Statistical Analysis Methods for Preclinical Studies

For the complete blood count (CBC) experiment with blood sampling at days 2 and 4, statistical significance was determined by 2-way ANOVA with Šidák’s multiple-comparison test. For FTY720 Q2Dx4 experiments (CBC and immunoprofiling), statistical significance was determined by 1-way ANOVA with Šidák’s multiple-comparison tests comparing vehicle vs. FTY720 Q2Dx4, vehicle vs. ATRi QDx3 plus RT, ATRi QDx3 plus RT vs. ATRi QDx3 plus RT plus FTY720, and FTY720 Q2Dx4 vs. ATRi QDx3 plus RT plus FTY720. For the ATRi QDx3 vs. vehicle cytokine/chemokine experiment, statistical significance was determined by unpaired, 2-tailed t test. For CT26 tumor response experiments involving vehicle, RT alone, RT plus anti–PD-L1, ATRi QDx3 plus RT plus anti–PD-L1, and ATRi QDx9 plus RT plus anti–PD-L1 treatment groups, the time (days) at which tumors reached 2 volume doublings was interpolated using Akima spline curves generated from tumor volume data, and significance for comparisons of the proportions of mice reaching 2 tumor volume doublings was determined by log-rank test with Holm-Šidák adjustment for multiple comparisons among all groups. For survival comparisons following treatment with RT alone, RT plus anti–PD-L1, ATRi QDx3 plus RT plus anti–PD-L1, or ATRi QDx9 plus RT plus anti–PD-L1, the survival endpoint was defined as the day when tumor volume exceeded 1,000 mm³, and significance was determined by log-rank test with Holm-Šidák adjustment for multiple comparisons among all non-vehicle treatment groups. For all other experiments, statistical significance was determined by ANOVA with Tukey’s multiple-comparison tests. A 95% confidence interval and significance of P less than 0.05 were used for all statistical tests. Data are reported as mean ± SD unless otherwise specified. Brackets are shown only for comparisons that were statistically significant unless otherwise specified. All statistical analyses were performed in GraphPad Prism 9.