Extended Data Fig. 7: p53 signaling induces a subset of immune checkpoint genes in chemotherapy-treated breast cancer cells.
a) GSEA analysis of sequenced single cells from clusters 0, 3, and 4 vs. cluster 1 from Fig. 2c was performed, and enrichment plots for indicated pathways are shown. b) 3642 tumor cells were transplanted into multiple wild-type or Ifng−/− C57Bl/6j mice and treated or not with doxorubicin. Tumors were harvested 48 h following the final treatment, mRNA prepared, and qPCR was performed for indicated checkpoint genes. Shown are individual data points representative of biologically independent tumors (Ifng−/− untreated: n = 4, Ifng−/− doxorubicin-treated: n = 6, WT untreated: n = 4, WT doxorubicin-treated: n = 6), mean, and SEM. c) FFPE tumor sections from untreated or doxorubicin treated mice were IF co-stained for PD-L1 (green) and p21 (red). Closed arrows indicate PD-L1+ cells but with weak nuclear p21 staining; open arrows indicate cells negative for PD-L1 but with strong, nuclear p21 staining. d) IF co-staining for CD80 (green) and p21 (red) on sections from Fig. Fig. 5. Closed arrows indicate CD80+ cells with strong nuclear p21 staining. Statistical significance was determined using one-way ANOVA with Tukey’s posttest for comparisons of three or more groups. e, f) As for Fig. 5j, k, multi-IHC staining was performed for PD-L1 (red), IRF1 (magenta), CD80 (green), p21 (yellow), γH2AX (orange), pStat3 (white) and DAPI (blue). e) Boxes show three individual areas that have been and expanded, with indicated channels separated. Arrowheads mark p21 + /CD80 + (closed yellow arrow) and IRF1 + /PD-L1+ cells (closed magenta arrow); γH2AX + /CD80+ cells (closed orange arrow) and γH2AX/PD-L1 negative cells (open arrow); p-Stat3+/PD-L1+ (closed arrow) and p-Stat3 + /CD80+ cells (open arrow). f) Untreated tumor stained and imaged in parallel. Scale bar=40 µm. qPCR and IF were repeated at least twice and representative data are shown.
