ cells leads to formation of periosteal SS tumours. E. Expression of hSS2 with concomitant stabilization of b-catenin in Prx1+ cells leads to formation of periosteal SS tumours. 11 Interestingly, when SS18-SSX2 was expressed earlier in embryonic development or at different points of differentiation in the myogenic lineage using various Cre drivers, it resulted in either embryonic lethality or myopathy, not the development of tumours64. These results support the hypothesis that SS tumour development requires not only expression of the fusion oncogene but also a permissive cell context, and the development of tumours using the Myf5-Cre driver led the authors to conclude that cells from the skeletal muscle lineage are a cell of origin for SS. While the use of a Myf5-Cre driver clearly led to successful generation of tumours, the conclusion that SS arises from committed myoblasts or satellite cells is not necessarily correct; in addition to its expression in satellite cells and myoblasts65,66, Myf5 is also expressed in somites early in embryonic development, prior to the divisions of dermatome, myotome, and sclerotome67, thus raising the possibility that Myf5-Cre-driven SS18-SSX2 expression causes neoplastic transformation in a population of cells that are not within the myogenic lineage. The same group later developed a similar model using a conditional allele of SS18-SSX1 (termed hSS1) and obtained comparable results in a Myf5-Cre/hSS1 model as with the Myf5-Cre/hSS2 model described above68. Two mouse models were developed with additional genetic alterations to study the role of WNT signaling in SS. One model incorporated a floxed Ctnnb1 allele (Ctnnb1Fl) into the Myf5-Cre/hSS2 model described above, thus generating a b-catenin-null background in the presence of SS18-SSX2 expression in Myf5+ cells54 (Figure 1.2B). Animals with biallelic loss of b-catenin did not develop tumours, indicating that WNT/b-catenin signaling is necessary for the development of SS. The other model assessed the effect of activated WNT signaling in SS development by generating mice carrying a conditional stabilized b-catenin allele (Ctnnb1Ex3Fl) 12 and the hSS2 allele55 (Figure 1.2C). Instead of using a Myf5-Cre driver, the authors used local injections of either an Adenovirus expressing Cre-recombinase (AdCre) or a TATCre protein. They found that compared to b-catenin-wildtype animals, mice with stabilized b-catenin developed tumours with a shorter latency period and a more invasive and aggressive phenotype, often demonstrating invasion into adjacent bone and quickly leading to humane endpoint. They also found that all tumours with stabilized b-catenin had a monophasic histologic appearance, leading the authors to conclude that WNT/b-catenin signaling blocks the mesenchymal-to-epithelial transition (MET) in SS. The most recent mouse model of SS used a tamoxifen-inducible Prx1-CreERT2 to conditionally express the hSS2 allele in early osteochondroprogenitors56 (Figure 1.2D). These mice developed SS tumours on their lower lip, and the addition of stabilized b-catenin in this model similarly enhanced tumour formation as seen in the previous study56 (Figure 1.2E). Taken together, these models highlight the importance of WNT/b-catenin signaling in SS and provide support for a mesenchymal progenitor (MP) cell of origin for SS. 1.3 Cell of origin of synovial sarcoma One significant obstacle to our understanding of SS is that while it is known to be a mesenchymal neoplasm, the exact cell of origin is unknown. Several cellular origins have been proposed including myogenic, neuronal, and MSC/MP origins, and it is possible that several cell types can give rise to SS in the presence of the SS18-SSX translocation. 13 The development of SS tumours in the Prx1-CreERT2/hSS2 model provides strong support for an MP cell of origin, in particular periosteal osteochondroprogenitors56. The Myf5-Cre/hSS1 and Myf5-Cre/hSS2 mouse models provide support for a myogenic origin, although given the expression of Myf5 in other cell types, it is possible that myogenic cells are not giving rise to SS in these models. Furthermore, in several experiments using the hSS1- and hSS2-based mouse models, injections of AdCre and TATCre were used instead of an endogenous Cre driver, thus no conclusions can be drawn regarding a cell of origin based on these studies55,68. The consistent neurodevelopmental expression profile found in SS and similarities with expression profiles of malignant peripheral nerve sheath tumours (MPNST)40 led to the hypothesis that SS, like MPNST, may arise from a neural crest cell of origin. Based on this hypothesis, Ishibe et al. (2008) performed in vitro differentiation assays using several SS cell lines and were able to induce neuronal differentiation in all examined cell lines, leading the authors to conclude that SS likely does arise from a neural crest cell of origin69. A more recent study using human pluripotent stem cell (hPSC)-derived human neural crest cells (hNCCs) and hPSC-derived human mesenchymal stem cells (hMSCs) found that expression of SS18-SSX in hNCCs, but not in hMSCs, resulted in epigenetic and transcriptional profiles similar to what is seen in SS cell lines70, again implicating NCCs as a possible cell of origin for SS. Other work has provided strong evidence for a multipotent MSC origin for SS. Naka et al. (2010) established two novel human SS cell lines and found that the cell lines possessed stem cell properties, including self-renewal and expression of stemness-related genes, and differentiated into osteogenic, chondrogenic, and adipogenic lineages upon silencing of SS18-14 SSX, suggesting that the loss of oncogene expression reverted the cells to a cellular identity consistent with multipotent MSCs10. Garcia et al. (2012) subsequently showed that expression of SS18-SSX in myoblasts and MSCs leads to cellular reprogramming, prevents normal differentiation, and induces a neural phenotype, further highlighting the ability of SS18-SSX to alter the behaviour of MSCs/MPs11. Along with the mouse models described above, these studies support an MSC/MP cell of origin for SS and show that MSCs/MPs are susceptible to neoplastic transformation driven by SS18-SSX. 1.4 Hic1+ Mesenchymal Progenitors 1.4.1 HIC1 Background Hypermethylated in cancer 1 (HIC1) was initially identified as a tumour suppressor which is hypermethylated in several types of cancer including brain, colon, and breast cancer71,72. HIC1 encodes for a Zinc finger transcription factor and is found at chromosome 17p13.3 in humans and chromosome 11 in mice and is ubiquitously expressed in normal tissues71,73 (Figure 1.3). Studies using genetically-engineered mouse models have shown that Hic1 heterozygous mice develop a variety of cancers including both carcinomas and sarcomas74 while Hic1 null animals are embryonic lethal and display an array of developmental defects73. 15 Figure 1.3 HIC1 gene structure The HIC1 gene is composed of three exons and contains a BTB/POZ domain and a central region both involved in transcriptional repression as well as five C2H2Zn fingers with sequence-specific DNA-binding motifs. Research in the Underhill laboratory using murine embryonic limb bud mesenchyme subsequently identified Hic1 as a possible marker for MPs and led to the development of conditional Hic1 knockout mice (Hic1Fl), conventional Hic1 knockout mice (Hic1LacZ), and an inducible Hic1-CreERT2 (Hic1CreERT2) mouse line to enable manipulation and lineage tracing of Hic1+ cells for further study (unpublished data). 1.4.2 Form and function of Hic1+ MPs Hic1+ cells have been characterized extensively in the Underhill laboratory and have been shown to identify mesenchymal progenitors within tissues (unpublished data). Hic1+ cells are primarily perivascular and are found in all tissues throughout the body to a varying degree. In vivo damage models and Hic1 knockout experiments suggest that Hic1 expression maintains quiescence in this population of MPs; after injury, Hic1 expression is downregulated and the MP population transiently expands to contribute to tissue repair, while Hic1 knockout leads to expansion of the MP population in the absence of damage. Lineage tracing experiments performed with the Hic1CreERT2 line and a Cre-dependent fluorescent marker (Hic1CreERT2; Rosa26tdTomato mice) have 16 shown that Hic1+ MPs are involved in regeneration, repair, and fibrosis after injury and can differentiate into osteogenic, adipogenic, chondrogenic, fibrogenic, and tenogenic lineages. Gene expression analysis and immunophenotyping of Hic1+ MPs show that this population can be broadly divided into two sub-populations: a larger population of fibro/adipogenic progenitors (FAPs) and a smaller population of pericytes. FAPs are tissue-resident MPs involved in homeostasis and repair, can contribute to fibrogenic, adipogenic, and osteogenic lineages, and express the cell surface markers platelet-derived growth factor receptor a (PDGFRA), cluster of differentiation 34 (CD34), and stem cell antigen 1 (Sca1)75,76. Pericytes are relatively poorly-understood perivascular cells found within the basement membrane of blood vessels and are believed to play a role in angiogenesis, hemodynamic regulation, and capillary permeability77. While there is a lack of consensus surrounding the exact identity and function of pericytes, platelet-derived growth factor receptor b (PDGFRB), regulator of G protein signaling 5 (RGS5), and melanoma cell adhesion molecule (MCAM) are known markers of pericytes78-80, and all three are consistently expressed in a sub-population of Hic1+ MPs found across various tissues. Hic1+ MPs do not express Cluster of differentiation 31 (CD31) or Cluster of differentiation 45 (CD45) and do not contribute to muscle, thus do not comprise endothelial, hematopoietic, or myogenic lineages. 1.5 Hic1+ MPs as cell of origin for synovial sarcoma Given that SS is a mesenchymal neoplasm and that there is strong evidence for an MSC/MP cell of origin, it is possible that Hic1+ MPs are a cell of origin for SS. The capability of SS cell lines to differentiate into osteogenic, chondrogenic, and adipogenic lineages upon silencing of SS18-17 SSX10 suggests reversion to a cell type with similar properties to Hic1+ MPs, and the ubiquitous distribution of Hic1+ MPs throughout tissues is consistent with the ability of SS to arise at any location in the body. Furthermore, PDGFRA is often expressed in SS compared to other sarcoma types81, which may be a reflection of the cell of origin and would again be consistent with a Hic1+ FAP-type MP. Based on the evidence supporting Hic1+ MPs as a candidate cell of origin for SS, as well as our current understanding of the mechanisms underlying the development of SS, we developed the following hypotheses: 1. Expression of SS18-SSX in Hic1+ MPs will lead to the development of tumours consistent with SS; 2. Expression of SS18-SSX and stabilization of b-catenin in Hic1+ MPs will cause development of SS tumours with a more aggressive phenotype including decreased latency period and increased tumour burden; 3. Expression of SS18-SSX and concomitant loss of Smarcb1 in Hic1+ MPs will cause development of SS tumours with a more aggressive phenotype including decreased latency period and increased tumour burden. We addressed these hypotheses by developing three unique mouse models wherein SS18-SSX was conditionally expressed in Hic1+ MPs alone or in combination with additional mutations. 18 Chapter 2: Methods 2.1 Mice 2.1.1 Genetics and Breeding All animals used in these experiments were in a C57Bl/6 background. Hic1CreERT2 mice were generated by genOway Inc. The Cre-recombinase is fused to a modified estrogen receptor with affinity for tamoxifen (TAM). In the absence of TAM, Cre remains in the cytoplasm, while in the presence of TAM, Cre translocates to the nucleus where it excises all floxed gene sequences present in the models described herein82. Rosa26hSS2 mice were generated by Dr. Mario Cappechi’s group and obtained from Dr. Kevin Jones. The hSS2 allele is driven by the endogenous Rosa26 promoter and generates an SS18-SSX2-IRES-EGFP bicistronic mRNA64. There is a floxed stop sequence between the promoter and SS18-SSX, therefore SS18-SSX and GFP are only expressed in the presence of an active Cre-recombinase64. Cre-dependent tandem dimer Tomato reporter mice (Rosa26tdTomato) were obtained from The Jackson Laboratory (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J - Jax stock number 007914). The tdTomato fluorescent reporter is driven by a CAG promoter and has an upstream floxed stop cassette which allows expression of tdTomato only after Cre-mediated recombination of the floxed sequence. Hic1CreERT2 mice and Rosa26hSS2 mice were bred to homozygosity at both loci to generate Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals. Upon treatment with TAM, excision of floxed sequences occurs and SS18-SSX and the GFP fluorescent reporter are expressed in all Hic1+ cells and their progeny (Figure 2.1). 19 Figure 2.1 Overview of Hic1CreERT2; Rosa26hSS2 synovial sarcoma model. Schematic of alleles and lineage tracing strategy. After treatment with TAM at 8 weeks of age, all Hic1+ cells express SS18-SSX-IRES-EGFP bicistronic mRNA. Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals were then interbred with Hic1CreERT2/CreERT2; Rosa26tdTomato/tdTomato animals to generate Hic1CreERT2/CreERT2; Rosa26hSS2/tdTomato mice. Upon treatment with TAM, excision of floxed sequences occurs and SS18-SSX, GFP, and tdTomato are expressed in all Hic1+ cells and their progeny in these animals. To generate the model with stabilized b-catenin, we obtained Ctnnb1Ex3Fl animals from Dr. Fabio Rossi’s lab in which exon 3 of the b-catenin gene is flanked by loxP sequences and excised in the presence of Cre83. Loss of exon 3 generates a stabilized form of b-catenin and causes activated WNT/b-catenin signaling in affected cells. Ctnnb1Ex3Fl animals were bred with Hic1CreERT2/CreERT2; Rosa26tdTomato/tdTomato animals to generate Hic1CreERT2/+; Rosa26tdTomato/+; Ctnnb1Ex3Fl/+ animals which were then interbred to yield Hic1CreERT2/CreERT2; Rosa26tdTomato/tdTomato; Ctnnb1Ex3Fl/Fl animals. These animals were then crossed with 20 Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals to obtain Hic1CreERT2/CreERT2; Rosa26tdTomato/hSS2; Ctnnb1Ex3Fl/+ experimental animals. Upon treatment with TAM, these animals express SS18-SSX, GFP, tdTomato, and stabilized b-catenin in all Hic1+ cells and their progeny (Figure 2.2). Figure 2.2 Overview of synovial sarcoma model with stabilized b-catenin. Schematic of alleles and lineage tracing strategy. After treatment with TAM at 8 weeks of age, all Hic1+ cells express SS18-SSX-IRES-EGFP bicistronic mRNA, tdTomato fluorescent reporter, and stabilized b-catenin. 21 To enable knockout of Smarcb1 from the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 synovial sarcoma model, we obtained a Smarcb1Fl allele wherein exon 1 is flanked by loxP sites and Cre-mediated recombination leads to inactivation of Smarcb184. We bred Smarcb1Fl/Fl animals with Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals to obtain Hic1CreERT2/+; Rosa26hSS2/+; Smarcb1Fl/+ mice, which were then interbred to generate both Hic1CreERT2/CreRT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl and Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/+ experimental mice. Upon treatment with TAM, these animals express SS18-SSX, GFP, and have biallelic or monoallelic loss of Smarcb1, respectively (Figure 2.3). Figure 2.3 Overview of synovial sarcoma model with loss of Smarcb1. Schematic of alleles and lineage tracing strategy. After treatment with TAM at 8 weeks of age, all Hic1+ cells express SS18-SSX-IRES-EGFP bicistronic mRNA and have mono- or biallelic loss of Smarcb1. 22 2.1.2 Husbandry and Genotyping All mice were housed in a conventional barrier pathogen-free transgenic mouse unit on a 12-hour light/12-hour dark schedule. Animals were fed ad libidum laboratory chow and water. Ear punches were obtained at 3-8 weeks of age for genotyping; DNA extraction and genotyping using polymerase chain reaction were performed by the Biomedical Research Centre Genotyping Facility using the primers listed in Table 2.1. Table 2.1 Genotyping Primers Gene Forward Primer (5’-3’) Reverse Primer (5’-3’) Cre GCG GTC TGG CAG TAA AAA CTA TC GTG AAA CAG CAT TGC TGT CAC TT GFP AAG TTC ATC TGC ACC ACC G TCC TTG AAG AAG ATG GTG CG TdTomato WT AAG GGA GCT GCA GTG GAG TA CCG AAA ATC TGT GGG AAG TC hSS2 GTT ATC AGT AAG GGA GCT GCA GTG G AAG ACC GCG AAG AGT TTG TCC TC AND GGC GGA TCA CAA GCA ATA ATA ACC Smarcb1 CAC CAT GCC CCC ACC TCC CCT ACA CAG GAA AAT GGA TGC AAC TAA GAT Ctnnb1 Ex3Fl GGT AGT GGT CCC TGC CCT TGA CAC CTA AGC TTG GCT GGA CGT AAA CTC Ctnnb1 WT GGT AGG TGA AGC TCA GCG CAG AGC ACG TGT GGC AAG TTC CGC GTC ATC C 2.1.3 Tamoxifen Treatment TAM (Sigma-Aldrich T5648) 25 mg/mL solution in corn oil (Sigma C8267) was prepared weekly and stored at 4°C. Animals were injected with 0.1 mL IP every 24 hours for 5 consecutive days beginning at 8 weeks of age. 23 2.1.4 EdU Treatment Fresh stock of 5-ethynyl-2’-deoxyuridine (EdU) (Sigma-Aldrich 900584) was prepared in phosphate-buffered saline (PBS) at a concentration of 2 mg/mL for each experiment and stored at 4°C. Animals were injected with 0.25 mL IP every 24 hours for 5 days prior to collection. Animals were collected 24 hours after the last EdU injection. All experimental (Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2) and control (Hic1CreERT2; Rosa26tdTomato) animals were injected with EdU beginning at 23 weeks of age (14 weeks post-tamoxifen treatment). 2.1.5 Tissue Collection Prior to tissue collection, all animals were anesthetized by IP injection of 0.5 – 0.8 mL of 25 mg/mL Avertin solution (2,2,2 – tribromoethanol, Sigma-Aldrich T48402 and tert-amyl alcohol, Sigma-Aldrich 152463). For collection of tissues for histology, we then performed transcardial perfusion with 10 mL of PBS with 2mM EDTA followed by 10 mL of 2% paraformaldehyde (PFA) by opening the right atrium and inserting a 25ga needle into the left ventricle. At this time, whole mount bright field and fluorescent images were taken using a Leica MZ6 fluorescent dissecting microscope. The desired tissues (tumours or control tongues) were then dissected and placed in 2% PFA for cryoprocessing (section 2.2) or 10% neutral buffered formalin for paraffin embedding (section 2.3). For scRNA-Seq experiments, the animals were not perfused and instead the desired tissues were dissected immediately and processed as described in section 2.5. 24 2.2 Immunofluorescent Staining 2.2.1 Cryoembedding Tissues were fixed in 2% PFA for 24-48 hours then washed 3 x 30 minutes in PBS. Tissues were then processed through a sucrose gradient starting in a 10% sucrose solution and increasing to a 50% sucrose solution in 10% increments and with a minimum of 24 hours at each concentration. Tissues were then cryoembedded in Tissue-Tek O.C.T. Compound (Sakura) and stored at -80°C. Tissues were sectioned at 6 µm with a Leica CM 3050 S Cryostat at -20°C and slides were stored at -20°C until use. 2.2.2 Antibody Staining Slides were thawed for 30 minutes at room temperature then washed 3 x 10 minutes in PBS followed by 45 minutes in 10 mg/mL sodium borohydride solution in PBS to reduce autofluorescence. Slides were then washed 3 x 5 minutes in PBS and placed in blocking solution (2.5% goat serum, Gemini Bio-products 100-109, and 2.5% Bovine Serum Albumin, Sigma Aldrich 7030-100G, in PBS) for 3 – 12 hours. Primary antibodies were diluted in blocking solution at the dilutions listed in Table 2.2 and slides were stained for 3 – 12 hours. After primary antibody staining, slides were washed 3 x 5 minutes in PBS then stained for 45 minutes with secondary antibodies in blocking solution at the dilutions listed in Table 2.3. Slides were then washed 3 x 5 minutes in PBS, counterstained with DAPI (1:500) in PBS for 5 minutes, rinsed in PBS, and coverslipped with Aqua-Poly/Mount mounting media (Polysciences Inc 18606-5). 25 Table 2.2 Primary Antibodies Antigen Manufacturer Catalogue # Dilution Species CD31 BD Biosciences 550274 1:50 Rat GFP Abcam ab13970 1:500 Chicken Laminin Abcam ab11575 1:100 Rabbit MUC1 Abcam ab15481 1:50 Rabbit TLE1 Abcam ab183742 1:100 Rabbit Table 2.3 Secondary Antibodies Fluorophore Manufacturer Catalogue # Dilution Species 488 Invitrogen A11039 1:500 Goat a Chicken 594 Invitrogen A11037 1:500 Goat a Rabbit 555 Invitrogen A21434 1:500 Goat a Rat 647 Invitrogen A21245 1:500 Goat a Rabbit 647 Invitrogen A21247 1:500 Goat a Rat 2.2.3 EdU Staining Slides of tissue from EdU-treated animals were thawed and washed in PBS as described in Section 2.2.2 then processed using the Click-iT EdU Alexa Fluor 647 Imaging Kit (ThermoFisher Scientific C10340) as per the kit protocol. After completion of the Click-iT reaction, the slides were stained with the desired immunofluorescent antibodies as described in Section 2.2.2, starting at the sodium borohydride step. 2.2.4 Fluorescent Microscopy Wide-field fluorescent imaging was performed on an Olympus BX63 microscope with an Olympus XM10 camera and confocal imaging was performed on a Nikon C2 Confocal microscope. Image collection was performed using the Olympus CellSens Program and the 26 Nikon Elements Program, and image processing was performed using Image J (Version 1.50i, National Institutes of Health). 2.3 Haematoxylin & Eosin Staining 2.3.1 Fixation, Paraffin Embedding, H&E Staining Tissues were fixed in 10% neutral buffered formalin for 24 – 72 hours, washed 3 x 30 minutes in PBS, then placed in 70% ethanol. Paraffin embedding, sectioning, and H&E staining were performed according to standard protocols by Ingrid Barta at the Biomedical Research Centre Histology Core. 2.3.2 Bright Field Imaging Bright Field Imaging was performed on an Olympus BX63 microscope with an Olympus DP72 camera. Image collection was performed using the Olympus CellSens Program. 2.4 Quantification 2.4.1 Vessel Association Tongues from Hic1CreERT2; Rosa26tdTomato control mice (n=3) were collected and processed for immunofluorescent imaging with CD31 antibody staining as described in section 2.2. Three 20X images were taken from separate, non-adjacent sections from each animal. All tdTomato+ cells were counted in each image and evaluated for their vessel association status. Cells were deemed vessel-associated when they were contacting a CD31-positive vessel and deemed non-vessel-associated when they had no clear contact with a CD31-positive vessel. The mean of all image 27 counts was calculated and vessel-association was expressed as a percentage of total number of tdTomato+ cells. 2.4.2 Proliferation Hic1CreERT2; Rosa26tdTomato control mice (n=3) and Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 experimental mice (n=6) were treated with EdU as described in section 2.1.4 and their tongues were collected and processed for EdU with subsequent antibody staining as described in section 2.2.3. Three 20X images were taken from separate, non-adjacent sections from each animal. For control animals, all tdTomato+ and tdTomato+EdU+ cells were counted in each image, the mean of all image counts (n=9) was calculated, and proliferation rate was expressed as a percentage of total number of tdTomato+ cells. For experimental animals, all GFP+ and GFP+EdU+ cells were counted in each image, the mean of all image counts (n=18) was calculated, and proliferation rate was expressed as a percentage of total number of GFP+ cells. 2.5 Single-cell RNA-Seq 2.5.1 Cell Isolation Tissues from Hic1CreERT2; Rosa26tdTomato control mice (tongue), Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 experimental mice (tongue and stifle masses), and Hic1CreERT2/CreERT2; Rosa26tdTomato/hSS2; Ctnnb1Ex3Fl/+ experimental mice (calvarial masses) were processed using the same protocol. After dissection, the tissues were minced with scissors and placed in activated Collagenase II digest cocktail (Sigma 6885, 500u/mL, activated with 10µL/mL 250mM CaCl2) for 30 minutes at 37°C. Tissues were then washed twice in ice-cold PBS by centrifugation at 120g for 1 minute at 4°C. After the second centrifugation step, the tissue pellets were 28 resuspended in activated Collagenase D/Dispase II digest cocktail [Collagenase D (Roche 11088882001, 1.5u/ mL)/Dispase II (Roche 04942078001, 2.4u/ mL, activated with 10µL/mL 250mM CaCl2] and rotated for 1 hour at 37°C with gentle vortexing every 15 minutes. After enzymatic digestion, the cells were washed in fluorescence-activated cell sorting (FACS) buffer (2% Fetal Bovine Serum in PBS with 2mM EDTA), strained through a 40 µm cell strainer (Fisherbrand 22363547), and centrifuged at 500g for 5 minutes at 4°C. The supernatant was discarded and the cell pellets were resuspended in antibody cocktail (in FACS buffer) for 30 minutes on ice. The antibody cocktails were prepared according to Table 2.4. After antibody staining, the cells were washed in FACS buffer, centrifuged at 500g for 5 minutes at 4°C, resuspended in FACS buffer containing Hoechst 33342 at 4 µM (Sigma B2261) and Propidium Iodide at 1.5 µM (Sigma P4864), and transferred to 5 mL polypropylene round-bottom tubes through the cell-strainer cap (Falcon 352063). Table 2.4: Antibodies for FACS Antigen Fluorophore Manufacturer Cat# Dilution Selection Experiment CD11b APC AbLab 67-0055-01 1:500 Negative All CD31 APC AbLab 67-0032-01 1:400 Negative All CD34 AF700 BD Biosciences 560518 1:50 Positive 4 wki tongues CD45 APC AbLab 67-0047-01 1:400 Negative All C-Kit APC Invitrogen 17-1172-82 1:500 Negative All F4/80 APC AbLab 67-0035-01 1:500 Negative All Sca-1 PE-Cy7 eBiosciences 25-5981-82 1:2000 Positive Control tongues TER119 APC AbLab AB00000305 1:200 Negative All Thy1 AF700 BioLegend 105320 1:250 Positive 4 wki tongues 29 2.5.2 Fluorescence-Activated Cell Sorting FACS was performed at the UBC Flow Cytometry Facility on a BD Influx. APC-labelled antibodies against lineage markers for erythrocytes, endothelium, and nucleated hematopoietic cells (Lin+) were used as negative selectors as listed in Table 2.4. Control tongues were sorted for tdTomato+/PE-Cy7+/Lin- cells. Calvarial masses from the stabilized b-catenin model were sorted for tdTomato+/Lin- cells. Endpoint tongue tumours, 8 wki tongue tumours, and stifle tumours were sorted for GFP+/ Lin- cells, and 4 wki tongue tumours were sorted for GFP+/AF700+/ Lin- cells. All samples except for the 4 wki tongues were then sorted a second time using the same parameters to increase purity. PI was added again between the first and second sort to identify dead and dying cells. After sorting, cells were visually evaluated on an Olympus BX63 microscope and manually counted using a hemocytometer prior to sequencing. 2.5.3 Sequencing Samples were loaded onto a Chromium Single Cell Controller (10X Genomics) to generate barcoded cDNA libraries and the libraries were sequenced using a NextSeq 500 Sequencer (Illumina) with paired end 42bp x 42bp reads. Sequencing data was demultiplexed using Illumina’s bcl2fastq2 and read sequences were aligned to the Mus musculus (mm10) reference sequence using STAR Aligner (https://www.ncbi.nlm.nih.gov/pubmed/23104886). ScRNA-Seq analysis was performed using Cell Ranger analysis pipelines (10X Genomics) and data visualization and evaluation was performed using Loupe Cell Browser (10X Genomics). Aggregates of multiple scRNA-Seq datasets were generated in Cell Ranger by Dr. Wilder Scott in the Underhill Lab. The control bone MP scRNA-seq dataset was collected and shared by Dr. Wilder Scott. 30 2.6 Statistical Analysis Kaplan-Meier plots were generated using Prism (Version 7) and t-tests were performed using Excel (MS Office). All statistical analyses for scRNA-Seq datasets were performed in Cell Ranger, and K-means clustering and t-SNE dimensionality reduction for data visualization was performed in Loupe Cell Browser. 31 Chapter 3: Results 3.1 Effects of conditional expression of SS18-SSX in Hic1+ cells To determine the effects of expression of the SS18-SSX fusion oncogene in Hic1+ cells, Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 and Hic1CreERT2/CreERT2; Rosa26hSS2/tdTomato animals were treated with TAM for five consecutive days beginning at 8 weeks of age. All hSS2 homozygous animals (n=69) developed macroscopic tumours with a mean latency period of 16.2 ± 2.5 weeks and all hSS2 heterozygous animals (n=33) developed macroscopic tumours with a mean latency period of 65.8 ± 16.0 weeks (Figure 3.1). Figure 3.1 Kaplan-Meier survival curve of Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 and Hic1CreERT2/CreERT2;Rosa26hSS2/tdTomato animals. Kaplan-Meier survival plot of Hic1CreERT2; Rosa26hSS2/hSS2 and Hic1CreERT2; Rosa26hSS2/tdTomato mice, and Hic1CreERT2 and Rosa26hSS2 controls. All mice with an hSS2 allele develop tumors; control mice do not develop tumors. 32 Animals heterozygous for hSS2 developed soft-tissue tumours at various locations including the face, dorsum, lateral and ventral thorax and abdomen, tail, and limbs, as well as occasional tumours arising in the tongue and gingival tissues. All hSS2 homozygous animals developed tumours in the tongue and in the fascia around the stifle, as well as a minimum of one other tumour at variable locations including the tail base, intercostal region, limbs and trunk (Figure 3.2). All tumours examined under blue light displayed green fluorescence as expected due to the IRES-EGFP marker gene in the hSS2 allele (Figure 3.2). 33 Figure 3.2 Tumour induction in Hic1+ MPs. Bright field and fluorescent whole mount images. A. Hic1+ MP-specific SS18-SSX expression leads to masses in the tongue. All mice (males and females; N=69) present with tumors with a mean latency period of 16.2 ± 2.5 weeks post TAM injection. B. Tongue tumors are GFP+ and mice also develop nascent GFP+ masses in the fascia surrounding the stifle (knee) joint. 34 To determine whether the resultant tumours were consistent with human synovial sarcoma, the tumours were examined histologically with H&E staining to assess cellular morphology and IF staining to assess marker expression. Examination of H&E-stained tumour samples revealed cellular morphology consistent with synovial sarcoma, with some tumours exhibiting the monophasic histologic subtype, with spindle cell/mesenchymal cellular morphology, and some tumours exhibiting the biphasic histologic subtype, containing both mesenchymal and epithelial cellular morphology (Figure 3.3). All tongue tumours examined with H&E exhibited monophasic morphology while tumours in other locations were variably monophasic or biphasic. Next, we evaluated expression of TLE1 and MUC1 in the mouse tumours. Strong nuclear TLE1 expression was found in both monophasic and biphasic tumours, with no TLE1 staining observed in adjacent normal tissues (Figure 3.4A, B). Biphasic tumours exhibited MUC1 expression on the apical surface of cells with epithelial morphology, while adjacent cells with mesenchymal morphology did not show MUC1 expression (Figure 3.4C). Endogenous GFP expression was detectable in all neoplastic cells (Figure 3.4A, C). 35 Figure 3.3 Histologic analysis of synovial sarcoma tumours from the Hic1CreERT2; Rosa26hSS2 mouse model. Hematoxylin and eosin stain showing characteristic biphasic and monophasic histologic subtypes A, B. Biphasic histologic subtype with gland-like epithelial structures in a cheek mass. C, D. Monophasic histologic subtype with uniform spindle cell morphology in a tongue tumour with no epithelial components evident (Frozen samples). Scale bars: A, C = 100µm, B, D = 50µm. 36 37 Figure 3.4 Immunofluorescent staining of synovial sarcoma tumours from the Hic1CreERT2; Rosa26hSS2 mouse model. A. Anti-TLE1 staining of a monophasic tongue tumour. Neoplastic cells exhibit nuclear TLE1 staining and expression of endogenous GFP. B. Anti-TLE1 staining of monophasic and biphasic tumours. Tumour borders are defined by white dashed lines. Neoplastic cells exhibit nuclear TLE1 staining whereas adjacent non-neoplastic cells are TLE1 negative. C. Anti-MUC1 staining on the apical surface of epithelial cells within a biphasic tumour and endogenous GFP expression. MF, myofibre; TE, tongue epithelium; OM, oral mucosa; E, epithelial morphology; M, mesenchymal morphology. Scale bars: A = 50µm, B (upper) = 50µm, B (lower) = 100µm, C (upper) = 100µm, C (lower) = 20µm. To assess the proliferation rate of neoplastic cells at clinical endpoint, we performed an EdU (5-ethynyl-2’-deoxyuridine) incorporation assay. Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 mice (n=6) were treated with EdU once every 24 hours for five days prior to collection and the tongue tumours were analyzed histologically. Proliferating tumour cells (GFP+ EdU+) were quantified as a proportion of total tumour cells (GFP+). Tongues from TAM-treated Hic1CreERT2; Rosa26tdTomato mice, where Hic1+ cells and their progeny are marked with a tdTomato fluorescent protein, were used as control tissues (n=3). Hic1CreERT2; Rosa26tdTomato mice underwent the same EdU treatment protocol, and proliferating tdTomato+ cells (tdTomato+ EdU+) were quantified as a proportion of total tdTomato+ cells. The proliferation rate of neoplastic cells at clinical endpoint (20.8 ± 2.0%) was significantly higher (p<0.0001) than the proliferation rate of control tdTomato+ cells (0.5 ± 0.3%) (Figure 3.5). 38 Figure 3.5 Cellular proliferation in mouse tumours. EdU incorporation assay with immunofluorescent staining in 16 wki Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tongue tumours. A. DAPI nuclear stain. B. Anti-GFP antibody staining of SS18-SSX-expressing cells. C. Composite image with DAPI, GFP, CD31, and EdU staining showing colocalization of GFP and EdU signals in neoplastic cells. D. Anti-CD31 antibody staining labelling endothelium. E. Nuclear EdU signal in proliferating cells. F. The proliferation rate of neoplastic SS18-SSX-expressing cells in synovial sarcoma tumours at clinical endpoint was 20.8 ± 2.0% which is significantly higher (p<0.0001) than the proliferation rate of control tdTomato+ cells (0.5 ± 0.3%). Scale bar = 100µm. 39 While none of the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 (n=69) and Hic1CreERT2/CreERT2; Rosa26hSS2/tdTomato (n=33) mice had macroscopic pulmonary lesions at clinical endpoint, lungs from a small group of Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 mice (n=3) were examined using serial histologic sections for the presence of microscopic pulmonary lesions. One animal exhibited microscopic pulmonary lesions characterized by irregular clusters of GFP+ cells located in the perivascular space around large pulmonary blood vessels (Figure 3.6). These lesions may represent additional primary lesions as they were not found in the peripheral locations typical of hematogenous metastatic lesions. 40 Figure 3.6 Pulmonary lesions in Hic1CreERT2; Rosa26hSS2 mice. Immunofluorescent staining in 16 wki Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 lungs. A. Composite image with DAPI, GFP, CD31, and laminin staining showing an irregular cluster of perivascular GFP+ cells. B. DAPI nuclear stain. C. Anti-Laminin antibody staining labelling extracellular matrix. D. Anti-CD31 antibody 41 staining labelling endothelium. E. Anti-GFP antibody staining of SS18-SSX-expressing cells. F. Pie chart showing proportion of examined Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals with pulmonary lesions at clinical endpoint (n=3). Scale bars = 50µm. Taken together, these results show that conditional expression of SS18-SSX in Hic1+ cells induces the formation of murine tumours with the histology and orthologous immunophenotype of human synovial sarcoma. Both hSS2 heterozygotes and homozygotes develop tumours with 100% penetrance, although homozygotes develop tumours within a consistent and significantly shorter latency period (p<0.0001) and always in the same locations (tongue and fascia surrounding stifles). As seen in the Capecchi/Jones models using the same hSS2 allele but with different Cre drivers56,64, the resulting tumours are histologically indistinguishable from human SS at the level of both architectural and cellular morphology, and in the expression of diagnostic immunophenotypic markers. Our results further demonstrate that these tumours exhibit an elevated cellular proliferation rate as expected for neoplastic tissue. 3.2 Identification of early and intermediate tumours Given the temporal and anatomic consistency of tumour development in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 mice, we endeavoured to identify early and intermediate microscopic tumours prior to the development of macroscopically visible tumours. We collected tongues from TAM-treated Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 mice at 4 wki (n=9) and 8 wki (n=4) and processed the tissue for histological analysis. None of the mice had macroscopic tumours at these time points, however seven out of nine 4 wki mice and all 8 wki mice had microscopic neoplastic lesions visible upon histologic sectioning. At 4 wki the lesions were small (< 100 µm diameter) and 42 were found mainly in the loose connective tissue between muscle bundles, often perivascularly, although they were also found dissecting between the myofibres within muscle bundles. These lesions were visible with both H&E staining (Figure 3.7) and IF staining using an anti-GFP antibody (Figure 3.8C). All animals had at least one lesion at 4 wki, although many animals had two or three lesions, suggesting that the tongue tumours at clinical endpoint are likely polyclonal. In addition to the small early tumours visible at 4 wki, IF staining using an anti-GFP antibody also revealed individual spindle-shaped, perivascular GFP+ cells (Figure 3.8B). By 8 wki, the neoplastic lesions were larger and already disrupting the normal architecture of the tongue parenchyma (Figure 3.8D) and by clinical endpoint (16 wki) the tumours had displaced the majority of normal tongue parenchyma (Figure 3.8E). The consistency of tumour development from as early as 4 wki suggests that expression of SS18-SSX causes neoplastic transformation of cells through a tightly controlled and predictable progression. 43 Figure 3.7 Histologic analysis of tongues from 4 wki Hic1CreERT2; Rosa26hSS2 mice. Hematoxylin and eosin stain showing early neoplastic lesions in the lingual connective tissue and surrounding myofibres. Dashed lines delineate margins of lesions. FFPE samples. Scale bar = 50µm. Figure 3.8 Immunofluorescent staining of early, intermediate, and endpoint tongue tumours. All images stained with DAPI nuclear stain, anti-CD31 antibody labelling endothelium, anti-GFP antibody labelling SS18-SSX-expressing neoplastic cells, and anti-laminin antibody labelling extracellular matrix. A. Control tongue from Hic1CreERT2; Rosa26tdTomato mouse. No GFP+ cells are present. B. 4 wki Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tongue with individual, perivascular GFP+ cells with normal MP morphology. C. 4 wki 44 Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tongue with a small early GFP+ neoplastic lesion. D. 8 wki Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tongue with a large GFP+ neoplastic lesion. E. 16 wki endpoint Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tongue showing complete infiltration of GFP+ neoplastic cells and loss of normal tongue architecture. Scale bar = 50µm. 3.3 Single-cell RNA-Sequencing analysis of mouse tumours To characterize the transcriptome of the mouse tumours, we performed single-cell RNA-Sequencing (scRNA-Seq) on endpoint tongue tumours at 16 wki, stifle tumours at endpoint, early tongue tumours at 4 wki, and intermediate tongue tumours at 8 wki. All tumour samples were generated from Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 mice. Animals were injected with TAM at 8 weeks of age, tumour cells were isolated by FACS at the desired time point and then sequenced. TdTomato+ MPs isolated from the tongues of 10-week-old Hic1CreERT2;Rosa26tdTomato were used as a control. Control tongues were sorted for tdTomato+/Lin- cells (Figure 3.9). Sixteen wki and 8 wki tumours were sorted for GFP+/Lin- cells (Figure 3.10), however this approach did not yield successful isolation of 4 wki lesions. 45 Figure 3.9 FACS strategy for Hic1+ MPs from control tongues. Hic1+ MPs and their progeny express a tdTomato fluorescent reporter. Lineage markers are labelled by APC-conjugated antibodies. Cells were gated sequentially from A-F. A. Selection based on cell size. B. Selection of singlets. C. Selection of living tdTomato+ cells (tdTomato+/PI-). D. Selection of mononucleated, Lin- events. E, F. Selection of tdTomato+/Lin- cells. 46 Figure 3.10 FACS strategy for neoplastic cells at 8 wki and 16 wki. Strategy used for 8 wki tongues and 16 wki tongues and stifles. Neoplastic cells express the GFP fluorescent reporter from the hSS2 allele. Lineage markers are labelled by APC-conjugated antibodies. Cells were gated sequentially from A-F. A. Selection based on cell size. B. Selection of singlets. C. Selection of mononucleated, Lin- events. D. Selection of GFP+ cells. E. Selection of GFP+/Lin- cells. F. Selection of living GFP+ cells (GFP+/PI-, P7). 47 To identify other possible isolation strategies for 4 wki lesions, we combined the scRNA-Seq datasets from the control MPs (n=1), the 8 wki tongue tumour sample (n=1, 5 mice pooled) and one 16 wki tongue tumour into an aggregate dataset. Using Loupe Cell Browser software from 10X Genomics, we performed K-means clustering and assessed the clusters visually for the expression of the tdTomato and GFP fluorescent markers and MP markers. Using this approach, we identified a group of 72 cells in the 8 wki sample that clustered separately from the majority of the 8 wki cells (Figure 3.11A). Like the rest of the 8 wki cells and the 16 wki cells, these cells show expression of GFP, indicating that they are expressing SS18-SSX (Figure 3.11B), however, they also express thymocyte differentiation antigen 1 (Thy1) and cluster of differentiation 34 (Cd34), two markers which are expressed in control MPs but whose expression is lost in tumour cells (Figure 3.11D, E). 48 Figure 3.11 Identification of an intermediate cell population using scRNA-Seq. Each dot represents a single cell. In B-E the coloured dots indicate presence of the desired transcript while grey dots indicate no expression of the transcript. A. scRNA-Seq analysis of 8 wki tumour cells, 16 wki tumour cells, and control tongue MPs. tSNE plots allow identification of a control MP population (population 0), an intermediate population from the 8 wki tumour sample (population 1), and a tumour population from the 8 wki and 16 wki tumour samples (population 2). B. Populations 1 and 2 express GFP, which confirms expression of the hSS2 allele. C. Population 1 does not express tdTomato, therefore it is not part of the control MP population. D, E. Control MPs (population 0) and population 1 express cell surface markers Thy1 and Cd34, while expression of these markers is lost in the tumour population (population 2), indicating that population 1 represents an intermediate state. 49 Based on these results, we sorted 4 wki tongues (n=1, 6 pooled tongues) for GFP+/Thy1+/CD34+/Lin- cells and successfully isolated 284 cells (Figure 3.12). We then combined the following scRNA-Seq datasets into an aggregate dataset: 16 wki tongue tumours (n=2), 16 wki stifle tumours (n=1, 2 mice pooled), 8 wki tongue tumours (n=1, 5 mice pooled), 4 wki tongue tumours (n=1, 5 mice pooled), and control tdTomato+ tongue MPs (n=1, 8 mice pooled). A total of 19, 351 tumour cells and 5951 control MPs were sequenced. 50 Figure 3.12 FACS strategy for neoplastic cells at 4 wki. Strategy used for 4 wki tongues. Neoplastic cells express the GFP fluorescent reporter from the hSS2 allele. Lineage markers are labelled by APC-conjugated antibodies. CD34 and Thy1 surface markers are labelled by AF700-conjugated antibodies. Cells were gated sequentially from A-F. A. Selection based on cell size. B. Selection of singlets. C. Selection of mononucleated events. D. Selection of AF700+/Lin- cells. E. Selection of AF700+/GFP+ cells. F. Selection of living GFP+ cells (GFP+/PI-, P6). 51 Using Loupe Cell Browser, we performed K-means clustering and analyzed tumour (GFP+) and control (tdTomato+) cell populations for the expression of MP markers and gene pathways known to be upregulated in human synovial sarcoma, including WNT signalling pathway components and neurodevelopmental genes. The clusters were assessed visually for the expression of the genes of interest. The following genes were analyzed: platelet-derived growth factor receptor a (Pdgfra), regulator of G protein signaling 5 (Rgs5), thymocyte differentiation antigen 1 (Thy1), cluster of differentiation 34 (Cd34), gastrulation brain homeobox 2 (Gbx2), zinc finger protein of the cerebellum 5 (Zic5), transducin-like enhancer of split 1 (Tle1), erythropoietin-producing hepatocellular A4 (Epha4), frizzled class receptor 1 (Fzd1), frizzled class receptor 10 (Fzd10), WNT family member 5A (Wnt5a), and axis inhibition protein 2 (Axin2) (Figure 3.13). 52 Figure 3.13 scRNA-Seq analysis of Hic1CreERT2; Rosa26hSS2 Tumours. Each dot represents a single cell. In B-E the coloured dots indicate presence of the desired transcript while grey dots 53 indicate no expression of the transcript. A. scRNA-Seq analysis of Hic1CreERT2; Rosa26hSS2 tumours at various time points including 4 wki (tongue), 8 wki (tongue), and 16 wki (tongue and stifle) and control tongue MPs. B. Tumour cells isolated from Hic1CreERT2; Rosa26hSS2 animals express GFP, which confirms expression of the hSS2 allele, while control cells express a tdTomato fluorescent marker. C. Control MPs express MP markers including Pdgfra, Rgs5, Thy1, and Cd34. Neoplastic cells maintain expression of Pdgfra but lose expression of Rgs5, Thy1, and Cd34. D. Neoplastic cells gain expression of genes involved in neural development including Gbx2, Zic5, Tle1, and Epha4. E. Neoplastic cells gain expression of genes involved in WNT signalling including Fzd1, Fzd10, Wnt5a, and Axin2. 54 Based on these analyses, tdTomato+ MPs in the tongue comprise at least two distinct populations: Pdgfra+ FAPs and Rgs5+ pericytes (Figure 3.13C). The tumour cells are Pdgfra+/Rgs5-, suggesting that the tumours arise specifically from the FAP subpopulation of Hic1-lineage cells, and not from pericytes (Figure 3.13C). Thy1 and Cd34, two cell surface markers present on MPs are expressed in the tdTomato+ cell population, as expected, however their expression is largely lost in the tumour cells (Figure 3.13C). Conversely, four genes involved in neural development were found to be uniformly expressed in the tumour cells and absent or expressed at lower levels in control MPs; expression of the transcription factors Gbx2 and Zic5 was absent from control MPs, while tyrosine-kinase receptor Epha4 and transcriptional co-repressor Tle1 showed increased expression in tumour cells compared to control MPs (Figure 3.13D). The expression of Fzd1, Fzd10, Wnt5a, and Axin2 - all components of the WNT signaling pathway - was also increased in tumour cells compared to control MPs (Figure 3.13D), suggesting that WNT pathway activation plays a role in the pathogenesis of murine synovial sarcoma tumours. We also specifically evaluated gene expression in the 4 wki cells compared to control MPs, 8 wki, and 16 wki tumours. We found that the 4 wki population contains two subpopulations: a population with high Rgs5 expression that clusters closely with the Rgs5+ control cells and likely represents pericytes, and a population with low Rgs5 expression which likely represents the early tumour cells (Figure 3.14). The cells with high Rgs5 expression were likely collected due to the use of Thy1 as a positive selector as they are also Thy1 positive (Figure 3.14B). 55 Figure 3.14 Identification of 4 wki subpopulations. A. tSNE plot of 4 wki, 8 wki, and 16 wki tumours and control tongue MPs shows that cells isolated from 4 wki Hic1CreERT2; Rosa26hSS2/hSS2 tongues cluster in two distinct populations, one representing pericytes and the other representing early tumour cells. B. The pericyte population shows high Rgs5 expression and clusters with Rgs5+ control cells, while the early tumour population shows low Rgs5 expression and clusters between control MPs and 8 wki and 16 wki tumour cells. 56 Analysis of the 4 wki early tumour cells showed that they do not yet express all the WNT pathway components and neural-associated genes that are found to be upregulated in the 8 wki and 16 wki tumours. Specifically, the 4 wki early tumour cells do not express Gbx2, Zic5, or Fzd10, and they show lower expression of Epha4, Fzd1, and Wnt5a compared to 8 wki and 16 wki tumours (Figure 3.15B, C). Expression of Tle1 and Axin2 is comparable in 4 wki, 8 wki, and 16 wki tumours (Figure 3.15B, C). These results show that the upregulated genes seen in the endpoint tumours are not upregulated simultaneously during sarcomagenesis and suggest that expression of some genes is likely required earlier than others to initiate tumour development. 57 Figure 3.15 Expression profile of early tumour cells. A. tSNE plot of 4 wki, 8 wki, and 16 wki tumours and control tongue MPs. B. Early tumour cells at 4 wki do not yet express the neural genes Gbx2 and Zic5 and show lower expression of Epha4 compared to intermediate (8 wki) and endpoint (16 wki) tumours. Expression of Tle1 is comparable in early, intermediate, and endpoint tumours. C. Early tumour cells at 4 wki do not yet express the WNT pathway component Fzd10 and show lower expression of Fzd1 and Wnt5a compared to intermediate and endpoint tumours. Expression of Axin2 is comparable in early, intermediate, and endpoint tumours. 58 Overall, the scRNA-Seq analysis of tumours derived from Hic1CreERT2/CreERT2;Rosa26hSS2/hSS2 mice shows that the mouse tumours are transcriptionally similar to human synovial sarcoma, with activation of the WNT signaling pathway and aberrant expression of genes associated with neural development. In addition, it shows that the tumours likely arise specifically from Hic1+ FAPs, not Hic1+ pericytes, highlights the phenotypic changes that occur during neoplastic transformation, including loss of MP cell surface markers, and provides evidence of variable timing of gene upregulation during sarcomagenesis. 3.4 Characterization of Hic1+ cells in the tongue Given the predilection for development of tongue tumours in the Hic1CreERT2/CreERT2;Rosa26hSS2/hSS2 mice, we sought to characterize Hic1+ MPs in the tongue. We used TAM-injected Hic1CreERT2;Rosa26tdTomato mice to fluorescently label Hic1+ MPs and their progeny in the tongue. The murine tongue is made up of skeletal muscle arranged in multiple orientations, allowing the tongue to move in many directions (Figure 3.16). The surface of the tongue is covered by stratified squamous epithelium underlain by lamina propria (Figure 3.16A), while the parenchyma of the tongue is mainly skeletal muscle bundles (Figure 3.16B) with areas of loose connective tissue between muscle bundles (Figure 3.16C). 59 Figure 3.16 Normal histology of the mouse tongue. Hematoxylin and eosin stain. A. Stratified squamous epithelium on the tongue surface with underlying skeletal muscle. B. Skeletal muscle in the tongue parenchyma. C. Loose connective tissue, vessels, and skeletal muscle arranged in multiple orientations. FFPE samples. E = epithelium, CT = connective tissue, LP = lamina propria, SM = skeletal muscle. Scale bar = 50µm. We performed immunofluorescent analysis of tongues from Hic1CreERT2; Rosa26tdTomato animals (n=3) using an anti-CD31 antibody to label vessels and quantified the proportion of Hic1+ MPs found on vessel and off vessel (Figure 3.17). We found that while the majority of Hic1+ MPs were on vessel, 29.2 ± 8.8% of Hic1+ MPs, mainly located in the connective tissue between muscle bundles, were off vessel (Figure 3.17E). This is a higher proportion than what has previously been found in our lab, as other work has shown that Hic1+ MPs are approximately 97% on vessel in skeletal muscle (unpublished data). 60 Figure 3.17 Vessel association of Hic1+ MPs in the tongue. Immunofluorescent staining in Hic1CreERT2; Rosa26tdTomato tongues. A. Composite image with endogenous tdTomato and DAPI and CD31 antibody staining showing both on-vessel (arrowheads) and off-vessel (arrows) tdTomato+ MPs. B. DAPI nuclear stain. C. tdTomato fluorescent reporter labelling Hic1+ MPs. D. Anti-CD31 antibody staining labelling endothelium. E. Plot showing proportion of on-vessel and off-vessel tdTomato+ MPs. Scale bars = 50µm. To evaluate the proliferation rate of Hic1+ MPs in the tongue during homeostatic conditions, we performed an EdU incorporation assay. Hic1CreERT2; Rosa26tdTomato mice (n=3) were treated with EdU once every 24 hours for five days prior to collection and proliferating tdTomato+ cells (tdTomato+ /EdU+) were quantified as a proportion of total tdTomato+ cells using immunofluorescent microscopy (Figure 3.18). The proliferation rate of tdTomato+ cells was 0.5 ± 0.3%. 61 Figure 3.18 Proliferation of Hic1+ MPs in the tongue. EdU incorporation assay and immunofluorescent staining in Hic1CreERT2; Rosa26tdTomato tongues. A. Composite image with DAPI, CD31, EdU, and endogenous tdTomato showing a proliferating tdTomato+/EdU+ MP (arrow). B. DAPI nuclear stain showing nucleus of proliferating MP (arrow). C. tdTomato fluorescent reporter labelling Hic1+ MPs. Arrow indicates proliferating MP. D. Anti-CD31 antibody staining labelling endothelium. E. EdU labelling showing nuclear EdU incorporation in proliferating MP (arrow). Scale bars = 50µm. 62 To further characterize Hic1+ MPs in the tongue we analyzed the scRNA-Seq data from the control tdTomato+ MPs isolated from the tongues of 10-week-old Hic1CreERT2;Rosa26tdTomato mice (n=1, 8 mice pooled) as described in section 3.3. Using this dataset alone, not aggregated with tumour samples, we performed K-means clustering in Loupe Cell Browser and analyzed expression of the following genes: Sca1, Cd34, Pdgfra, Pdgfrb, Rgs5, and Mcam. As shown in Figure 3.13, Hic1+ MPs in the tongue broadly consist of two populations: FAPs and pericytes. All cells express the tdTomato fluorescent reporter (Figure 3.19B). In addition to expression of Pdgfra, the FAP clusters express Cd34 and Sca1, both known FAP markers (Figure 3.19C), while the pericyte clusters express Rgs5, Pdgfrb, and Mcam (Figure 3.19D). 63 Figure 3.19 scRNA-Seq analysis of control tongue Hic1+ MPs. Each dot represents a single cell. In B-D the coloured dots indicate presence of the desired transcript while grey dots indicate 64 no expression of the transcript. A. scRNA-Seq analysis of Hic1CreERT2; Rosa26tdTomato tongue MPs. B. All cells express the tdTomato fluorescent marker. C. The majority of MPs isolated from the tongue express FAP markers including Sca1, Cd34, and Pdgfra. D. A smaller subset of tongue MPs express the pericyte markers Pdgfrb, Rgs5, and Mcam. Overall, analysis of Hic1+ MPs in the tongue confirmed the presence of two populations, FAPs and pericytes, which express known markers of these populations. Hic1+ MPs in the tongue were found to have a baseline proliferation rate of 0.5 ± 0.3% and a higher proportion of off-vessel cells (29.2 ± 8.8%) than expected based on what has been found in other tissues. 3.5 Effects of addition of stabilized b-catenin to Hic1CreERT2; Rosa26hSS2 mouse model To assess the effect of activated WNT/b-catenin signaling in the context of SS18-SSX expression, we generated Hic1CreERT2/CreERT2; Rosa26hSS2/tdTomato; Ctnnb1+/Ex3Fl animals that express one copy of the hSS2 allele, the tdTomato fluorescent reporter, and a single copy of stabilized b-catenin causing constitutive activation of the canonical WNT signaling pathway in Hic1+ cells. These animals were treated with TAM for five consecutive days beginning at 8 weeks of age and all animals (n=56) developed macroscopic tumours and reached clinical endpoint with a mean latency period of 6.3 weeks (Figure 3.20). This latency period was significantly shorter (p<0.0001) than the latency period of Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals that did not express stabilized b-catenin (Figure 3.20). Animals with one copy of stabilized b-catenin but that did not express SS18-SSX did not develop tumours (Figure 3.20). 65 Figure 3.20 Kaplan-Meier survival curve of Hic1CreERT2/CreERT2; Rosa26tdTomato/hSS2; Ctnnb1+/Ex3Fl animals. Kaplan-Meier survival plot of Hic1CreERT2/CreERT2; Rosa26tdTomato/hSS2; Ctnnb1+/Ex3Fl and Hic1CreERT2; Rosa26hSS2/hSS2 mice, and Hic1CreERT2; Rosa26tdTomato; Ctnnb1+/Ex3Fl controls. Animals that express SS18-SSX and have stabilized b-catenin develop tumours with a mean latency period of 6.3 ± 1.9 weeks. All mice with an hSS2 allele develop tumors; control mice with stabilized b-catenin alone do not develop tumors. In contrast to the soft-tissue tumours seen in the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 model, all tumours in the b-catenin model arose along the surface of bones. Each animal had over 20 tumours, while some had over 50, ranging in size from under 2 mm to 1 cm in diameter. Tumours arose in multiple locations, including the femur, tibia, fibula, scapula, humerus, radius, ulna, vertebrae, ribs, and skull (Figure 3.21). As expected, the majority of tumours exhibited tdTomato fluorescence (Figure 3.21), however occasional tumours were tdTomato-. This 66 suggests that each tumour likely arose from a distinct clone, and that the multiple tumours were not a result of metastasis from one primary tumour. Although all tumours would be expected to express tdTomato based on genotype, it is possible that the tdTomato- tumours had incomplete excision of the stop sequence in the conditional tdTomato allele. Figure 3.21 Tumour induction in Hic1+ MPs with stabilized b-catenin. Bright field and fluorescent whole mount images. Hic1+ MP-specific SS18-SSX and tdTomato expression in the presence of stabilized b-catenin leads to multifocal periosteal masses throughout the body including the calvaria, femur, and ribs. All mice (males and females; N=56) present with tumors with a mean latency period of 6.3 ± 1.9 weeks post TAM injection. To determine whether the tumours in this model were also consistent with human synovial sarcoma, the tumours were examined histologically with H&E staining to assess cellular 67 morphology. Immunofluorescent analysis was also performed to assess tdTomato expression. Examination of H&E-stained tumour samples revealed cellular morphology consistent with synovial sarcoma, with tumours exhibiting the monophasic histologic subtype with spindle cell morphology (Figure 3.22). No tumours exhibited the biphasic histologic subtype. On histologic section, the tumours appeared to arise from the periosteal surface of affected bones and were markedly invasive, often demonstrating destruction of cortical bone and exuberant bone formation (Figure 3.22). Figure 3.22 Histologic analysis of synovial sarcoma tumours with stabilized b-catenin. Hematoxylin and eosin stain showing the monophasic histologic subtype in all tumours. A-C. Mid-diaphyseal femoral mass showing marked exuberant bone formation. Dashed line demarcates femoral diaphysis. D-F. Calvarial mass with exuberant bone formation. FFPE samples. Scale bars: A, D = 100µm, B, C, E, F = 50µm. 68 Multiple animals exhibited tdTomato+ tumours directly adjacent to tdTomato- tumours. Immunofluorescent analysis of two adjacent calvarial masses – one tdTomato+ and one tdTomato- (Figure 3.23A) – showed that in the tdTomato+ tumour, all neoplastic cells were tdTomato+ (Figure 3.23B), while in the tdTomato- tumour, all neoplastic cells were tdTomato- (Figure 3.23C). These findings further suggest that each tumour arose from a distinct clone. 69 Figure 3.23 Immunofluorescent analysis of synovial sarcoma tumours from the Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1+/Ex3Fl mouse model. DAPI nuclear stain and endogenous tdTomato fluorescent reporter shown. A. Adjacent tdTomato+ and tdTomato- calvarial masses. B. Magnification of tdTomato+ calvarial mass showing tdTomato+ neoplastic cells. C. Magnification of tdTomato- calvarial mass showing tdTomato- neoplastic cells. Occasional tdTomato+ cells within the mass likely represent non-neoplastic perivascular MPs. Scale bars: A = 100µm, B, C = 50µm. 70 We next sought to characterize the expression profile of tumour cells from this model using scRNA-Seq. Animals were injected with TAM at 8 weeks of age and calvarial tumours at clinical endpoint were sorted by FACS for tdTomato+/Lin- cells (Figure 3.24). We generated two scRNA-seq datasets from Hic1CreERT2/CreERT2; Rosa26hSS2/tdTomato; Ctnnb1+/Ex3Fl calvarial tumours as well as a control Hic1CreERT2;Rosa26tdTomato bone MP dataset. We then combined these with the previously described Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tumour scRNA-Seq datasets and control tdTomato+ tongue MP dataset into one aggregate dataset. 71 Figure 3.24 FACS strategy for neoplastic cells from b-catenin-stabilized tumours. Cells sorted from calvarial tumours. Neoplastic cells express a tdTomato fluorescent reporter. Lineage markers are labelled by APC-conjugated antibodies. Cells were gated sequentially from A-E. A. Selection based on cell size. B. Selection of singlets. C. Selection of living tdTomato+ cells (tdTomato+/PI-). D. Selection of mononucleated, Lin- events. E. Selection of tdTomato+/Lin- cells (P5). 72 Using Loupe Cell Browser, we performed K-means clustering and analyzed the clusters for the fluorescent markers (GFP, tdTomato), neural genes (Gbx2, Zic5, Tle1, and Epha4) and WNT pathway components (Fzd1, Fzd10, Wnt5a, and Axin2) previously analyzed in the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tumours. We also assessed the tumours from the b-catenin model for differential gene expression compared to the original model. As expected, the b-catenin tumours expressed both GFP and tdTomato (Figure 3.25B), as well as the neural genes (Figure 3.25C) and WNT pathway components (Figure 3.25D) that are upregulated in the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 model. However, in contrast to the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tumours, the b-catenin tumours expressed several genes associated with bone including osteocrin (Ostn), alkaline phosphatase (Alpl), and bone morphogenetic protein 3 (Bmp3) (Figure 3.25D). Upregulation of these genes in the b-catenin-stabilized tumours and not in the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tumours likely reflects the periosteal MP subpopulation from which they arise, as opposed to the soft-tissue MPs in the tongue and stifle which give rise to the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 tumours. 73 Figure 3.25 scRNA-Seq analysis of Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ Tumours. Each dot represents a single cell. In B-E the coloured dots indicate presence of the desired 74 transcript while grey dots indicate no expression of the transcript. A. scRNA-Seq analysis of Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ endpoint calvarial tumours, Hic1CreERT2; Rosa26hSS2 tumours at various time points including 4 wki (tongue), 8 wki (tongue), and 16 wki (tongue and stifle), control tongue MPs, and control bone MPs. B. Tumour cells isolated from Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ animals express tdTomato and GFP fluorescent markers. C. Neoplastic cells from Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ tumours also gain expression of the neural genes Gbx2, Zic5, Tle1, and Epha4. D. Neoplastic cells from Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ tumours also gain expression of WNT pathway genes Fzd1, Fzd10, Wnt5a, and Axin2. E. In contrast to Hic1CreERT2; Rosa26hSS2 tumours, b-catenin-stabilized tumours also express bone genes Ostn, Alpl, and Bmp3. This model shows that activation of WNT/b-catenin signaling in the context of SS18-SSX expression leads to a far more aggressive neoplastic phenotype, with a shorter latency period and increased tumour burden. In addition, it alters the location of tumour development, shifting tumours from soft tissue locations (tongue) to periosteal locations, and leads to a modified transcriptional profile likely reflective of the cell of origin. Taken together, these results support a role for WNT/b-catenin signaling in the pathogenesis of synovial sarcoma. 3.6 Effects of loss of Smarcb1 in Hic1CreERT2; Rosa26hSS2 mouse model To assess the effects of loss of Smarcb1 in the context of SS18-SSX expression, we modified the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 synovial sarcoma model with the addition of a conditional Smarcb1 knockout allele. We generated mice with homozygous Smarcb1 loss (Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl) and heterozygous Smarcb1 loss 75 (Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/+) and all animals were treated with TAM for five consecutive days beginning at 8 weeks of age. All animals with biallelic Smarcb1 loss (n=32) developed macroscopic tumours with a mean latency period of 12.4 ± 4.8 weeks and all animals with monoallelic Smarcb1 loss (n=42) developed macroscopic tumours with a mean latency period of 16.7 ± 2.7 weeks (Figure 3.26A). Biallelic Smarcb1 loss resulted in a significantly shorter latency period compared to the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals with wildtype Smarcb1 (p<0.05), whereas monoallelic Smarcb1 loss did not cause a significant change in latency period (p>0.05). In addition, animals with biallelic Smarcb1 loss presented with very large soft-tissue masses that were not seen in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals and only occasionally presented with tongue tumours (Figure 3.26B). Animals with monoallelic Smarcb1 loss showed the same clinical presentation as Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals, with every animal developing a tongue tumour and small tumours in the connective tissue around the stifle. 76 Figure 3.26 Synovial sarcoma model with loss of Smarcb1. A. Kaplan-Meier survival plot of Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl, Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/+, and Hic1CreERT2; Rosa26hSS2/hSS2 mice, and Hic1CreERT2 controls. Animals with biallelic loss of Smarcb1 develop tumours with a significantly shorter latency period than animals with wildtype Smarcb1 (p<0.05), whereas animals with monoallelic loss of Smarcb1 show no change in latency period (p>0.05). Control mice without SS18-SSX expression do not develop tumours. B. Whole mount image of a typical large mass on the external thoracic wall of an animal with biallelic Smarcb1 loss. Next, we analyzed the Smarcb1-deficient tumours histologically with H&E staining to assess cellular morphology and IF staining to assess GFP expression. As seen in the Hic1CreERT2; Rosa26hSS2 model, examination of H&E-stained tumour samples revealed cellular morphology consistent with synovial sarcoma, with some tumours exhibiting the monophasic histologic 77 subtype, with spindle cell/mesenchymal cellular morphology, and some tumours exhibiting the biphasic histologic subtype, containing both mesenchymal and epithelial cellular morphology (Figure 3.27). We then performed immunofluorescent analysis of tumours using antibodies for CD31 (vessels), laminin (extracellular matrix), and GFP; as expected, all neoplastic cells in the tumours were GFP+, confirming expression of SS18-SSX (Figure 3.28). Figure 3.27 Histologic analysis of Smarcb1-deficient synovial sarcoma tumours. Hematoxylin and eosin stain showing characteristic biphasic and monophasic histologic subtypes A, B. Biphasic histologic subtype with gland-like epithelial structures in a thoracic wall mass. C, D. Monophasic histologic subtype with uniform spindle cell morphology in a tongue tumour with no epithelial components evident (FFPE samples). Scale bars: A, C = 100µm, B, D = 50µm. 78 Figure 3.28 Immunofluorescent analysis of Smarcb1-deficient synovial sarcoma tumours. Immunofluorescent staining of a Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl synovial sarcoma tumour. A. Composite image with DAPI, GFP, CD31, and laminin staining. B. DAPI nuclear stain. C. Anti-CD31 antibody staining labelling endothelium. D. Anti-GFP antibody staining of SS18-SSX-expressing cells. E. Anti-Laminin antibody staining labelling extracellular matrix. Scale bars = 50µm. In contrast to the models with wildtype Smarcb1, one animal with biallelic Smarcb1 loss was found to have macroscopic pulmonary lesions upon dissection. These lesions were nodular and peripherally located, which suggests that they represent metastasis of the primary tumour (Figure 3.29A). To determine the frequency of microscopic pulmonary lesions, we examined serial histologic lung sections from Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl animals (n=20). Microscopic pulmonary lesions were found in 65% of examined animals (Figure 3.29B). 79 Figure 3.29 Pulmonary lesions in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl mice. A. Whole mount bright field image of macroscopic pulmonary lesions. B. Pie chart showing proportion of examined Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl animals with pulmonary lesions at clinical endpoint (n=20). We evaluated the macroscopic and microscopic pulmonary lesions histologically, using immunofluorescent staining and H&E staining. We performed immunofluorescent staining using antibodies for CD31 (vessels), laminin (extracellular matrix), and GFP. The macroscopic pulmonary lesions were circular and located in the periphery of the lung (Figure 3.30A, C), while the microscopic pulmonary lesions were characterized by clusters of GFP+ cells found in perivascular and peribronchial locations (Figure 3.30B, C). The morphology of the microscopic lesions again suggests that they may represent additional primary lesions as opposed to metastatic lesions. 80 81 Figure 3.30 Histologic analysis of pulmonary lesions in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl mice. A. Immunofluorescent image of peripherally-located macroscopic pulmonary lesion. (a) Composite image with DAPI, GFP, CD31, and laminin staining showing a nodular mass of GFP+ cells. an irregular cluster of perivascular GFP+ cells. (b) DAPI nuclear stain. (c) Anti-CD31 antibody staining labelling endothelium. (d) Anti-Laminin antibody staining labelling extracellular matrix. (e) Anti-GFP antibody staining of SS18-SSX-expressing cells. B. Immunofluorescent image of a perivascular/peribronchial microscopic pulmonary lesion. (a’) Composite image with DAPI, GFP, CD31, and laminin staining showing an irregular cluster of perivascular/peribronchial GFP+ cells. (b’) DAPI nuclear stain. (c’) Anti-CD31 antibody staining labelling endothelium. (d’) Anti-Laminin antibody staining labelling extracellular matrix. (e’) Anti-GFP antibody staining of SS18-SSX-expressing cells. C. Hematoxylin and Eosin staining of pulmonary lesions. (a”) Peripherally-located macroscopic pulmonary lesion. (b”) Perivascular pulmonary lesion. Scale bars = 50 µm. These results show that loss of Smarcb1 in the context of SS18-SSX expression in Hic1+ MPs causes the development of tumours which are histologically consistent with the synovial sarcoma tumours seen in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2 animals. Biallelic Smarcb1 loss results in a shorter latency period and may also confer a higher metastatic potential compared to SS18-SSX expression alone, while monoallelic loss of Smarcb1 has no effect on the timing or location of tumour development. 82 Chapter 4: Discussion 4.1 Hic1+ MPs are a cell of origin for synovial sarcoma While the causative mutation underlying SS has been known for decades and several driving mechanisms have been proposed for the development of SS, identification of the cell of origin has remained elusive. There is evidence for neural crest cell, mesenchymal stem/progenitor cell, and myogenic origins10,11,56,64,69,70, with the strongest in vivo evidence suggesting an MP cell of origin in the Prx1-CreERT2/hSS2 model56. The first mouse model of SS used a Myf5-Cre driver to target myogenic cells and resulted in the development of tumours with 100% penetrance64, although the broad expression of Myf5 early in embryogenesis means that these tumours may not in fact be arising from myogenic cells. Here we show that expression of SS18-SSX in Hic1+ MPs in a genetically-engineered mouse model leads to formation of tumours with 100% penetrance and that these tumours are histologically, immunophenotypically, and transcriptionally consistent with SS, thus demonstrating that Hic1+ MPs are a cell of origin for SS. While Hic1+ MPs comprise both FAPs and pericytes, SS appear to arise specifically from the FAP subpopulation as they maintain an FAP-like expression profile (Pdgfra+/Rgs5-). These findings are consistent with previous work showing that SS cell lines differentiate into adipocytes, osteocytes, and chondrocytes upon silencing of SS18-SSX10, suggesting reversion to a cellular identity consistent with Hic1+ FAPs, not pericytes. It is not clear why the tumours only arise from Hic1+ FAPs, not Hic1+ pericytes, since SS18-SSX is expressed in both cell populations after TAM treatment. Furthermore, each animal develops only a small number of tumours and in specific locations, specifically the tongue, despite the ubiquitous distribution of Hic1+ MPs throughout the body. It is likely that SS18-SSX requires a specific cell context in 83 order to successfully drive formation of SS, and that only Hic1+ FAPs in certain tissues are permissive of SS18-SSX-driven neoplastic transformation. While the tongue is an unusual location for tumour formation, the head and neck regions are relatively common locations for SS in humans3 and several case reports describe SS in the tongue85-87. One possible explanation for the propensity for SS development in the tongue in this model is that the Hic1+ cells in the tongue are inherently different than Hic1+ cells elsewhere. We did find that the tongue had a higher proportion of off-vessel Hic1+ cells compared to other tissues, which may suggest a slightly different phenotype or level of activation among Hic1+ cells in this tissue. Alternatively, it may be an effect of timing if the Hic1+ cells in the tongue are at a different developmental stage than other Hic1+ cells at the time of TAM injection. Comparison of transcriptomic profiles between Hic1+ cells in the tongue and other tissues would help clarify any underlying differences between these cells. In addition to the tongue tumours, the presence of small tumours in the fascia surrounding the stifle joint in all Hic1CreERT2; Rosa26hSS2 mice effectively recapitulates human SS, as the most common location for human SS is near the knee joint. The accuracy of the model is further supported by the histology and immunophenotype of the murine tumours; all tumours displayed either monophasic histologic morphology, with tightly-packed spindle cells with uniform nuclei, or biphasic histologic morphology, with both spindle cell components and epithelial, gland-like components. We examined the mouse tumours for expression of TLE1 and MUC1 and found that they do express both of these common markers of SS, and we also found that while Cd34 is 84 expressed in Hic1+ MPs, its expression is lost upon neoplastic transformation, which is consistent with the finding that SS is typically CD34 negative3. Synovial sarcoma is known to have a unique transcriptomic profile characterized by the expression of neural genes involved in embryogenesis and development, and it has been hypothesized that this is reflective of a neural crest cell of origin for SS. Previous work in our lab has shown that embryonic Hic1+ cells show some similarities with NCCs, including spatial distribution and expression of some NCC markers, however they are not wholly consistent with NCCs as they do not express NCC markers Sox10, Wnt1, and Foxd3, thus the exact embryonic origin of Hic1+ cells is unclear (unpublished data). Here we show that consistent with human SS data, the Hic1CreERT2; Rosa26hSS2 tumours express several neurodevelopmental genes including Gbx2, Tle1, Zic5, and Epha4. These genes are not expressed in Hic1+ MPs prior to the induction of SS18-SSX expression, thus their expression is induced at some point during SS18-SSX-driven neoplastic transformation. One hypothesis is that these genes are bivalently marked with both H3K27me3 and H3K4me3 histone marks in Hic1+ MPs and that the expression of SS18-SSX induces epigenetic changes resulting in the loss of H3K27me3 and de-repression of these loci, essentially reverting the cells to a more embryonic-like phenotype. Histone ChIP-Seq of both wildtype Hic1+ cells and Hic1CreERT2; Rosa26hSS2 tumour cells could be used to test this hypothesis, and further work to elucidate the embryonic origins of Hic1+ cells would clarify the significance of the neurodevelopmental signature seen in these tumours. 85 Although SS is known to be a genetically stable neoplasm with a low rate of secondary mutations24,61-63, it would be worthwhile to evaluate the murine tumours in this model for the presence of any additional mutations that may contribute to sarcomagenesis. In a separate study we found that a subset of Hic1CreERT2; Rosa26hSS2 tumours have a chromosome 6 copy number gain88 and hence amplification of the hSS2 allele, which likely contributes to SS18-SSX-driven sarcoma development. This finding was not unexpected as copy number variation of chromosome 6 was previously shown to occur in the Myf5-Cre/hSS1 and Myf5-Cre/hSS2 models as well68. We also show that Hic1CreERT2; Rosa26hSS2 tumours express WNT pathway components including Fzd1, Fzd10, Wnt5a, and Axin2 at a higher level than in Hic1+ MPs prior to neoplastic transformation, which is again consistent with previous findings in human SS40,43,50. This finding suggests that as in human SS, activated WNT/b-catenin signaling plays a role in the pathogenesis of tumour development in this model, however further experiments will be required to elucidate the mechanisms underlying this pathway activation. One of the most valuable aspects of this model is the temporal and spatial consistency with which the tumours arise. Not only were we able to consistently identify early tumours at 4 wki, we were able to isolate these cells for scRNA-Seq analysis and demonstrate altered levels of expression of neural and WNT pathway genes compared to more advanced tumours, suggesting a step-wise process of gene expression during the development of SS. The ability to analyze early tumours in comparison to advanced tumours is unique to this model among previously described murine models of SS and should allow us to clarify what occurs at transcriptional and 86 epigenetic levels early in sarcomagenesis. Another unique advantage of this model is that it enables direct comparison between tumours and normal tissues; since the tumours arise in Hic1+ MPs, wildtype Hic1+ MPs are an ideal control with which we can accurately assess the changes that occur to give rise to SS. 4.2 Activated WNT/b-catenin signaling potentiates synovial sarcomagenesis To further assess the effect of activated WNT/b-catenin signaling in SS, we added a stabilized b-catenin allele to our model to generate Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ animals. Previous work using a similar strategy showed that addition of stabilized b-catenin in the presence of the hSS2 allele generates highly invasive periosteal tumours with a shorter latency period compared to animals with wildtype b-catenin55,56. In one such model, addition of stabilized b-catenin was able to induce tumour formation in preosteoblasts with 100% penetrance, whereas hSS2 expression alone did not result in tumour formation using the same Cre driver, suggesting that activation of WNT/b-catenin signaling can have a powerful effect on SS18-SSX-mediated cellular transformation56. Unsurprisingly, our results were comparable, with all animals developing multiple, invasive periosteal tumours with a mean latency period of only 6.3 wki, compared to 16.2 wki in the Hic1CreERT2; Rosa26hSS2 model. Single-cell RNA-Seq analysis of Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ tumours showed that in addition to expression of neural genes and WNT pathway components, tumours with stabilized b-catenin also expressed markers associated with bone including Ostn, Alpl, and Bmp3. It is likely that expression of these genes is reflective of the periosteal subpopulation of 87 Hic1+ MPs that give rise to the tumours in this model, although it is not clear why stabilization of b-catenin results in a shift in tumour development from soft tissue locations to an exclusively periosteal location. One possibility is that activated WNT/b-catenin signaling causes a more “activated” phenotype and/or enhances endogenous WNT signaling in periosteal Hic1+ MPs, making them more permissive to SS18-SSX-induced neoplastic transformation. Indeed, canonical WNT/b-catenin signaling plays a central role in bone development and maintenance89, and stabilization of b-catenin has been shown to cause osteoclast and osteoblast dysfunction and result in increased deposition of bone90,91. Further research comparing wildtype periosteal Hic1+ MPs and periosteal Hic1+ MPs with stabilized b-catenin would help highlight the fundamental effects of stabilized b-catenin on this cell population and the changes that occur to make them permissible to neoplastic transformation. Overall, this model shows that WNT/b-catenin signaling plays an important role in development of SS, driving faster tumour development and local tissue invasion. While the effects of WNT/b-catenin signaling in SS are still unclear, further study of this model will provide a more complete understanding of this pathway in SS. 4.3 Biallelic loss of Smarcb1 potentiates synovial sarcomagenesis In vitro studies in SS cell lines suggests that SS18-SSX disrupts BAF complexes by displacing the SMARCB1 subunit from the complex resulting in its degradation and altered BAF complex targeting23. In addition, SMARCB1 is a well-known tumour suppressor: in humans, biallelic SMARCB1 mutations are associated with rhabdoid tumours, an aggressive, childhood neoplasm 88 that arises in kidneys and the central nervous system92,93, and epithelioid sarcoma, a high-grade soft-tissue sarcoma that affects adolescents and young adults94. Mice with biallelic loss of Smarcb1 develop lymphoma or rhabdoid tumours with 100% penetrance84, and a subset of mice with monoallelic Smarcb1 loss develop rhabdoid tumours95. To assess the role of SMARCB1 in SS in an in vivo setting, we incorporated a conditional Smarcb1 null allele in the Hic1CreERT2; Rosa26hSS2 SS model and found that biallelic loss of Smarcb1 resulted in a decreased latency period and enhanced SS tumour formation in altered locations, whereas monoallelic Smarcb1 loss had no effect. These results are interesting because in theory, one would expect that if SS18-SSX displaces SMARCB1 and leads to its degradation, genetic loss of Smarcb1 should have no effect on tumourigenicity. Furthermore, if SS18-SSX acts in a competitive manner to eject SMARCB1 from BAF complexes, monoallelic loss of Smarcb1 should result in enhanced tumour formation by decreasing the concentration of SMARCB1 by 50%. Thus, these results suggest that the potentiation of SS tumour formation with biallelic loss of Smarcb1 seen in this model occurs through a mechanism other than strictly SS18-SSX-mediated loss of SMARCB1 from BAF complexes. Smarcb1 loss has been shown to have many effects, including activation of canonical WNT signaling96 and the sonic hedgehog pathway97, therefore it is likely that one or a combination of other mechanisms are at play in Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl mice. 89 Another interesting finding in the Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl model was that one mouse was found to have macroscopic pulmonary lesions, likely metastases from the primary tumour, something which was not seen in any Hic1CreERT2; Rosa26hSS2 mice. It is therefore possible that loss of Smarcb1 also confers a higher metastatic potential in this model, perhaps through the same mechanisms that enhance primary tumour formation. Clearly, a larger sample size is needed to confirm whether this is a repeatable finding or whether it was an isolated event in this particular animal. Further experiments are necessary both to evaluate the metastatic potential in this model and also to elucidate the mechanisms underlying SS formation in the context of Smarcb1 loss – scRNA-Seq of primary tumours and metastatic lesions from this model would allow better understanding of the pathways involved, and comparison with data from Hic1CreERT2; Rosa26hSS2 tumours would help clarify the effects of Smarcb1 loss. In addition to the one animal found with macroscopic pulmonary lesions, over fifty percent of examined Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl mice had microscopic pulmonary lesions, characterized by clusters of peribronchial and/or perivascular GFP+ cells. These lesions likely represent additional primary tumours, as they are not in the peripheral locations typical of metastases. One Hic1CreERT2; Rosa26hSS2 animal also had microscopic pulmonary lesions with a similar distribution, although again a larger sample size is needed to accurately assess the presence of lung lesions in this model. Additional experiments are needed to confirm whether the macroscopic and microscopic pulmonary lesions in the Hic1CreERT2; Rosa26hSS2 and Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl models are primary lesions or metastases. One possible approach is to employ a 90 cellular barcoding technique where next-generation sequencing can be used to determine whether the pulmonary lesions arise from the same clone as the primary tumours or whether they are unique clones. Both models could be modified to include a Cre recombinase-based endogenous barcoding technique, such as Polylox98, where each Cre-expressing cell can be labelled with a unique DNA barcode. Alternatively, a retroviral technique99 could be used where a retroviral barcode library is injected in the developing primary tumour, however this technique would only be suitable for the Hic1CreERT2; Rosa26hSS2 model where the location and timing of the primary tumour is consistent. 4.4 Preclinical applications of Hic1CreERT2; Rosa26hSS2 mouse model The Hic1CreERT2; Rosa26hSS2 model is not only useful for identifying the mechanisms underlying the development of SS, it is also a valuable tool for evaluating potential therapeutics. The remarkable temporal consistency of the model is ideal for preclinical drug testing, as the animals can be treated prior to their known humane endpoint (16 wki) and overall survival can be used as an outcome measure. Currently, xenograft models are often used for preclinical evaluation of therapeutics for SS; while this approach still provides useful information, an animal model with tumour development from endogenous expression of SS18-SSX provides a far more accurate representation of human disease and allows evaluation of drugs in the context of a functioning immune system. Many potential therapeutics are being investigated for efficacy in SS, including anti-FZD10 antibodies and other WNT pathway inhibitors, EZH2 inhibitors, HDAC inhibitors, and tyrosine kinase inhibitors, and the Hic1CreERT2; Rosa26hSS2 model could be used for further testing for a 91 wide variety of these agents. In particular, several in vitro and in vivo studies have identified HDAC inhibitors and proteasome inhibitors as potentially useful therapeutics for SS32,34,100-102, and this model is an ideal platform to further test the efficacy of these drugs. One drawback to this model is that the development of tumours in the tongue means that the animals reach humane endpoint as soon as a macroscopic tumour is present, as the animals are unable to eat. Thus, treatment must be initiated prior to the development of macroscopic disease, a scenario which would not occur in human disease. This model is also not useful for evaluating treatment of metastatic disease as we do not know if the animals develop metastatic lesions. 4.5 Conclusion 4.5.1 Strengths and limitations The main strength of this work is the successful development of an accurate animal model of SS and the identification of a cell of origin for SS. A known cell of origin is profoundly important for a complete understanding of neoplastic disease since it allows us to compare the normal, untransformed cells with the neoplastic cells and identify the changes that occur to drive oncogenesis. This model has a well-defined cell of origin – Hic1+ MPs – which has been extensively studied by the Underhill Laboratory and whose properties are well known. The remarkable consistency of the Hic1CreERT2; Rosa26hSS2 model, both in tumour location and latency period, and the ability to identify small cell populations early after induction of SS18-SSX expression is another tremendous strength of this work as any alterations in tumour development, as well as early events in tumourigenesis, can be accurately assessed. 92 In this work we used several robust approaches to evaluate the three models including histology, immunofluorescence, and scRNA-Seq. Furthermore, we not only characterized the tumours at clinical endpoint, but also throughout tumour development from as early as 4 wki. The Hic1CreERT2; Rosa26hSS2/tdTomato; Ctnnb1Ex3Fl/+ and Hic1CreERT2/CreERT2; Rosa26hSS2/hSS2; Smarcb1Fl/Fl models highlight our ability to easily modify the Hic1CreERT2; Rosa26hSS2 model through the addition of other genetic perturbations and also provide insight into the mechanisms underlying synovial sarcomagenesis. In addition to the various limitations of the model discussed above in other sections, one notable limitation of this work is the scRNA-Seq data analysis. All scRNA-Seq data was evaluated qualitatively at a superficial level by visual inspection of t-sne plots. A more thorough analysis of the scRNA-Seq data is warranted, including quantitative comparison of expression levels of genes of interest, and would provide a deeper understanding of the observed changes. 4.5.2 Future directions In addition to the future research mentioned above and the preclinical avenues discussed, there are several possible alterations to the Hic1CreERT2; Rosa26hSS2 model that may further elucidate the pathogenesis of SS. In this model we have shown that SS likely arises from Pdgfra+ FAPs; to confirm this, we could generate PdgfraCreERT2;Rosa26hSS2 animals and compare their phenotype to Hic1CreERT2; Rosa26hSS2 animals. For further evaluation of the role of WNT signaling in SS, a conditional null allele for Wntless103 – a protein necessary for WNT signaling – could be added to the Hic1CreERT2; Rosa26hSS2 model. Alternatively, Hic1CreERT2; Rosa26hSS2 animals could be treated with small-molecule WNT inhibitors after induction of SS18-SSX expression. 93 Given the various epigenetic perturbations that are believed to drive synovial sarcomagenesis, evaluation of histone marks by ChIP-Seq and chromatin accessibility by ATAC-Seq in both normal Hic1+ MPs and Hic1CreERT2; Rosa26hSS2 tumour cells should certainly be performed. This will complement previous work done in SS cell lines and could potentially identify additional epigenetic targets for treatment of SS. Finally, functional gene analysis using the Hic1CreERT2; Rosa26hSS2 model would also identify possible treatment targets for SS. Several different approaches could be used including shRNA or CRISPR/Cas9 screens. One possible experimental design would be to transfect Hic1CreERT2; Rosa26hSS2 MPs prior to expression of SS18-SSX and evaluate which cells are subsequently unable to undergo neoplastic transformation upon treatment with TAM. Alternatively, SS18-SSX-expressing Hic1CreERT2; Rosa26hSS2 neoplastic cells could be isolated, transfected and then evaluated for apoptosis and/or growth arrest. Overall, we have described the successful generation of a unique mouse model of SS which effectively recapitulates human disease. We have also generated two modified versions of this model: one to evaluate the role of activated WNT/b-catenin signaling in SS and another to study the effect of loss of the core BAF complex subunit SMARCB1. Of the three models, the original Hic1CreERT2; Rosa26hSS2 model provides the simplest and most robust platform for further study of the mechanisms underlying SS. The consistency of this model is unique among previously generated mouse models of SS – indeed among most animal models of cancer – and gives us the opportunity to compare neoplastic SS cells with their wildtype precursors. 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Cell and Developmental Biology
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Generation and characterization of a novel mouse model of synovial sarcoma
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