Fig 1: A+CAFs induce AR-V7 expression and ENZ resistance in cancer cells.(A) Immunofluorescence staining and fluorescence intensity quantification of A−CAFs and A+CAFs showing the expression of APCDD1 (green) and COL1A1 (red). Scale bars: 50 μm. (B and C) Schematic of the Boyden chamber coculture system and colony formation assays used to indirectly coculture C4-2 and LNCaP cells with A+CAFs or A−CAFs with or without ENZ treatment. Quantification of colonies is shown on the right (n = 3). (D) Images of PDOs cocultured with A+CAFs or A−CAFs, with and without ENZ treatment. Quantification of organoid size is shown on the right (n = 30). Scale bars: 100 μm. (E) Schematic of in vivo experiments. C4-2 cells were co-injected with A+CAFs or A−CAFs into castrated NSG mice, followed by ENZ treatment. Tumor growth analysis confirms that A+CAFs significantly enhanced tumor growth under ENZ treatment (n = 5 for each group). (F) CCK-8 assay showing increased cell viability in C4-2 cells treated with A+CTM, but not A−CTM, under ENZ treatment. (G and H) Gene ontology enrichment and gene set enrichment analysis of differentially expressed genes in C4-2 cells treated with A+CTM versus A−CTM, highlighting RNA splicing–related pathways (n = 5 for each group). (I) RT-PCR analysis of AR and AR-V7 expression in C4-2 cells treated with A+CTM, showing an increase in AR-V7 levels. (J) CCK-8 assay showing the reversal of A+CTM-induced ENZ resistance in C4-2 cells treated with an AR-V7 PROTAC. Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test [(B) to (D)] and two-way ANOVA [(E), (F), and (J)] followed by a multiple comparisons test. **P < 0.01, and ***P < 0.001. ns, not significant.