Fig. 2.

TIME modulation renders tumors responsive to αPD-1/αCTLA-4 with radiation and promotes immunologic memory. a Established mEER tumors were treated with CTX/L-NIL (2 doses of CTX at 2 mg/mouse delivered weekly and L-NIL 2 mg/mL continuously delivered in the drinking water). Tumor were harvested at day 23 of treatment and immune cell type enrichment scores from Nanostring whole tumor immune-related RNA expression was compared to untreated control tumors (Two-way ANOVA with Sidak correction; N = 1; n = 9/group). c and d Established mEER tumors were treated with CTX/L-NIL immunomodulation combined with αPD-1/αCTLA-4 and tumor directed radiation (collectively called the “CPR” regimen) according to schedule in (b), mice were euthanized when tumors reached 225 mm². c Average tumor area until time of first mouse euthanization (Tukey’s multiple comparison test; N = 1 representative of 2; n = 6–8/group). d Kaplan Meier survival curves comparing different treatment combinations (Log-rank test; N = 2; n = 12–16/group). e CPR treated mice which rejected primary mEER tumor challenge were rechallanged approximately 100 days after primary rejection using 5-fold the original mEER tumor inoculum on the opposing flank. Data shows individual mouse tumor area compared to age-matched naïve control mice in gray (N = 2; n = 10/group). f Similar to 2E, CPR mice which rejected primary mEER tumor challenge were rechallanged simultaneously with MOC2 tumor cells and MOC2 tumor cells expressing HPV E6 and E7 on the opposing flank. Data shows average tumor area for MOC2 tumors (right) and MOC2 E6/E7 tumors (left) statistically compared to age-matched naïve control mice at time of first mouse euthanization (Tukey’s multiple comparison test; N = 2; n = 10/group). Fractions next to growth curves indicate the number of mice which fully rejected rechallange. **p < 0.01; ***p < 0.001; ****p < 0.0001, n.s. indicates not significant