Fig. 1

Immune checkpoint inhibition, with or without radiation, fails to clear established mEER tumors. a Flow cytometry immune profiling of untreated mEER tumors harvested at day 23 of tumor growth. Left shows a representative histogram for PD-L1 (top) and PD-L2 (bottom) within the non-immune tumor fraction (CD45 negative cells after gradient separation). Right shows cumulative flow cytometry scatterplots of PD-1 levels on tumor infiltrating CD8⁺ T cells (top) and CTLA-4 levels on splenic CD8⁺ T cells (bottom) (percentage show mean ⁺/− SD; N = 1 representative of 2; n = 5 aggregate samples per group). (b top) Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with 6 total doses of αPD-1 (250 μg/dose) and/or αCTLA-4 (100 μg/dose). (b bottom) Individual tumor area for each ICI treated mouse subset (N = 1 representative of 2; n = 6–8/group). c-e Mice bearing established mEER tumors were treated with αPD-1 and αCTLA-4 alone or in combination with localized tumor irradiation (2 X 10 Gy with one dose given each week) according to the schedule in (c), and euthanized when tumors reached 225 mm². d Average tumor area until time of first mouse euthanization (Tukey’s multiple comparison test; N = 1 representative of 2; n = 6–9/group). e Kaplan Meier curves comparing survival of mice treated with immune checkpoint inhibitors with and without tumor-directed irradiation (Log-rank test; N = 2; n = 12–18/group). f Pie-chart showing tumor-infiltrating lymphoid and myeloid subsets as a fraction of total CD45⁺ cells on day 23 of treatment (N = 2; n = 10–16/group). g Log2 fold-change of key immune subsets comparing αPD-1/αCTLA-4+ RT vs. αPD-1/αCTLA-4 at day 23 of treatment (Tukey’s multiple comparison test; N = 2; n = 10–12/group). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001