Journal: bioRxiv

Article Title: EWSR1::ETS-low cells promote metabolic reprogramming of the tryptophan-kynurenine-AHR axis, immunosuppression, and poor outcome in Ewing sarcoma

doi: 10.1101/2025.05.16.654502

Figure Lengend Snippet: a) Contingency table showing distribution of IPASS score and AHR activity score-based clusters in EwS tumors (n=166). Two-tailed Fisher’s exact test. b) Comparative analysis of inferred pro-inflammatory immune cell infiltration and c) immunosuppressive MDSCs and regulatory T cells infiltration between AHR-low (K1) and -high (K2) activity scoring EwS tumors. Horizontal bars represent the mean and whiskers the SEM. P -values were determined by Wilcoxon test. n=166. d) Relative cell viability MHH-ES-1/TR/shEF1 and TC-106/TR/shERG and e) fold change of interferon (IFN)-γ levels of MHH-ES-1/TR/shEF1 after 24 h co-culture with NK-92 cells. Horizontal bars represent the mean and whiskers the SEM. n=5 independent biological experiments. P- values were determined by two-tailed Mann-Whitney test. f) Relative AHR mRNA and g ) protein expression after establishing single cell clones with independent DOX-inducible shRNA targeting AHR (255, 286) in MHH-ES-1/TR/ and TC-106/TR/ cells (AU = arbitrary units). n=4−6 biologically independent experiments. Relative densitometry is shown, calculated from western blots of lysates from n=4 biologically independent experiments. Two-tailed Mann-Whitney test. h) Relative cell viability MHH-ES-1/TR/shAHR255 bulk and single clone #4 after 24 h co-culture with NK-92 cells. Horizontal bars represent the mean and whiskers the SEM, n=6 biologically independent experiments. P -values were determined by two-tailed Mann-Whitney test.

Article Snippet: AHR silencing (transcript ID: Human NM_001621.5) was achieved by cloning the shRNA oligonucleotide sequences into the Tet-pLKO-puro backbone (Addgene #21915, USA).

Techniques: Activity Assay, Two Tailed Test, Co-Culture Assay, MANN-WHITNEY, Expressing, Clone Assay, shRNA, Western Blot