Synovial Sarcoma Tissue Research Articles

1944P MAGE-A4 and NY-ESO-1 expression analysed in a synovial sarcoma tissue micro-array

1944P MAGE-A4 and NY-ESO-1 expression analysed in a synovial sarcoma tissue micro-array

Abstract 174: Organoids developed from synovial sarcoma patient-derived xenografts

Abstract Background: Soft tissue sarcoma (STS) is a heterogeneous family of rare malignancies. Despite this heterogeneity, systemic treatment options for patients with advanced STS are rather uniform and have very limited efficacy. There is an unmet medical need to develop more active, subtype-specific treatment options. Three-dimensional (3D) cell culture models (organoids) have been successfully used for drug testing in carcinoma research. However, the development of STS organoids is lagging behind. We attempted to identify the best growth conditions for organoids derived from synovial sarcoma (SySa), which is one of the more common translocation-related subtypes of STS. Methods: Human synovial sarcoma tissue was implanted subcutaneously into immunodeficient mice and grown to create a patient-derived xenograft (PDX). Fresh tumor tissue from the PDX model was collected and enzymatically and mechanically dissociated into small cell clusters. The cells were seeded into a 3D scaffold with advanced Dulbecco's modified Eagle’s medium (DMEM)/F12 and different growth factors were added. In order to obtain optimal results, different scaffolds and growth factors were compared. The growth of the 3D cultures was evaluated by bright-field microscopy and a cell viability assay using PrestoBlue. The organoids were characterized by histopathological stainings and the presence of the SSX-SS18 translocation was molecularly confirmed. Finally, we attempted to create organoids from PDX-derived material stored in liquid nitrogen. Results: A scaffold consisting of 50% collagen type 1, 30% Matrigel, 20% DMEM 10x and sodium hydroxide was superior to Matrigel alone. The addition of human recombinant epidermal growth factor (EGF, 10 ng/ml), human insulin-like growth factor 1 (IGF-1, 20 ng/ml), human recombinant fibroblast growth factor 2 (FGF-2, 10 ng/ml), and N-acetylcysteine (NAC, 500 mmol) to advanced DMEM/F12 stimulated the growth of SySa organoids the best. The Rho-kinase inhibitor Y-27632 was added to prevent apoptosis during early passages. So far, attempts to create organoids from frozen material have been unsuccessful. Conclusions: At present, the success rate of establishing organoids from synovial sarcoma cells is limited if the culture conditions are not adapted to their needs. We identified that growth factors such as EGF, FGF-2, IGF-1, and NAC are beneficial for growth. The availability of fresh samples plays an important role in the development of these 3D models. Despite these limitations, organoids can be an interesting tool for studying this rare disease and aid in the development of new drugs. Citation Format: Lore De Cock, Agnieszka Wozniak, Kimberly Verbeeck, Patrick Schöffski. Organoids developed from synovial sarcoma patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 174.

Interdependence of SS18-SSX-driven YAP1 and β-Catenin Activation in Synovial Sarcoma.

This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and β-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

Association of SMC4 with prognosis and immune infiltration of sarcoma.

This study was performed to explore the prognostic relevance of structural maintenance of chromosomes 4 (SMC4) in pan-cancer and explore the association between SMC4 and immune infiltration of sarcoma. Elevated expression of SMC4 was detected in cancer tissues compared to normal tissue, which was confirmed in synovial sarcoma tissues with immunohistochemistry (IHC). Additionally, higher expression of SMC4 was connected to worse outcomes of sarcoma, gastric cancer, breast cancer, liver cancer or ovarian cancer. Moreover, SMC4 was positively connected to immune cell infiltrates in sarcoma. In addition, infiltrating immune cell markers including monocyte, TAM, M1 and M2 presented different SMC4-associated immune infiltration patterns. The results from our study showed that SMC4 was positively related to the prognosis and immunological status of sarcoma. SMC4 could be a potential biomarker for prognosis and immune cell infiltrates in sarcoma. Several databases including ONCOMINE, GEPIA, and Kaplan-Meier Plotter were adopted to explore the expression pattern of SMC4 in sarcoma, which was confirmed by IHC. The GEPIA and TIMER datasets were adopted to investigate the associations between SMC4 and prognosis in various cancers, especially in sarcoma.

GPR64, Screened from Ewing Sarcoma Cells, Is a Potential Target for Antibody-Based Therapy for Various Sarcomas.

Simple SummaryNew strategies for immunotherapy have led to an increased interest in tumor-specific antigens on the cell surface in the field of oncology. Identifying markers in sarcomas is difficult because their tumor mutation burden is less than that of carcinomas. We assumed that a target protein may be acceptable as a therapeutic target, even if it is only expressed in the epididymis along with the tumor, because the epididymis has special barriers, known as the blood–epididymis barrier (BEB). We identified GPR64 as a therapeutic target for Ewing sarcoma via next-generation RNA-sequencing. GPR64 is located on the apical membranes of efferent ductules and separated from antibodies by the BEB. This study revealed, for the first time, that anti-GPR64 antibodies accumulate in various sarcomas and avoid targeting GPR64 in the epididymis in vivo. Furthermore, GPR64 is widely expressed in various sarcomas and is, therefore, a potential antibody-based therapeutic target for sarcomas.Ewing sarcoma is an aggressive and the second most common bone tumor in adolescent and young adult patients. The 5-year survival rate is 60–70% for localized disease but 30% for patients with metastases. Here, we aimed to identify a therapeutic target for Ewing sarcoma and evaluate antibody-based therapeutic agents using in vitro and in vivo models. We identified G protein-coupled receptor 64 (GPR64) as a therapeutic target for Ewing sarcoma via next-generation RNA-sequencing. GPR64v205 mRNA was expressed in HTB166, A673, MG63, 143B, HS-Sy II, and HT1080 cell lines as well as in Ewing sarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, dedifferentiated liposarcoma, and synovial sarcoma tissues. GPR64 expression was observed in 62.5% of sarcoma cases and was overexpressed in 33.9% cases. GPR64-specific monoclonal antibodies were tested as near-infrared probes for in vivo imaging using subcutaneous tumor mouse xenografts. Fluorescence intensity was stronger for the AF700-labeled anti-GPR64 antibody than that for the AF700-labeled isotype control antibody. GPR64 was detected in engrafted tumors of A673, 143B, HT1080, and the epididymis but not in other resected tissues. The anti-GPR64 antibody showed excellent binding to GPR64-positive tumors but not to healthy tissues. This antibody has potential for drug delivery in the antibody-based treatment of sarcomas.

SS18-SSX-Dependent YAP/TAZ Signaling in Synovial Sarcoma.

Synovial sarcoma is a soft tissue malignancy characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein acts as a transcriptional dysregulator representing the major driver of the disease; however, the signaling pathways activated by SS18-SSX remain to be elucidated to define innovative therapeutic strategies. Immunohistochemical evaluation of the Hippo signaling pathway effectors YAP/TAZ was performed in a large cohort of synovial sarcoma tissue specimens. SS18-SSX dependency and biological function of the YAP/TAZ Hippo signaling cascade were analyzed in five synovial sarcoma cell lines and a mesenchymal stem cell model in vitro. YAP/TAZ-TEAD-mediated transcriptional activity was modulated by RNAi-mediated knockdown and the small-molecule inhibitor verteporfin. The effects of verteporfin were finally tested in vivo in synovial sarcoma cell line-based avian chorioallantoic membrane and murine xenograft models as well as a patient-derived xenograft. A significant subset of synovial sarcoma showed nuclear positivity for YAP/TAZ and their transcriptional targets FOXM1 and PLK1. In synovial sarcoma cells, RNAi-mediated knockdown of SS18-SSX led to significant reduction of YAP/TAZ-TEAD transcriptional activity. Conversely, SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ-dependent signals, mechanistically mediated by an IGF-II/IGF-IR signaling loop leading to dysregulation of the Hippo effectors LATS1 and MOB1. Modulation of YAP/TAZ-TEAD-mediated transcriptional activity by RNAi or verteporfin treatment resulted in significant growth inhibitory effects in vitro and in vivo. Our preclinical study identifies an elementary role of SS18-SSX-driven YAP/TAZ signals, highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis, and provides evidence for innovative therapeutic approaches.

Abstract 2315: Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma

Abstract Synovial sarcoma is a rare and aggressive form of soft tissue cancer that affects the extremities of the arms or legs, for which current chemotherapeutic agents have not been proven to be very effective. The cyclin-dependent kinase 4/6-retinoblastoma protein (CDK4/6-Rb) pathway is aberrant in a large proportion of cancer. Recent evidences on pre-clinical application of CDK4 inhibitors have been implicated in many types of human cancers, and the FDA has approved the CDK4 selective inhibitor for the treatment of breast cancer. However, the expression and therapeutic potential of CDK4 in synovial sarcoma remain unclear. In the present study, we examined the expression of CDK4 in synovial sarcoma cell lines by western blot and immunofluorescence assay, and in synovial sarcoma tissue microassays by immunohistochemical analysis. Cell viabilities were determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor. Flow cytometry analysis and wound healing assay were conducted to determine the mechanisms underlying the cytotoxic effects of the selective CDK4 inhibitor. CDK4 specific small interference RNA was used to validate the effect of targeting CDK4 by the selective CDK4 inhibitor in synovial sarcoma cells. We found that CDK4 was highly expressed in human synovial sarcoma, and was related to clinical stage and TNM grade in synovial sarcoma patients and poor prognosis in sarcoma patients. Cell viabilities determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor showed that this selective CDK4 inhibitor repressed synovial sarcoma cell proliferation and growth in a dose- and time- dependent manner. The selective CDK4 inhibitor inhibited the CDK4/6-Rb signaling pathway and promoted cell apoptosis without influence on the expression of CDK4/6, suggesting that the selective CDK4 inhibitor only repressed the hyperactivation, not the production of CDK4/6. The inhibition effect of the selective CDK4 inhibitor was confirmed by knockdown of CDK4 with specific small interference RNA. Flow cytometry analysis revealed that the selective CDK4 inhibitor induced G1 cell-cycle arrest by targeting CDK4/6-Rb pathway in synovial sarcoma cells. Furthermore, the wound healing assay exhibited that inhibition of CDK4/6-Rb pathway with the use of the selective CDK4 inhibitor significantly decreased synovial sarcoma cell migration in vitro. Our data highlight the role of dysregulated CDK4/6-Rb pathway and current selective CDK4/6 inhibitor may be a potential promising therapeutic agent in the targeted treatment of human synovial sarcoma. Citation Format: Xiaoyang Li, Cassandra Garbutt, Francis John Hornicek, Zhenfeng Duan. Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2315.

Immunoreactivity of a Monoclonal Antibody to SS18-SSX Fusion Gene Product in Formalin-fixed Paraffin-embedded Synovial Sarcoma Tissue Section.

Synovial sarcoma is an aggressive sarcoma with specific reciprocal chromosomal translocation of SS18 (also known as SYT) and SSX genes. In the present study, we aimed to detect the SS18-SSX fusion gene product in routinely processed pathologic synovial sarcoma tissue section. Monoclonal antibodies to peptide QRPYGYDQ-IMPKKPA, which covered the fusion region of SS18-SSX, were newly established and subsequently characterized by enzyme-linked immunosorbent assay, Western-immunoblotting, and immunohistochemical staining. A monoclonal antibody designated BG35, reacted with boundary region of SS18-SSX chimera protein in a specific manner, when C-terminal or neighboring region of QRPYGYDQ-IMPKKPA is structurally exposed. Immunoreactivity of BG35 was localized in nucleus of synovial sarcoma cells, but not in other sarcoma cells, examined. Interestingly, synovial sarcoma cells with epithelial differentiation exhibited much strong BG35 immunoreactivity than synovial sarcoma cells with sarcomatous differentiation. BG35 may be useful for evaluating molecular kinetics of SSX-SS18 gene product in situ.

Radioimmunotherapy of solid tumors targeting a cell-surface protein, FZD10: therapeutic efficacy largely depends on radiosensitivity

Frizzled homolog 10 (FZD10) is expressed at high levels on the cell surface of almost all synovial sarcoma tissues, but is absent in most normal organs. In a previous study, yttrium-90 ((90)Y)-labeled anti-FZD10 antibody (MAb 92-13) showed considerable therapeutic efficacy in synovial sarcoma cell-bearing mice. The purpose of the present study was to elucidate the factors associated with this therapeutic efficacy of (90)Y-MAb 92-13. FZD10 expression levels of SYO-1 (FZD10-overexpressing synovial sarcoma cell line) and DLD-1/FZD10 (FZD10-transfected DLD-1 cell) were determined by the cell binding assay, and their radiosensitivity was evaluated by incubation with (90)Y-MAb 92-13 in vitro. Biodistribution study of indium-111 ((111)In)-MAb 92-13 was performed in SYO-1 and DLD-1/FZD10 tumor-bearing mice. For therapeutic studies, SYO-1 and DLD-1/FZD10 tumor-bearing mice were treated with (90)Y-MAb 92-13 (100, 150, and 200 muCi), after which the change in tumor volume was measured. Immunohistochemical staining was performed on the excised tumor. Expression level of FZD10 on DLD-1/FZD10 was much greater than that on SYO-1. The accumulation of (111)In-MAb 92-13 was much higher in DLD-1/FZD10 tumor-bearing mice than in SYO-1 tumor-bearing mice (49.0 +/- 4.2 and 22.0 +/- 4.5% ID/g, respectively, at 48 h after administration). In SYO-1 tumor, substantial tumor size reduction was observed in all mice treated with (90)Y-MAb 92-13 (tumor volume decreased to less than 0.1 cm(3) at 11 days after treatment) and tumor regrowth was not observed in most of them. In contrast, only slow progression was observed in DLD-1/FZD10 tumor. When incubated with (90)Y-MAb 92-13, high radioactivity was needed to damage DLD-1/FZD10. Immunohistochemical study indicated apoptosis of SYO-1 tumor. The therapeutic efficacy of RIT seems to largely depend on the tumor radiosensitivity.

The Synovial Sarcoma SYT-SSX2 Oncogene Remodels the Cytoskeleton through Activation of the Ephrin Pathway

Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway.

The SYT-SSX Fusion Protein Down-Regulates the Cell Proliferation Regulator COM1 in t(x;18) Synovial Sarcoma

Chromosomal translocations are frequently associated with soft-tissue sarcomas. Fusion proteins generated by such translocations often play critical roles in tumorigenesis. Therefore, it is important to understand the function of the fusion protein to develop therapeutic interventions. The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in a fusion between the SYT gene on chromosome 18 and an SSX gene on the X chromosome. Although SYT-SSX fusion proteins appear to trigger synovial sarcoma development, little is known about the downstream targets of SYT-SSX. We found that the SYT-SSX fusion protein produces a dominant-negative function for SYT, which is a transcriptional coactivator. We then analyzed the gene expression profiles of SYT-SSX1-expressing HeLa cells using oligonucleotide microarrays and found that the SYT-SSX1 fusion protein directly down-regulated the expression of COM1, a regulator of cell proliferation. COM1 was found to be expressed at relatively low levels in synovial sarcoma tissues and cell lines. We then investigated the impact of conditional COM1 expression in the synovial sarcoma cell line. Increased COM1 expression resulted in induced apoptosis and in reduced cell growth and colony formation activity. Our results suggested that restoration of COM1 expression may be of therapeutic benefit in synovial sarcoma.

Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression

The synovial sarcoma translocation t(X;18)(p11.2; q11.2) results in the fusion of the SYT gene on chromosome 18 to exon 5 of either SSX1 or SSX2 genes on chromosome X. We recently reported that the SSX4 gene is also involved in such a translocation. In the present investigation we cloned and sequenced the full-length cDNA of SYT/SSX1, SYT/SSX2 and SYT/SSX4 from synovial sarcoma tissues. We isolated a novel fusion transcript type variant involving the fusion of SYT with exon 6 of the SSX4 gene (SYT/SSX4v). The SYT/SSX4 and SYT/SSX2 open reading frame also differed from previously reported SYT/SSX sequences by an in-frame addition of 93bp exon located in the junction between exon 7 and 8 of the SYT. This exon is identical to that reported for the murine SYT but has not been previously found in the human transcript. Two SYT transcripts, with and without the 93 bp exon, were co-expressed in mouse NIH3T3 cells, human malignant cells and human testis tissue, but not in human normal fibroblasts. Stable transfection of an SYT/SSX4 expression vector into human and murine cell lines correlated with a down-regulation of SYT transcripts. This was also observed in a synovial sarcoma tumor expressing SYT/SSX4. This suggests that the SYT/SSX fusion gene may regulate SYT expression from the normal allele and as such alter the normal function of SYT.

SYT-SSX fusion proteins in synovial sarcomas: detection and characterization with new antibodies

To identify and characterize the SYT-SSX fusion proteins in synovial sarcomas, we developed two polyclonal antibodies against the N-terminal part and for the C-terminal part of the SYT-SSX2 protein. Specificity was demonstrated on COS-7 cells transfected with two subtypes of SYT-SSX fusion genes, SYT-SSX1 and SYT-SSX2. Both antibodies recognized a single protein of 61 kDa in an immunoprecipitation of the transfected COS-7 cell lysates. These antibodies also detected the native protein of 61 kDa in the lysate of a human synovial sarcoma cell line (HS-SY41) with immunoprecipitation, and in extracts of human synovial sarcomas with western blot analysis. An immunohistochemical study, using human synovial sarcoma tissues, demonstrated that the SYT-SSX fusion proteins localized in the nucleus of the tumor cells. These antibodies provide a useful method for studying the expression of the SYT-SSX fusion proteins.

Epithelial expression of glycodelin in biphasic synovial sarcomas

Glycodelin is a 28 kDa glycoprotein of the lipocalin family that was previously considered to be specific for the reproductive tract. Glycodelin is found in the secretory glandular epithelium of endometrium and in seminal vesicles. Given the cyclic differentiation of normal endometrial epithelium, we studied by immunohistochemistry a possible expression of glycodelin in other tissues displaying stroma-to-epithelium maturation. We report here that 11/11 biphasic synovial sarcomas expressed glycodelin in the cells exhibiting epithelial or glandular differentiation while the sarcomatous spindle cells remained negative. Glycodelin was also found in secreted material in the lumina of the gland-like structures. In only 1 of the 7 monophasic synovial sarcomas studied, focal glycodelin reactivity was seen in some flattened spindle cells. The expression of glycodelin in biphasic synovial sarcoma tissue was further verified by the demonstration of glycodelin mRNA by reverse transcriptase-polymerase chain reaction. Considering the monoclonal origin of synovial sarcomas, our findings raise the intriguing possibility that activation of expression of the glycodelin gene is involved in the molecular regulation of mesenchyme-to-epithelium differentiation. Int. J. Cancer 76:487–490, 1998.© 1998 Wiley-Liss, Inc.

Epithelial expression of glycodelin in biphasic synovial sarcomas.

Glycodelin is a 28 kDa glycoprotein of the lipocalin family that was previously considered to be specific for the reproductive tract. Glycodelin is found in the secretory glandular epithelium of endometrium and in seminal vesicles. Given the cyclic differentiation of normal endometrial epithelium, we studied by immunohistochemistry a possible expression of glycodelin in other tissues displaying stroma-to-epithelium maturation. We report here that 11/11 biphasic synovial sarcomas expressed glycodelin in the cells exhibiting epithelial or glandular differentiation while the sarcomatous spindle cells remained negative. Glycodelin was also found in secreted material in the lumina of the gland-like structures. In only 1 of the 7 monophasic synovial sarcomas studied, focal glycodelin reactivity was seen in some flattened spindle cells. The expression of glycodelin in biphasic synovial sarcoma tissue was further verified by the demonstration of glycodelin mRNA by reverse transcriptase-polymerase chain reaction. Considering the monoclonal origin of synovial sarcomas, our findings raise the intriguing possibility that activation of expression of the glycodelin gene is involved in the molecular regulation of mesenchyme-to-epithelium differentiation.

Interphase cytogenetic analysis of distinct X‐chromosomal translocation breakpoints in synovial sarcoma

Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2). Two distinct X-chromosomal breakpoints occur in different synovial sarcoma tumour samples. These breakpoints are located within two related genomic regions containing ornithine aminotransferase-like sequences, termed OATL1 and OATL2. Preliminary observations indicated the potential correlation of OATL1-associated breakpoints with biphasic tumours and OATL2-associated breakpoints with monophasic fibrous tumours. The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples. Two-colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp. One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2-associated breakpoint, while a biphasic tumour revealed a hybridization pattern indicating a breakpoint within the OATL1 region. These results confirm our previous suggestion of a relationship between alternative breakpoints in Xp11.2 and different histological phenotypes observed in synovial sarcomas. They also demonstrate the utility of the two-colour hybridization approach for the identification of chromosomal changes in interphase nuclei isolated from frozen tissues.