Fig. 3

(R)-crizotinib had anti-cancer vaccination effects on NSCLC. Wild type (WT) TC1 cells were treated with mitoxantrone (MTX; 4 µM), cisplatin (CDDP; 150 µM), mitomycin C (MitoC; 150 µM) alone or in combination with (R)-crizotinib (Criz, 10 µM) for 24 h. Then the cells were collected and subcutaneously (s.c.) injected (10⁶ cells per mouse) into the left flank of immunocompetent C57BL/6 mice (a). PBS was used as control. Two weeks later all mice were rechallenged with living TC1 cells (2 × 10⁵ per mouse) in the right flank. The evolution of tumor incidence over time was reported as Kaplan–Meier curves (b–d). Statistical significance was calculated by means of the Likelihood ratio test. ***p < 0.001 compared to PBS group; n = 10 per group. Final tumor size distribution at endpoint is shown in e. Statistical significance was calculated by means of the ANOVA test for multiple comparisons, ***p < 0.001 as compared to the PBS group. f, g ANXA1-deficient (Anxa1^−/−) or HMGB1-deficient (Hmgb1^−/−) TC1 cells treated 24 h with (R)-crizotinib/CDDP combination or receiving an equivalent volume of PBS were s.c. inoculated in the flank of WT C57BL/6 mice, which were 2 weeks later rechallenged in the opposite flank with living TC1 cells. h, i WT TC1 cells treated 24 h with the combination of (R)-Criz and CDDP were incubated with chicken αCALR antibody or isotype antibody (2.5 µg per 10⁶ cells) for 30 min at room temperature; or an ATP diphosphohydrolase apyrase (5 IU per 10⁶ cells) for 30 min at room temperature before being inoculated s.c. into the flank of WT C57BL/6 mice, which were 2 weeks later rechallenged in the opposite flank later with living TC1 cells. Statistical significance was calculated by means of the Likelihood ratio test. ***p < 0.001 compared to PBS group; ^###p < 0.001 as comparing indicated groups, n = minimum of 6 mice per group