Initiation approximation: synovial sarcoma model optimization

Synovial sarcoma is a malignant soft tissue tumor that presents with a chromosomal translocation resulting in abnormal fusion genes, SS18-SSX1 or SS18-SSX2 in most cases. This mutation is known as a somatic mutation because it forms in some cells of the body during the individual's lifetime and thereby it is not inherited. Also, different molecular biological signaling including PI3' lipid signaling have been associated in the progression of synovial sarcoma. Synovial sarcoma affects both young adults and adolescents, and occurs more frequently in males than females. The incidence of synovial sarcoma in a year is estimated to be 900 cases in the United States. Furthermore, synovial sarcoma can spread easily to other parts of the body through blood circulation, and sometimes through lymph nodes. Metastasis is when the cells can break away from the primary tumor and travel to the bloodstream, and when synovial sarcoma becomes metastatic it is commonly found in the lung. Additionally, the cell of origin of synovial sarcoma is poorly understood, and there has been no therapeutic improvement recently. Here we describe a pre-clinical model that showed predictable location of the tumor. Genetically engineered mouse models homozygous for hSS2 (SS18-SSX2), one of the 9 variants of the translocations (SSX1-9), has been used to observe and test synovial sarcoma tumorigenesis. In this project, TATCre injections initiate tumor formation as a result of hSS2 expression in one allele at the Rosa26 locus. Our hypothesis was investigating whether the loss of Snf5 gene would increase the rate of tumorigenesis along with TATCre protein injection in mice. The measurements of the tumorigenesis volume rate in SSM2 +/+; Snf5 fl/fl supported our hypothesis, demonstrating that the loss of Snf5 in mice iii along with TATCre injection does increase the rate of tumorigenesis. However, having one copy of the Snf5 does not cause a significant difference in increasing the rate of tumorigenesis. After each injection, the volume of each tumor was measured. Later, mouse models were sacrificed to harvest tumors and search for metastases in other relevant organs. Using this model, we have generated a localized and short latency model of synovial sarcoma. This model will serve as a great pre-clinical model to design and evaluate new therapeutic methods.

INITIATION APPROXIMATION: SYNOVIAL SARCOMA MODEL OPTIMIZATION By Asal Kareem A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biology Approved: ______________________________ Kevin B. Jones, MD Thesis Faculty Supervisor _____________________________ M. Denise Dearing, Ph.D. Chair, Department of Biology _______________________________ Michael Bastiani, Ph.D Honors Faculty Advisor _____________________________ Sylvia D. Torti, PhD Dean, Honors College December 2019 Copyright © 2019 All Rights Reserved ABSTRACT Synovial sarcoma is a malignant soft tissue tumor that presents with a chromosomal translocation resulting in abnormal fusion genes, SS18-SSX1 or SS18-SSX2 in most cases. This mutation is known as a somatic mutation because it forms in some cells of the body during the individual’s lifetime and thereby it is not inherited. Also, different molecular biological signaling including PI3’ lipid signaling have been associated in the progression of synovial sarcoma. Synovial sarcoma affects both young adults and adolescents, and occurs more frequently in males than females. The incidence of synovial sarcoma in a year is estimated to be 900 cases in the United States. Furthermore, synovial sarcoma can spread easily to other parts of the body through blood circulation, and sometimes through lymph nodes. Metastasis is when the cells can break away from the primary tumor and travel to the bloodstream, and when synovial sarcoma becomes metastatic it is commonly found in the lung. Additionally, the cell of origin of synovial sarcoma is poorly understood, and there has been no therapeutic improvement recently. Here we describe a pre-clinical model that showed predictable location of the tumor. Genetically engineered mouse models homozygous for hSS2 (SS18-SSX2), one of the 9 variants of the translocations (SSX1-9), has been used to observe and test synovial sarcoma tumorigenesis. In this project, TATCre injections initiate tumor formation as a result of hSS2 expression in one allele at the Rosa26 locus. Our hypothesis was investigating whether the loss of Snf5 gene would increase the rate of tumorigenesis along with TATCre protein injection in mice. The measurements of the tumorigenesis volume rate in SSM2 +/+; Snf5 fl/fl supported our hypothesis, demonstrating that the loss of Snf5 in mice ii along with TATCre injection does increase the rate of tumorigenesis. However, having one copy of the Snf5 does not cause a significant difference in increasing the rate of tumorigenesis. After each injection, the volume of each tumor was measured. Later, mouse models were sacrificed to harvest tumors and search for metastases in other relevant organs. Using this model, we have generated a localized and short latency model of synovial sarcoma. This model will serve as a great pre-clinical model to design and evaluate new therapeutic methods. iii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 13 RESULTS 16 DISCUSSION 21 REFERENCES 26 iv INTRODUCTION Synovial sarcoma, also known as a malignant synovioma, is a soft tissue sarcoma that is a rare and aggressive form of cancer (Herzog, 2005). Sarcomas arise from the mesenchyme tissue and can appear in any region in the body (Figure 1) (Genadry et al., 2018). Synovial sarcoma arises from soft tissues near the joints and rarely within the joints (Abbasi et al., 2016). Regardless of its name and location near the joints, synovial sarcoma has no relationship or connection with the synovial membrane (de Necochea-Campion et al., 2017). The synovial membrane, also referred to as the synovium, is connective tissue that lines the inner surface of the joint and where the synovial fluid is produced (Vincent, 1960). Hence the name of this type of sarcoma cancer is a misnomer (Miettinen et al., 1984). Synovial sarcomas most commonly occur in the arms, thighs, knees, and near the joints such as the wrist or ankle, and lungs are common metastatic site for synovial sarcoma (Abbasi et al., 2016). Additionally, synovial sarcoma account for the most common malignant sarcoma of the foot (Latt et al, 2010). Synovial sarcoma occurs at any age, but it is seen more often in adolescents and young adults aged 15-35 (Hale et al., 2019). The annual incidence rate of synovial sarcoma is 2-3 per 100,000, which accounts for 1% of all malignant tumors and 2% of all cancer-related deaths (Ferrari et al., 2011). In addition, synovial sarcoma represents 10% of all soft tissue sarcomas (Ferrari et al., 2011). Also, synovial sarcoma affects more males than females (12 males for every 10 females) (Murphey et al., 2007). The overall prognosis of synovial sarcoma is poor, whereas the 5-year survival rate is 50% and the 10-year survival rate is 25% (National Cancer Institute, 2019). According to the Sarcoma Foundation of America, the poor prognostic factors include:  Distant metastasis  Age older than 25 years  Tumor size greater than 5 cm  Poorly differentiated area seen in histology Thus, the survival rate of synovial sarcoma depends on the prognosis factors that varies between each patient (National Cancer Institute, 2019). Figure 1. Regions of soft tissue sarcoma in the human body. Reprinted from “Soft Tissue Sarcoma Overview,” by Dana -Farber Cancer Institute. 2 According to research studies, 90% of synovial sarcoma cases have been associated with a chromosomal translocation. It is characterized by translocation of chromosome X and chromosome 18 that results in fusion of the SS18 (formerly known as SYT) gene to SSX genes, which produces an SS18-SSX oncogene (Turc-Carel et al., 1987; Clark et al., 1994; de Leeuw et al., 1995). Genes in the SSX gene family are predicted to function as a transcriptional repressors (Jones et al., 2016). Whereas, SS18 gene is a member of the mammalian SWI/SNF complex (Jones et al., 2016). Epigenetic analyses inferred that SS18-SSX fusion oncoproteins serve as regulators of transcription in synovial sarcoma (Jones et al., 2016). These analyses predicted that SS18-SSX fusion genes were a remarkable driver in synovial sarcomagenesis (Sandberg and Bridge, 2002). However, another study directly compared SS18-SSX2 to other sarcomagenic fusion oncogenes, EWSR1-ATF1 and ASPSCR1-TFE3 indicated that SS18-SSX2 resulted in fewer sarcomas at longer latencies. (Goodwin et al., 2014). This indicated that there is a secondary genetic factor that drives SS18-SSX2 sarcomagenesis by altering the SWI/SNF complex (Jones et al., 2016). Previous research has shown that the loss of Snf5/Smarcb1, a gene that codes for Baf47, plays a pivotal role in the pathological emergence of synovial sarcoma. (Kadoch and Crabtree, 2013). SWI/SNF complex, also known as the BAF complex, is composed of 15 subunits. The BAF complex is an ATP dependent chromatin remodeling complex that disrupts the interaction between DNA and histones (Yoo and Crabtree, 2009). This disrupts the chromatin forming a nucleosome that opens and makes the transcription binding domain more accessible, thus increasing transcription (Yoo and Crabtree, 2009). In most cases, SS18 fuses with SSX1gene (SS18-SSX1), or with SSX2 gene (SS18-SSX2), and rarely SS18 fuses with SSX4 gene (Amary et 3 al., 2007). Hence, a fusion oncogene (SS18-SSX2) will contribute into the development of synovial sarcoma (Xing et al., 2018). The oncogenic potential of SS18-SSX1 and SS18-SSX2 was investigated by generating a mouse allele that conditionally expressed the SS18-SSX1 cDNA from the Rosa26 locus (described as hss1) as shown figure 2a (Jones et al., 2016). The hss1 allele matched the hss2 allele for SS18-SSX2 by using identical primers to capture the coding sequence and the early 3’ untranslated region (Jones et al., 2016). From each model, mouse embryonic fibroblasts (MEF) isolated and exposed in culture to TATCre (Fig. 2b). Additionally, both hSS1 and hSS2 damaged embryogenesis following expression induced by Hprt-Cre (Fig. 2d) (Jones et al., 2016). Thus, hSS1 and hSS2 were equally conditional and toxic to cells. Furthermore, mice were bred in the Myf5-Cre lineage to activate hss1. Based on the green florescent protein (GFP), tumors appeared and expressed the fusion gene as shown in figure 2a and 2d (Jones et al., 2016). MSS and BSS of human synovial sarcoma and hss1 and hss2 derived tumors were also compared and demonstrated histological types (Fig. 2f) (Jones et al., 2016). Furthermore, mice groups of hss1 and hss2 were each induced by Myf5-Cre, Rosa26CreER, or TATCre protein. In each model, hss1 was expressed less frequently than hss2 in tumor samples (Jones et al., 2016). The genetic independence of each fusion oncogene was tested by sequencing Myf5-Cre-initiated tumor exomes and were compared to the germline control group (Jones et al., 2016). Results of the exome sequencing showed that allele fraction and gene transcription levels derived synovial sarcomagenesis (Jones et al., 2016).The additional genetic change that was observed beyond the activation of the hss1 and hss2 fusion gene, was the amplification of chromosome 6 (Jones et al., 2016). 4 Additional secondary genetic factors that have been correlated in the human synovial sarcoma, is the loss of the phosphatase and tensin homologue on chromosome 10 (PTEN) gene (Barretina et al., 2010; Jones et al., 2016). The function of PTEN gene is explained to act as a tumor suppressor gene that regulates cell division by keeping cells from growing or dividing rapidly in uncontrolled environment. PTEN gene also function as a negative regulator of the phosphatidyl inositol 3′-lipid (PI3′-lipid) signaling, it has been demonstrated that in most cancer types, PTEN gene has been silenced or lost (Ali et al., 1999; Vivanco and Sawyers, 2002; Oda et al., 2005; Zbuk and Eng, 2007; Chalhoub and Baker, 2009; Jones et all, 2016). Mutation of the PTEN gene increases signaling of PI3′-lipid that causes activation of downstream pathways, such as phosphorylation and activation of protein kinase B (known as pAKT). Thus, leading to proliferation, cell survival, and preventing apoptosis (Tamura et al., 1998; Patel et al., 2001; Chow and Baker, 2006). Based on histopathology, synovial sarcoma is divided into three subtypes: biphasic, monophasic, and poorly differentiated (Asher, van Schalkwyk and Bali, 2011). Biphasic synovial sarcoma (BSS) as shown in figure (2f) contains epithelial and spindle cell (fibrous/mesenchymal cells) components and it accounts for 60% of tumors with large metastases (Qi et al., 2015). Whereas, monophasic synovial sarcoma (MSS) showing in figure (2f) contain spindle cells only (fibrous/mesenchymal cells) (Qi et al., 2015). MSS is less common, and presents no metastases, and accounts for 40% of tumors (de Leeuw et al., 1994; Kawai et al., 1998; Ladanyi et al., 2002; Renwick et al., 1995). MSS and BSS of human synovial sarcoma and hss1 and hss2 derived tumors were compared and demonstrated histological types (Fig. 2f) (Jones et al., 2016). 5 6 Figure 2. Comparison of synovial sarcomagenesis from S18-SSX1 and SS18-SSX2. Adapted from “The impact of chromosomal translocation locus and fusion oncogene coding sequence in synovial sarcomagenesis,” by Jones et al, 2016. In this figure, 2a shows the genetic scheme of the fusion oncogene transcripts SS18SSX2 and SS18-SSX1, which shows the insertion of the latter into a Cre-loxP that is conditionally expressed at the Rosa26 locus (Fig. 2b). Reverse transcriptase polymerase chain reaction (RTPCRs) with SS18-SSX primers, showing expression in both hSS2 and hSS1 mouse embryo fibroblast (MEFs) 16 hours after administration of the TATCre protein (Fig. 2c). Fluorescence associated flow cytometry shows increased apoptotic and dead cell fractions 72 hours after TATCre administration compared to hSS1 and hSS2 MEFs in vitro controlled conditions (Fig. 2e) GFP images of tumors in hSS1 and hSS2 Myf5Cre mice (Fig. 2f). The monophasic fibrous (MSS, Left) and biphasic (BSS, right) glandular and fibrous areas of tumors originating from human and mouse hSS1 and hSS2 are shown to be descriptive hematoxyl and eosin histopathology. (Length of the scale bars is 10 μm). 7 The poorly differentiated synovial sarcoma showing in figure (3) shares features of both monophasic and biphasic sarcoma, but it lack the spindle cells that has been found in both monophasic and biphasic synovial sarcoma (Rijn, et al. 1999). Also, the poorly differentiated synovial sarcoma comprises small round cells that are similar to Ewing’s sarcoma. All of the synovial sarcoma subtypes are associated with chromosomal translocation (Rijn, et al. 1999). Figure 3. Histology image from a piece of cutaneous biopsy displaying the poorly differentiated synovial sarcoma. Adapted from “Poorly differentiated synovial sarcoma in the wrist - Case report,” by Maia, D., Menezes, C., Bastos, T., Ferreira, L. and Francesconi, F. 8 Research studies show that, despite an increased knowledge of potentially targeting biolo gy in synovial sarcoma, there has been no clinical advancement of therapeutic treatment options over the last two decades (Weitz, 2003). While it is the most common soft-tissue sarcoma in the adolescent and young adult population, synovial sarcoma is rare enough that no single center develops a sufficient case load to qualify for synovial-sarcoma-specific research. This places greater emphasis on validation of every treatment strategy before the clinical trial, so that any therapy progressing towards the clinical trial stage will have promise and priority. However, effective preclinical animal models are also lacking. The features of an ideal pre-clinical animal model include the efficient formation of a tumor which corresponds histologically with the human disease, in relation to gene expression and location and also in anatomy and in a host animal which is healthy enough to stand imaging and drug tests. The aim of the synovial sarcoma model optimization, was to test the rate of tumorigenesis in different genotype groups based on TATCre injection administered at a different interval. Meaning that I have focused on measuring the rate of growth not the differences in growth. In this experiment, two groups of mice each with three different genotypes of interest were used, which include (Rosa26 +/+; Snf5 wt/fl, Rosa26 +/+; Snf5 fl/fl, Rosa26 +/+; Snf5 wt/wt). Group (1) mice was injected at day 8, and group (2) was injected at day 28 post birth. Both groups were injected with a TATCre (10 uL) with the same dosage into the quadriceps muscle of each mouse in the targeted genotypes. Further details of the genetic breeding and analysis of the tumorigenesis rate are included in the methods and results sections. After observable tumorigenesis, mice were sacrificed and organs were collected for further 9 testing. Additionally tumors were sent off for histological, immunohistochemistry, and gene expression analysis. The first genetic mouse model of synovial sarcoma was developed based on the conditional expression of human SS18-SSX2, as identified by a group of researchers studying the transforming function of the fusion protein in chosen tissues (Haldar, 2007). The cell of origin for synovial sarcoma still unknown, but the occurrence of this disease has been associated with skeletal muscles that led the researchers to study the skeletal muscle lineage as a potential source of synovial sarcoma (Haldar, 2007). The chosen target cell was skeletal muscle specific Myf5 lineage. The mice formed SS that models human SS based on transcription profiling, histology, and immunohistochemistry (Haldar, 2007). To generate the target mouse lines, researchers used SS18-SSX2 cDNA generated from RNA extract of the human synovial sarcoma tumor for targeting into the mouse Rosa26 locus on chromosome 6 (Haldar, 2007). The ROSA promoter is known to be ubiquitously active, thereby allowing transcription of the fusion protein in any chosen tissue following Cre-dependent recombination (Soriano, 1999; Zambrowicz et al., 1997). Furthermore, researchers assigned two groups of mice, SSM1 and SSM2 (synovial sarcoma mouse 1 and 2 respectively) using a marker gene (IRES-EGFP) only in SSM2 (Haldar, 2007). The marker gene is used because it’s important to know where and when Cre protein is produced (Haldar, 2007).Because it was in the presence of Cre, SS18-SSX2 transcription occurs, but in the absence of Cre, did not express SS18-SSX2 (Haldar, 2007). Myf5 Cre was used as a driver to induce the Cre expression within myoblast that produces the myogenic regulatory factor Myf5. Then, the conditional mice (SSM1 and SSM2) were bred with the Myf5-Cre mice. 10 The Myf5-Cre/SSM mice were born smaller as compared to their siblings, and multiple tumors were detected. Most of the detected tumors were located near skeletal muscles since the fusion protein was stimulated within the skeletal muscle Myf5 lineage (Haldar, 2007). Histological and immunohistochemical analysis of the mice tumors were compared with human synovial sarcoma profile for similarities. Both analyses showed that there were similarities to human synovial sarcoma of both the biphasic and monophasic subtypes. However, the occurrence of the monophasic subtype was observed more than the biphasic subtype (Haldar, 2007). The development of human SS18-SSX2 in Myf5-expressing cells used initial genetic models of synovial sarcoma confirmed as excellent human tumor recapitulations (Haldar, 2007). Because this mouse model induces oncogene fusion transcription in a whole lineage of developmental cells — most of which are apoptoses and some of which transform — the mice bear Myf5 lineage ablation's developmental toxicity. While this exposure is survivable by simple growth, it has serious adverse effects on adult mice that have extreme kyphosis of the thoracic spine, defects of the lung, and eventual failure to thrive. By the period at which Myf5-Cre stimulated the development of SS18-SSX2 produces observable tumors, mice are already usually unable to survive and do not withstand anesthesia or even moderate therapeutic toxicity. In fact, these mice generate tumors by variable timing and anatomy. The second mouse model of synovial sarcomagenesis triggered by Rosa26-CreER led to SS18-SSX2 expression in dispersed cells through most mouse tissues (Haldar, 2009). This avoided the toxicity of the development of a whole lineage ablation. Nevertheless, these mice often developed tumors through inconsistent anatomical sites and variable latencies which made 11 their use very difficult for pre-clinical testing (Jones et al, 2019). In fact, in this second model, the average period for the development of observable sarcomas is over a year, increasing the cost of any pre-clinical study (Jones et al, 2019). A locally mediated model of synovial sarcomagenesis became an important goal to reduce the toxicity of the environment and improve predictability with respect to tumor pathology, which could allow early imaging. Kirsch et al. (2007) identified the development of pleiomorphic soft-tissue sarcomas using an adeno-associated virus as a tool for transmitting a Cre-recombinase (AdCre) expression vector into mice's limbs with Kras and Trp53 dependent disorders. The median sarcoma forming period was 3 months (Kirsch, 2007), but two significant genetic hits were needed. Some human data was helpful in determining which additional genetic manipulations might improve synovial sarcomagenesis. Analysis of 47 pairs of tumor/human DNA for 6 subtypes of soft tissue sarcoma including synovial sarcoma, Barretina, et al., showed that phosphatase loss and the gene of tensin homolog (PTEN) was the most common genetic modification from normal other than synovial sarcoma fusion-generating translocation (Barretina, 2010). 12 METHODS Mouse Information Mouse studies were carried out in compliance with legal and ethical guidelines with the authorization of the University of Utah Institutional Animal Care Committee and Use Committee. The hSS1, hSS2, and Pten fl/fl mice mentioned previously have been maintained on a mixed strain basis, C57BL/6 and SvJ. (Groszer et al., 2001; Haldar et al., 2007; Jones et al., 2016). TATCre injections were 10 µl vol of 42 µM into the quadriceps. Breeding Scheme Homozygous wild type CreER mouse was crossed with a homozygous wild type Rosa26 (termed as SSM2) mouse producing the wild type offspring of Rosa26 CreER +/+ mouse. Then, Rosa26 CreER +/+ crossed a mutated heterozygous Rosa26 +/+; Snf5 wt/- mouse producing the three genotypes of interest that includes: Rosa26 +/+; Snf5 wt/fl within a phenotypic frequency of 50%, Rosa26 +/+; Snf5 fl/fl within a phenotypic frequency of 25%, and Rosa26 +/+; Snf5 wt/wt within a phenotypic frequency of 25%. 13 Figure 4. Breeding scheme of Snf5 mouse model as explained above showing the three genotypes of interest in the synovial sarcoma optimization model. 14 Figure 5. TATCre injection was administered into the three different genotypes. Group (1) within the three genotypes was injected at day 8 of their date of birth. Mice in group (2) also contain the three different genotypes that were injected at day 28. TATCre injection was given to the targeted genotypes in both groups. Mice in group (1) were injected with TATCre post birth at day 8 into their quadriceps muscle. Similarly, Group (2) were also injected with same dosage of TATCre (10 uL) and anatomical location, but injection was administered at day 28 post birth. Statistical Analysis The tumor rate of growth was measured during the life period of each mouse by using a caliper. The length and the width of the tumor was measured in each genotypes for both groups. Then, the volume of the tumor was calculated using this equation [volume= (width^2)*(length)/2]. The rate of tumorigenesis in the targeted genotypes was plotted vs. post day injection by using excel software (Fig. 6 and 7). Additionally, T-Test was performed to test the significance of the volume rate between each genotype showing in table. 1. T-Test was calculated by using the excel program. 15 RESULTS A localized model of synovial sarcomagenesis in the mouse Building on the achievements of Kirsch et al., we tried to initiate SS18-SSX2 expression in mice through AdCre limb injection. From the injections of a few pilot mice, two tumors (< 10% incidence) formed over a period of almost a year were induced When we have extended this study to insert seven mice in a consistent position in bilateral lower extremities (tibialis anterior muscle and pretibial periosteum), there were no tumors over a 12-month duration. Certain studies with other sarcomas directed us to use TATCre as a biologically more stable option rather than AdCre. TATCre is a protein variant of Cre-recombinase that reaches cells and their nuclei for direct action due to a human immunodeficiency virus TAT-tag (Straessler, 2013). This also led to more frequent tumor formation when SS18-SSX1 or SS18-SSX2 were initiated. Tumors have regularly formed at the local injection site and about a year interval. During their development and ageing, the Mice hosts these induced tumors were otherwise healthy before tumor growth itself became morbid. The measurements of tumorigenesis were taken via caliper measuring the tumor length (top to bottom) and tumor width (side to side) during the lifetime of the mice. The tumor volume of each mouse was calculated using the volume equation: (width^2)*(length)/2. Measurements of each mouse from the three different genotypes were taken on average of 5 days of both injections at day 8 (Fig. 6) and day 28 (Fig. 7). The mice group at day 8 injection (Fig. 6), showed an early tumorigenesis with in a period of approximately one month. However, the mice group injected at day 28, tumorigenesis was visible after almost three months. This suggest that tumorigenesis could start showing when the mice are injected early in their lifetime. However, 16 the mice group that was injected early (at day 8) did develop tumorigenesis in a short time period as compared to the mice group that was injected at day 28. Additionally, SSM2 +/+; Snf5 fl/fl showed the most tumor volume out of the three different genotypes at day 8 and day 28. To test the significance of the tumor volume in each genotype, we have generated a T-Test shown in table (1). Based on the t-test values of injection at day 8, result revealed that differences in genotypes were not significant in speeding the rate of tumorigenesis. However, the t-test values at day 28 showed that the genotype does matter in increasing the tumorigenesis showing in table (1). T-test of SSM2 +/+; Snf5 fl/fl (Day 28) to SSM2 +/+; snf5 wt/wt (Day 28) were significant as (t=4.69566E-08), and Snf5 fl/fl (Day 28) to Snf5 wt/fl (Day 28) were also significant as (t=1.21301E-07). 17 1.2 Injection At Day 8 1 Volume (IN^3) 0.8 SSM2 +/+; Snf5 fl/fl 0.6 SSM2 +/+; Snf5 wt/wt 0.4 SSM2 +/+; Snf5 fl/wt 0.2 0 0 50 100 150 200 250 300 Days Post Injection Figure 6. Three different types of genotypes of mice were injected with TATCre at day 8. Measurements were taken during the lifetime of mice developing tumorigenesis. In the Sn5f fl/fl genotype, there were two mice that were born at a different date. One was born in September, 2018 and the other in January, 2019. The mouse that was born in September was ready to sacrifice in February, and the other mouse that was born in January was found dead on May, 25th. Similarly, there were two mice born at a different time, each in Snf5 fl/wt and Snf5 wt/wt. The additional mice that were born at a different time were also measured and added to the graph, and this may have skewed the graph of the results. However, mice within the Snf5 fl/fl genotype showed a significantly earlier tumorigenesis as compared to Snf5 wt/wt and Snf5 fl/wt. Whereas, mice within the Snf5 fl/wt genotype showed tumorigenesis but with a slower growth rate as compared to Snf5 fl/fl. 18 0.9 Injection AT DAY 28 0.8 0.7 Volume (IN) 0.6 0.5 SSM2 +/+; Snf5 fl/wt 0.4 SSM2 +/+; Snf5 wt/wt 0.3 SSM2 +/+; Snf5 fl/fl 0.2 0.1 0 0 50 100 150 200 Days Post Injectoin 250 300 Figure 7. Three different types of genotypes of mice injected at day 28 using TATCre protein. Unlike injection at day 8, the mice used in the three different genotypes at day 28 injection were all born at the same date. Mice in the Snf5 fl/fl genotype also showed a significantly earlier tumorigenesis as compared to Snf5 wt/wt and Snf5 fl/wt. Additionally, mice within the Snf5 fl/fl genotype were found dead at day 124, although the mice of the same genotype injected at day 8 were sacrificed at day 133 post injection. The Snf5 fl/fl displayed similar results in tumorigenesis as predicted at day 8 (Fig. 6) and day 28. Here, Snf5 wt/wt mice showed tumorigenesis later in their lifetime. Whereas, Snf5 wt/wt injected at day 8, displayed minimal tumorigenesis (Fig. 6). 19 Genotypes Of Each Mouse Group T-Test Value Snf5 fl/fl (Day 8) and Snf5 wt/wt (Day 8) 0.136105 Snf5 fl/fl (Day 8) and Snf5 wt/fl (Day 8) 0.455842 Snf5 fl/fl (Day 28) and Snf5 wt/fl (Day 28) 1.21301E-07 Snf5 fl/fl (Day 28) and Snf5 wt/wt (Day 28) 4.69566E-08 SSM2 +/+; Snf5 fl/wt of Day 8 and Day 28 0.012671 SSM2 +/+; Snf5 fl/fl of Day 8 and Day 28 0.75264 SSM2 +/+; Snf5 wt/wt of Day 8 and Day 28 5.71111E-10 Table 1. T-Test Value of The Three Different Genotypes. Tumor volume of each genotype was used in the t-test at both day 8 and day 28. 20 DISCUSSION Synovial sarcoma is one of the most common types of soft tissue sarcoma (STS) and it is a distinct type of cancer, once synovial sarcoma become metastatic it is fatal and incurable (Jones et al, 2016). As compared to the other types of soft tissue sarcoma, synovial sarcoma has been resistant to most therapies, but it is more reactive to cytotoxic chemotherapy than other types of sarcoma (Jones et al, 2016). Synovial sarcoma is an interesting type of STS that can occur at any age and everywhere in the body, within the range of 15-30 years old that accounts for 10% of all STS (Desar et al, 2018). It is characterized by chromosomal translocation t(X;18), resulting in the fusion of SS18 gene on chromosome X to one of the synovial sarcoma genes (SSX) on chromosome X, causing the formation of the SS18-SSX fusion oncogenes (Desar et al, 2018). As for prognosis factors, studies have showed that survival rate of synovial sarcoma has been associated with the age, size of tumor, and histological subtype (Desar et al, 2018). The survival rate in children is better than of the adults due to genomic instability in older individuals (Desar et al, 2018). The common treatment of synovial sarcoma include surgery, radiation therapy, and chemotherapy. When the tumor is localized, the primary goal in treating synovial sarcoma is the surgical removal of the tumor (Desar et al, 2018). Similarly, surgery is the base option in treating localized recurrent synovial sarcoma tumors (Eilber and Dry, 2008). It is possible to see the tumor recurrence in a short period of time which indicates that it is biologically aggressive, and it requires additional treatment, such as neoadjuvant systemic therapy (Eilber and Dry, 2008). Within metastatic cases of synovial sarcoma, surgical option is limited because it requires careful patient selection, since patients are selected by the responses to the systematic chemotherapy and 21 free interval of recurrence (Eilber and Dry, 2008). When lungs become metastatic, a complete resection is required for a better survival (Eilber and Dry, 2008). As for radiation therapy, adjuvant radiation therapy has been used in patients with a tumor size of ≥5 cm (Eilber and Dry, 2008). There are two different types of radiation therapy, neoadjuvant or adjuvant that can be administered to patients with synovial sarcoma (Eilber and Dry, 2008). Both types of the radiation therapy showed to improve the local rate of in patients with synovial sarcoma (Eilber and Dry, 2008). Adjuvant radiation therapy has failed to prove the survival benefits in localized synovial sarcoma, therefore it is not the best standard of treatment. (Desar et al, 2018). However, patients with high risk primary extremity STS treated with neoadjuvant (ifosfamide based) had improved survival than those who with doxorubicin (contain no ifosfamide) (Eilber et al., 2001). Thus, neoadjuvant chemotherapy (ifosfamide based) treatment should be beneficial for patients with extremity synovial sarcoma (Eilber and Dry, 2008). Alternative option in stabilizing the response rate of synovial sarcoma was achieved by the only targeted oral drug, pazopanib (Desar et al, 2018). Pazopanib is a multi-targeted tyrosine kinase inhibitor that inhibits both VEGFR (vascular endothelial growth factor) and PDGFR (platelet-derived growth factor) that way blocking tumor growth and inhibiting angiogenesis (Desar et al, 2018). The mentioned cytotoxic therapies, including ifosfamide and pazopanib provide limited benefits for patients with synovial sarcomas. Even after surgery and radiation therapies, patients remain at high risk of tumor recurrences and early or late metastases. Even with the use of different therapies that include surgical removal of the tumor and following neoadjuvant chemotherapy, the mortality rate remains high of 50% within 10 years of diagnosis 22 (Laporte et al., 2017). Therefore, additional targeted therapies are needed to improve the survival rate of synovial sarcoma. We developed a short latency, localized synovial sarcoma model. This model matched the expected features of an excellent pre-clinical testing model, as the tumors were fully penetrant and more rapidly developed in a healthy host and anatomically predictable location (Jones et al, 2019). Before 6 months, almost every injection in Pten fl /fl mice resulted in tumorigenesis (Jones et al, 2019). The tumors resembled histologically previous mouse models and human synovial sarcomas, including even biphasic histologic subtypes (Jones et al, 2019). There are two important considerations that restrict the utility of this more effective preclinical design (Jones et al, 2019). Firstly, since the model directly removes a major node in the PI3 kinase and AKT pathway, it would seem inappropriate to specifically test drugs that are targeted by this model (Jones et al, 2019). Second, a large portion of the tumors resulting from Pten's homozygous loss showed osteogenesis areas (Jones et al, 2019). An osteogenic histological variant has been identified in human synovial sarcoma (Hisaoka, 2009; Milchgrub, 1993). The main concern is that in these SS18-SSX-induced tumors, the osteogenic areas do not seem to consist solely of reactive bone produced by regular osteoblasts (Jones et al, 2019). Rather, some of the osteogenic cells exhibit nuclear pleiomorphism and cellular atypia, indicating that they have changed (Jones et al, 2019). While other efforts in synovial sarcomagenesis suggest the potential of pre-osteoblasts to develop from fusion oncogene expression, it requires special circumstances and additional genetic disturbances (Jones et al, 2019). 23 When Pten loss simply provides these additional hits that allow osteoblast or preosteoblast to transform in addition to fusion gene expression, it raises a concern about human synovial sarcoma's external validity (Jones et al, 2019). On the other hand, if Pten loss alone can produce osteosarcomas, we can form osteosarcomas adjacent to synovial sarcomas (Jones et al, 2019). In the form of TATCre injections into the limbs of Pten fl/fl mice that lack conditional expression of SS18-SSX1, further investigation of this possibility is underway (Jones et al, 2019). There are two possible methods of mitigation available when osteosarcomas are formed adjacent to synovial sarcomas (Jones et al, 2019). First, following treatment with any specific therapy, histological testing of residual tumor tissue will measure the relative prevalence of synovial sarcoma and osteosarcoma tissue as an indicator of the relative response to the therapy being investigated (Jones et al, 2019). The other possible intervention consists of seeking inductive injections into anatomically isolated regions, such as the thigh. These experiments are in progress. Synovial sarcoma pre-clinical models are critical for the evaluation of treatment methods because no center has a significant case load in the trials of synovial sarcoma (Jones et al, 2019). The Pten-null, SS18-SSX1 expressing mouse tumors resemble human sarcoma and will provide an effective pre-clinical treatment trial with the caution that we will not directly use this mouse in order to test inhibitors of Pten / PI3K / AKT pathway, and further research/characterization is required to better understand the adjacent production of a malignant osteoid and possibly reduce it (Jones et al, 2019). Therefore, the model we generated will encourage scientists to study the tumorigenesis rate, and further analyze the effect of Snf5 gene in synovial sarcoma. It is important to check the 24 rate of tumorigenesis so it better models human sarcomas to have a quicker turn round when learning about synovial sarcoma. 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