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. Author manuscript; available in PMC: 2023 Jun 8.
Published in final edited form as: Nat Cancer. 2022 Dec 8;3(12):1513–1533. doi: 10.1038/s43018-022-00466-y

Figure 5. p53 activation induces expression of CD80 and other checkpoint genes in breast cancer cells.

Figure 5.

A) GSEA was performed for all HALLMARK pathways on single cells from clusters 0, 3 and 4 vs. cluster 1 (Fig. 2C); shown is enrichment plot for HALLMARK_P53_PATHWAY. B) sc-Seq feature plots for indicated p53 target genes. Clusters 0, 3, 4 are indicated by red, turquoise, and dark blue dashed lines, respectively. C) Doxorubicin treated or proliferating (+/−) 4226 cells were treated with IFNγ or not (+/−) as in Fig. 3H, and then qPCR performed for indicated genes. Data points are biologically independent cell harvests (n=3), with mean and SEM, untreated (−/−) normalized to 1. D) qPCR for indicated genes was performed on tumors harvested from Ifng−/− and wild-type C57BL/6j mice. Data points represent biologically independent tumors (Ifng−/− untreated: n=4, Ifng−/− doxorubicin-treated: n=5, WT untreated: n=4, WT doxorubicin-treated: n= 4), with mean and SEM. E) Immunoblots for CD80 and actin on 4226 cells treated with doxorubicin or Nutlin-3a and harvested after indicated timepoints F) 4226 cells were treated with doxorubicin or Nutlin-3a for 4h, and then qPCR performed for indicated genes. Data points are biologically independent (n=3), with mean and SEM. G) ChIP with p53 or no antibody was performed on 3642 tumors from untreated (−) or doxorubicin-treated (+) mice in triplicate. qPCR was performed for indicated genes (Chrnb4 is a negative control). H) IF co-staining for PD-L1 (green) and p21 (red) on 3642 tumor sections from doxorubicin-treated or untreated mice. Closed arrows indicate PD-L1+/p21 low cells; open arrows indicate PD-L1 low/p21+ cells. I) IF co-staining for CD80 (green) and p21 (red) on sections from (H). Closed arrows indicate CD80+/p21+ cells. J-K) Distinct cell populations in treated tumors express PD-L1/IRF1, vs. CD80/p21/γH2AX. Multi-IHC staining was performed for PD-L1 (red), IRF1 (magenta), CD80 (green), p21 (yellow), γH2AX (orange), pStat3 (white) and DAPI (blue). J) Orange box has been expanded and indicated channels separated for informative comparisons. 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). K) Untreated tumor stained and imaged in parallel. Scale bar=40µm. Western blots, qPCR and IF were repeated at least twice and representative data are shown.