Fig. 9: AR inhibition suppresses tumorigenicity of BRAFi-resistant melanoma cells.

a RT-qPCR of the indicated genes, normalized to RPLP0, in YUMM1.7 mouse melanoma cells, parental (P), or selected for DAB resistance (BR). Mean ± SD, n(biological replicates)=3, unpaired, two-tailed t-test, (AR)**p = 0.0098; (EGFR)**p = 0.00086; (SERPINE) *p = 0.017. b WB of AR, PAI-1and H3 in (P) versus (BR) YUMM1.7 cells. c RT-qPCR of the indicated genes in YUMM1.7-BR cells treated with AZD3514 or ARCC4 AR inhibitors (ARi), or DMSO. Mean ± SEM, n(biological replicates)=3, one-way ANOVA, ****p < 0.0001. d Live-cell imaging proliferation assays (IncuCyte) of YUMM1.7-BR cells treated with DAB versus AR inhibitors. n(dishes)= 3, mean ± SD, Pearson r correlation test, ****p < 0.0001. e Tumor volume quantification 14 days after intradermal injection into immunocompetent mice (C57BL/6 J) of YUMM1.7-BR cells, pretreated (12 hours) with either AZD3514 or ARCC4 AR inhibitors (ARi) or DMSO. n(tumors) = 5, paired, two-tailed t-test, **p = 0.0014. f Tumor size (volume) in immunocompetent mice (C57BL/6 J) injected with YUMM1.7-BR cells followed by gavage with the indicated AR inhibitors or DMSO control (CNTRL), starting day 3 after injection. n(tumors)= 5, mean ± SD, two-tailed Pearson r correlation test, ***p = 0.0001. g RT-qPCR expression analysis of the indicated genes, normalized to Rplp0, in tumors as in f. Median, box (25–75%) and whiskers (5-95%), n(tumors) = 5. h Ki67 IF (green) of tumors as in f, with DAPI (blue). Representative images and quantification of Ki67⁺ cells per tumor. Mean ± SEM, n(tumors)= 3, unpaired, two-tailed t-test, **p = 0.0096. Scale bar: 250 µm. i Cleaved-caspase3 IF (magenta) of tumors as in f, with DAPI (blue). Representative images and quantification of cleaved-caspase3⁺ areas per tumor. Mean ± SEM, n(tumors)= 3, unpaired, two-tailed t-test, **p < 0.01. Scale bar: 50 µm. j CD4+ (blue) and CD8+ (white) IF of sections from tumors made as in f and 3D reconstruction analysis by Imaris software. Additional tumors are in Supplementary Fig. 8a. Scale bar: 30 µm. k Cleaved-caspase3 (magenta), granzyme B (cyan), CD8 (gray) IF of tumors made as in f, with DAPI (yellow), followed by a 3D analysis. Additional tumors are in Supplementary Fig. 8b. Scale bar: 30 µm. l, m Quantification of the tumor areas occupied by CD8⁺ cell clusters (n (cells/cluster) >5), as detected by IF of tumors formed in mice with gavage l or by ARi-treated cells m, as those from f, e, respectively. Mean ± SEM, unpaired, two-tailed, t-test in l, Veh n(tumors)=4; ARi n(tumors)=8; *p = 0.0188, and paired, two-tailed t-test in m Veh n(tumors)=8; ARi n(tumors)=8, ***p < 0.0009. Images used for quantifications are in Supplementary Fig. 9c–e.