Fig. 6. Aspirin improves chemotherapy in an immune system and autophagy-dependent fashion.

a, b WT immunocompetent and athymic (nu/nu) mice were subcutaneously injected with MCA205 cells and when tumors became palpable, were divided into four groups and intraperitoneally treated with 5.17 mg/kg mitoxantrone (MTX) (a) or 10 mg/kg oxaliplatin (OXA) (b), alone or in combination with daily 100 mg/kg i.p. aspirin administration, or an equivalent volume of PBS. From left to right: (1) means ± s.e.m. tumor growth curves of WT mice treated with aspirin alone or in combination with MTX or OXA (n = 8 mice per group); (2) tumor size distribution at day 19 (MTX) or day 18 (OXA) of data shown in (1); (3) individual growth curves from mice treated with MTX or OXA alone or combined with aspirin of data shown in (1); (4) averaged ± s.e.m. tumor growth curves from immunodeficient nu/nu mice subjected to aspirin administration alone or in combination with MTX (n = 5 mice/group) or OXA (Ctrl, OXA and aspirin with n = 5 mice/group, OXA + aspirin n = 6 mice/condition). PBS and aspirin groups are shared in (a) and (b) for the nu/nu mice experiment. c Extracellular ATP levels (n = 7/8 replicates) were measured in the supernatants of autophagy competent or Atg5^KD MCA205 cells treated with 2 mM MTX and/or 5 mM sodium salicylate (Salic). d WT mice were inoculated subcutaneously with autophagy competent cells expressing a scrambled control shRNA (left panel) or Atg5^KD MCA205 cells (middle panels) or with CD39 overexpressing MCA205 cells (right panel). When tumors became palpable, mice were treated as in (a) (n = 8 mice/condition). Data are shown as means ± s.e.m. for tumor growth curves, as individual curves of each mice in the group, or as box and whisker plots, which show median, first and third quartiles, and maximum and minimum values. Circles in the graphs (a, b) indicate each mouse used in the experiment, in (c) indicate each replicate. Statistical analysis was performed by linear mixed effect modeling (over the whole-time course) and linear modeling for the single time point (*p < 0.05, **p < 0.01, ***p < 0.001 for MTX or OXA versus MTX + aspirin or OXA + aspirin; ^#p < 0.05, ^###p < 0.001 for PBS versus MTX or OXA alone). In (c) statistical analysis was done by two-way ANOVA Tukey’s multiple comparisons test (***p < 0.001 MTX versus MTX + salicylate, ^###p < 0.001 PBS versus MTX). Asp aspirin, d day, MTX mitoxantrone, OXA oxaliplatin, Salic salicylate.