Figure 6: Distinct ShcA signalling networks differentially sensitize mammary tumours to immunotherapies.

(a) Mammary fat pad injection of FVB mice with the indicated cell lines. Starting on day 5, mice were treated with 100 μg of a neutralizing PD1 (α-PD1) antibody or its corresponding isotype control IgG and every 3 days thereafter (n=10 tumours each). Data represented as mean±s.e.m. (b) FVB mice received three intraperitoneal injections (days 0, 7 and 14) with PBS or mitomycin C-treated breast cancer cells of the indicated genotypes. On day 21, mammary fat pad injections were performed with breast cancer cells of the same genotype used for vaccination (n=9–11 mice each). (c) Schematic diagram illustrating the relationship between ShcA-driven, STAT1/STAT3 activation and sensitivity to PD1 immune checkpoint inhibitors or tumour vaccination strategies. (d) Primary breast tumours from the TCGA RNAseq data set (n=1,215) were equally stratified into four quartiles based on gene expression signatures that are either unique to loss of the Y239/240 (2F) or Y313 phosphorylation site (313F), or shared in both groups (double mutant, DM). ssGSEA was used to rank order each tumour based on acquisition of a DM, 2F or 313F-like ShcA signature. Tumours in the first quartile resemble those that possess elevated phosphotyrosine-dependent ShcA signalling, whereas those in the fourth quartile are reminiscent of the lowest degree of ShcA-dependent transcriptional responses. The average GZMB, CD8A and PD-L1 mRNA levels were evaluated in each quartile. The same tumours were stratified based on relative expression levels of STAT1 or STAT3 target genes. The average STAT1 and STAT3 ssGSEA levels were determined for tumours in each quartile. The data are shown as average expression levels±s.e.m. (comparing quartiles 1 and 4). (e) Tumours (n=320) were stratified by STAT1^Low (first quartile)/STAT3^Low (first quartile), n=110 or (34.4%); STAT1^Low (first quartile)/STAT3^High (fourth quartile), n=58 (18.1%); STAT1^High (fourth quartile)/STAT3^Low (first quartile), n=61 (19%); or STAT1^High (fourth quartile)/STAT3^High (fourth quartile), n=91 (28.4%) ssGSEA signatures. Relative GZMB, CD8A and PD-L1 expression levels are plotted (±s.e.m.). Significance was determined using multiple t-test with Holm–Sidak method for a (*P<0.05 and **P<0.01) and unpaired two-tailed Student’s t-test for e,d.