Fig. 3: Tumors induce Cyp11a1 expression in T cells in vivo.

a B16-F10 cells were injected subcutaneously into the Cyp11a1-reporter mice. After 12 days brachial lymph node (LN), blood, and tumor tissues were analyzed by flow cytometry. Gating strategy: Singlets>Live cells>CD45,Cyp11a1-mCherry. N = 5. CD45⁺Cyp11a1-mCherry⁺ cells were further gated to show T helper cell (CD4⁺CD3e⁺) expression of Cyp11a1. b Splenic cells were purified from tumor-bearing Cyp11a1-reporter mice, and restimulated in vitro using PMA/ionomycin and analyzed by flow cytometry. N = 9. c, d B16-F10 cells were injected subcutaneously into the Cyp11a1-mCherry reporter mice. After 5, 7, and 12 days tumor tissues were analyzed by flow cytometry to detect the CD4⁺ T cells (c), CD8⁺ T cells (d) N = 4. All cell>Singlets>Live cell>CD45⁺>CD4⁺CD3e⁺ or CD8⁺CD3e⁺. e–g EO771 cells were injected into the mammary fat pad of Cyp11a1-reporter mice. After 15 days, tumor tissues and tumor-draining LN were analyzed by flow cytometry to detect the Cyp11a1-mCherry expression in CD4⁺ T cells (e, f) CD8⁺ T cells (g). Gating: All cell>Singlets>Live cell>CD45⁺>CD4⁺TCRb⁺ or CD8⁺TCRb⁺. e Representative FACS profile of Cyp11a1⁺CD4⁺ T cells. f, g Representative graphical presentation of one experiment showing Cyp11a1-expressing CD4 and CD8 T cells. N = 4. h B16-F10 tumor-infiltrating leukocytes (TIL) and splenocytes were purified from tumor-bearing mice on post-inoculation day-12, cultured for 48 h, and the supernatant was analyzed by ELISA to measure pregnenolone. N = 12, pooled analysis of three independent experiments. i Metastasized lungs were harvested 10 days post-B16-F10 intravenous injection in C57BL/6 mice, dissociated into single-cell suspension, cultured for 48 h, and the supernatant was analyzed by ELISA. Naïve uninfected lungs (normal lung) were used as control. N = 6, pooled analysis of two independent experiments. j Hierarchical clustering of steroidogenic genes and IL4 mRNA expression across 44 melanoma patient samples (GEO: GSE19234). k Schematics showing glucocorticoid synthesis pathway. l Frequency distribution histogram showing CYP11A1 mRNA expression (normalized read counts, log10 scale) across 22 melanoma patients’ tumor-infiltrating CD4⁺ T cells (EGAD00001000325). Individual data points for CYP11A1 expression are shown in Supplementary Fig. 3i. All error bars in this figure represent mean with s.d. P-values were calculated by unpaired two-tailed t-test. N represents biologically independent animals.