Journal: bioRxiv

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1101/2025.06.11.653876

Figure Lengend Snippet: ( A ) scSeq data from 6 EwS cell lines with CAF-like subpopulations. Cells scored as EWS::FLI1 “true low” (below median expression of activated/above median expression repressed targets ) or “hybrid” (above median expression of activated and repressed targets), other cells termed “high”. ( B ) Single cell TGFBR2 expression grouped by EWS::FLI1 activity state (n=6 cell lines). ( C ) Flow cytometry of TGFBR2 and CD73. n=3 ( D ) TGFBR2 expression by GeoMx digital spatial profiling of 12 regions of CHLA10 xenografts in NSG mice, previously scored as EWS::FLI1 “low”, “hybrid”, or “high” activity . ( E ) 40 EwS patient tumors from stroma-poor or stroma-rich (ý30% non-tumor stroma) samples . Genes ranked by mean stroma-rich/mean stroma-poor sample values. Dashed lines = top/bottom 5% of genes. ( F ) Ranked enrichment in stroma-rich vs. stroma-poor samples in 40 patient tumors of EWS::FLI1 activated or repressed target genes (0 = not enriched). P-value = Mann-Whitney test. ( G ) Immunoblots of phospho vs. total SMAD2 and EWS::FLI1 in CHLA10 cells transduced with dominant negative TGFBR2 vs. empty vector control -/+ 24 hrs treatment with TGFβ1. Representative of n=2. A549 = positive control for TGFβ induced pSMAD2 and negative control for EWS::FLI1. Loading control = GAPDH. ( H ) RT-qPCR of TNC and COL1A1 in parent, empty vector, or dominant-negative TGFBR2 transduced CHLA10 cells -/+ 24 hrs treatment with TGFβ1. n=3, p-values = unpaired t-tests. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The transfer plasmids utilized in this study included pLenti CMV Blast DEST (Addgene #17451), pLenti CMV Blast DN-TGFBR2-HA (Addgene #130888), pLKO.1 shFLI1 (Sigma TRCN0000005322), and pLKO.1 non-silencing control (Sigma SCH002).

Techniques: Expressing, Activity Assay, Flow Cytometry, MANN-WHITNEY, Western Blot, Transduction, Dominant Negative Mutation, Plasmid Preparation, Control, Positive Control, Negative Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1101/2025.06.11.653876

Figure Lengend Snippet: (A ) % GFP+ after transduction with SBE-GFP SMAD reporter-/+ TGFβ1 (48 hours, n=2). ( B ) TGFB1 and TGFB2 expression in CAF-like (top 30% expression CAF-like gene signature) vs. non-CAF-like cells by single cell sequencing (9 EwS lines). ( C ) ELISAs of TGFβ1 in conditioned media (3 days of culture). n=3. Dashed line indicates TGFβ1 level detected in media only controls. ( D ) ELISAs of secreted total TGFβ2 in conditioned media (3 days of culture). n=3. TGFβ2 not detected in media only controls. ( E ) Genes significantly (padj < 0.05) upregulated by TGFβ1 or TGFβ2 in A673, CHLA10, and PDX305 (bulk RNAseq, n=3). ( F ) Gene ontology (GO:BP) of the 365 genes commonly significantly induced (padj<0.05) by TGFβ1 and TGFβ2 in A673, CHLA10, and PDX305 cells. ( G ) ELISAs of secreted total TGFβ2 after 3 days of culture of CHLA10 dominant negative TGFBR2 or empty vector transduced cells. n=3. p-value = unpaired t-test. ( H ) Expression of TNC , TGFB1 , and TGFB2 in PDX305 cells treated with TGFβ1 (10 ng/mL) -/+ vactosertib, TGFβ2 (10 ng/mL) -/+ vactosertib, or vactosertib alone (1 μM), n=2, p-values = unpaired t-tests. ( I ). Expression of TGFβ induced genes in CHLA10 cells treated with TGFβ ligand blocking antibodies (10 ug/mL, 4 days). ( J ) IF of TNC in CHLA10 cells treated with TGβ ligand blocking antibodies vs. IgG control (10 ug/mL, 4 days). ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The transfer plasmids utilized in this study included pLenti CMV Blast DEST (Addgene #17451), pLenti CMV Blast DN-TGFBR2-HA (Addgene #130888), pLKO.1 shFLI1 (Sigma TRCN0000005322), and pLKO.1 non-silencing control (Sigma SCH002).

Techniques: Transduction, Expressing, Sequencing, Dominant Negative Mutation, Plasmid Preparation, Blocking Assay, Control

Journal: bioRxiv

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1101/2025.06.11.653876

Figure Lengend Snippet: (A ) GSEA NES values of EWS::FLI1 target genes in TGFβ2 treated cell lines (bulk RNAseq, n=3, p-values = padj from GSEA). ( B ) TGFBR2 expression (RNA-seq) of cell lines treated with TGFβ1, TGFβ2, or vactosertib (n=3, error bars = standard error, p-values = padj). ( C ) Expression of TGFBR2 , TGFB1 , TGFB2 in shFLI1 vs. shNS CHLA10 cells by RT-qPCR (n=3). ( D ) RT-qPCR of TGFB2 and TGFBR2 in CHLA10 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with vactosertib (1μM, 24 hrs). n=3. ( E ) RT-qPCR of TGFB2 and TGFBR2 in CHLA10 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with TGFβ2 (10 ng/mL, 24 hrs). n=3. ( F ) RT-qPCR of EWS:FLI1 and its direct activated targets SOX2 and NR0B1 in CHLA10 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with TGFβ2 (10 ng/mL, 24 hrs). Normalized to housekeeping genes then to the average of shNS samples. n=3. P-values = unpaired t-tests. ( G ) RT-qPCR of TGFB2 and TGFBR2 in TC71 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with TGFβ2 (10 ng/mL, 24 hrs). n=3. ( H ) Model of the positive feedback loop between TGFBR2 activity, TGFβ2 expression, and de-repression of EWS::FLI1 downregulated targets in permissive EWS::FLI1 “low” or EWS::FLI1 “hybrid” states vs. EWS::FLI1 “high” cells. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The transfer plasmids utilized in this study included pLenti CMV Blast DEST (Addgene #17451), pLenti CMV Blast DN-TGFBR2-HA (Addgene #130888), pLKO.1 shFLI1 (Sigma TRCN0000005322), and pLKO.1 non-silencing control (Sigma SCH002).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Transduction, Activity Assay