Effects of AZD8186 in combination with anti-PD1 on syngeneic models. (A) Xenograft of murine melanoma BP cells. BP cells were subcutaneously implanted into BALB/c mice. Mice bearing BP xenografts were treated with vehicle, AZD8186 at 50 mg/kg daily, anti-PD1 at 200 μg, twice a week or AZD8186 in combination with anti-PD1 for 14 days. Tumor volume was measured at the indicated time points. Mean ± SEM values of tumor volume were obtained from 5 mice in each treatment group from single experiment. The tumor volumes at the conclusion of experiment were analyzed by two-way analysis of variance to determine statistical significance. *p values (combination vs control: p < 0.001; combination vs AZD8186: p = 0.002; combination vs anti-PD1: p = 0.003). (B) Mass cytometry analysis. Tumors were harvested from the same mice above at the end point. Tumors were digested and stained with mass cytometry staining antibody panel including CD8 antibody. Samples were then analyzed using a CyTOF2 or Helios mass cytometer. Values are presented as mean ± SEM of percentage of CD8+ TILs of tumors. Data were analyzed by multiple t-tests to determine statistical significance. *p = 0.02 (AZD8186 vs control); #p = 0.016 (anti-PD1 vs control); *#p values for combination (combination vs control: p = 0.001; combination vs AZD8186: p = 0.01; combination vs anti-PD1: p = 0.014). (C, F) Xenografts of BP cells (C) and CT26 cells (F). BALB/c mice bearing xenografts were treated with vehicle, AZD8186 at 50 mg/kg daily, anti-PD1 at 200 μg, twice a week, paclitaxel at 10 mg/kg, once a week, and double or triple combinations.