(P 448) CAF-LIKE EWING SARCOMA CELLS DEPOSIT PRO-TUMORIGENIC EXTRACELLULAR MATRIX IN RESPONSE TO TGF-BETA SIGNALING

Objective: Despite limited genetic and histologic heterogeneity, Ewing sarcoma (EwS) tumor cells are transcriptionally heterogeneous. Recently we identified subsets of highly mesenchymal EwS cells that deposit pro-tumorigenic extracellular matrix proteins. Due to their role in ECM remodeling, which is typically carried out by cancer-associated fibroblasts (CAFs) in carcinomas, we termed them CAF-like cells. In this study we sought to further characterize the transcriptional heterogeneity and upstream drivers of these CAF-like states.

Methods: Single cell proteogenomic-sequencing (CITE-seq) of EwS cell lines was performed and integrated with patient tumor transcriptomic data. Tumor cell subpopulations were evaluated in cell line or patient-derived xenografts and patient tumor biopsies using multiplex immunofluorescence (mIF) staining. Digital spatial profiling and human whole transcriptome analysis interrogated spatial transcriptomic heterogeneity in EwS xenografts. Cell subpopulations were isolated by FACS for assessment of gene expression, proliferation, and migration. TGF-beta signaling was induced with recombinant TGFB1 and inhibited via CRISPR-Cas9 knockout of TGFBR2, lentiviral expression of a dominant negative TGFBR2 construct, or addition of small molecule TGFBR1/ALK4 inhibitor SB505124. Invasion was measured by invasive strand formation of tumor spheroids cultured in 3D collagen-I rich gels.

Results: CITE-seq of 9 EwS cell lines identified the cell surface protein CD73 (NT5E) as a biomarker of CAF-like EwS cells in vitro. CAF-like EwS cells deposited extracellular matrix (ECM) proteins including Tenascin-C (TNC), COL1A1, Biglycan, and SPARC in vitro and CD73+ cells co-localized with areas of ECM-deposition in xenografts and patient tumor samples in vivo. Gene ontology (GO) analysis of genes that were reproducibly upregulated in NT5E+ vs. NT5E- cells by single cell sequencing showed highly significant enrichment of GO terms “TGF-beta regulation of extracellular matrix” (NCATS BioPlanet, p.adj = 5.3E-11) and SMAD2 binding (ChEA, p.adj = 4.4E-14), suggesting that TGF-beta may regulate the CAF-like state. In support of this, flow cytometry of EwS cell lines revealed a 3-fold increase of TGFBR2 surface expression in CD73+ vs. CD73- cells. TGFB1 treatment of EwS cells in vitro induced expression of NT5E as well as CAF-like signature genes BGN, COL1A1, SPARC, and TNC (p. adj < 8.5E-5). In contrast, knockout of TGFBR2 or expression of a dominant negative TGFBR2 led to reduced expression of these genes. mIF of patient tumors and xenografts revealed the presence of subpopulations of phospho-SMAD2 S465/467+ tumor cells adjacent to areas of TNC deposition. Phenotypically, modulation of TGF-beta signaling had minimal impact on EwS cells grown in 2D culture, but TGF-beta inhibition reduced invasion and viability in 3D collagen I-rich culture.

Conclusion: EwS tumors generate subpopulations of tumor cells that deposit pro-tumorigenic ECM, functioning analogously to CAFs. EwS cells have been reported to be non-responsive to TGF-beta signaling due to EWS::FLI1-mediated repression of TGFBR2. Here, we report that CAF-like EwS cells express TGFBR2 and respond to TGF-beta ligands. We find that TGF-beta signaling is a master regulator of CAF-like states and ECM deposition in Ewing sarcoma. This raises the possibility of TGF-beta inhibition as a therapeutic strategy to inhibit tumor progression by targeting the tumor microenvironment.