Fig. 1

Malignant epithelial cells with increased self-renewal properties are located adjacent to the stroma of Hh-expressing cancers. a Scheme depicting the purification of epithelial and mixed stromal populations from disaggregated M6 murine tumor models. b Expression of genes significantly downregulated (blue) or upregulated (red) by RNA-Seq analysis (cut-off log₂ (Fold Change) ≥ 2; vertical lines) in the epithelium of M6-Hh tumors compared to the epithelium of M6-Ctrl or M6-Hh tumors treated with the SMOi, GDC-0449 (100 mg/kg/bid), plotted against FDR values (horizontal lines indicate −log¹⁰ (FDR) > 2). Each symbol represents the transcriptome from five biological replicates per treatment group. c GSEA analysis reveals significant enrichment for genes encoding stemness and invasion in M6-Hh primary cells (FDR q value < 0.05). d Heat map showing relative CSC genes expression in the epithelium of M6-Hh tumors compared to M6-Ctrl and M6-Hh tumors + SMOi. Data show normalized row Z-score (n = 5 biological replicates for each treatment group). e Representative FACS dot plots showing the expression of CSC markers CD61 and CD29 within the EpCAM⁺/GFP⁺/CD24⁺ population of M6-Ctrl and M6-Hh tumors (n = 3 biological replicates per group). f Relative expression of key stemness genes in M6-Ctrl and M6-Hh whole tumors ± SMOi (100 mg/kg/bid). n = 3 biological replicates per treatment group; statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. g Kaplan−Meier and h tumor penetrance curves of mice injected with 250 primary M6-Ctrl (blue) or M6-Hh cells (violet); n = 10 biological replicates. Six tumors from the M6-Hh and two tumors from the M6-Ctrl models were detectable, respectively. Statistical significance was determined using log-rank test. i Primary M6-Ctrl and M6-Hh tumor cells were isolated by FACS and transplanted at various dilutions into recipient mice. Limiting dilution analysis demonstrating higher in vivo tumor-forming capacity in M6-Hh cells compared to M6-Ctrl cells. n = 10 mice per condition. j Representative images and quantification of CK6-progenitor and phospho-Histone H3-positive cancer cells at the tumor−stromal interface of M6 tumor models. Scale bars: 100 μm for CK6 and 200 μm for phospho-Histone H3. n = 3 biological replicates per treatment group. Statistical significance was determined using unpaired two-tailed Student’s t test; *P < 0.05; **P < 0.01. Bars represent mean ± s.e.m.