Extended Data Fig. 1: p95HER2 promotes tumor growth by suppressing anti-tumor immunity.
(a) Cell proliferation assay evaluating the effect of expressing FL-HER2 or p95HER2 on cell growth in culture. Cell proliferation was monitored in real time using the IncuCyte imaging system. N = 4 replicate wells per cell line; data are expressed as mean ± SEM; results are representative of n = 3 biological repeats. (b) Growth curves of PB2 tumorgrafts in both immunodeficient NSG mice and immunocompetent C57BL/6 mice. PB2 cells expressing either FL-HER2 or p95HER2 under the control of doxycycline were implanted into the mammary fat pads of mice which were then fed doxycycline diet. Empty vector (EV) PB2 tumorgrafts served as a control. Growth for each tumor type is plotted normalized to the initial (baseline) EV tumor size. EV: left n = 10, right n = 14; FL-HER2: left n = 10, right n = 13; p95HER2: left n = 10, right, n = 13. Data represent mean ± SEM. Statistical analyses by ordinary one-way ANOVA with Tukey’s correction for multiple comparisons. P values are indicated within the plots. (c) Individual growth trajectories for PB2 tumors expressing either p95HER2 or FL-HER2 in C57BL/6 mice, analogous to panel b, but without normalization (FL-HER2 n = 10, p95HER2 n = 10). Statistical analysis by unpaired, two-tailed Student’s t test (p = 0.0133). (d) Extended growth trajectories for individual PB2 tumors expressing p95HER2 in C57BL/6 mice, along with associated Kaplan Meier curve depicting Event Free Survival (EFS) which includes mandated sacrifice as an event. (e) qRT-PCR demonstrating the effect of p95HER2 on expression of immune-related genes in PB2 tumors grown 21 days in the mammary glands of immunocompetent C57BL/6 mice (n = 8 for p95HER2+ tumor-bearing mice compared to n = 10 for EV tumor-bearing mice). Data represent mean ± SEM. Statistical analysis by unpaired, two-tailed Student’s t test with Welch’s correction. P values are indicated within the plot. (f) Gene Set Enrichment Analysis (GSEA) of RNA-seq data from PB2 tumors expressing p95HER2 versus control PB2 tumors, performed using GOBP and KEGG databases. (g) Enrichment plots of gene sets that are involved in IFN-γ response and antigen processing and presentation. Both gene sets are found to be downregulated in p95HER2+ PB2 tumors grown in immunocompetent C57BL/6 mice. GSEA was performed against the GOBP database. The significance is reported as a Normalized Enrichment Score (NES) and adjusted for multiple testing using False Discovery Rate (FDR) control. (h) Ingenuity pathway analysis (IPA) of the same RNAseq data. The top 20 pathways that are significantly altered in p95HER2+ PB2 tumors and have absolute z-score = or >2 are included. Pathways downregulated in association with p95HER2 are in blue, while those upregulated in association with p95HER2 are in orange. Statistical analyses by a right-tailed Fisher’s exact test adjusted with false discovery rate control. (i) Proportion of CD45+ and CD8+ immune cells positive for IFN-γ in PB2 tumors that express either p95HER2 or FL-HER2 (n = 4 EV tumors and n = 7 p95HER2+ and HER2+ tumors). Data represent mean ± SEM. Statistical analysis by unpaired, two-tailed Student’s t test. P values are indicated within the plot.
