Fig. 5: ALK inhibition activates the immune system.

As illustrated in the phagocytosis assay scheme (A), murine tumor cells were, first, stained with the green cell tracker (CMFDA), then, treated with different drugs, while bone marrow-derived dendritic cells (BMDCs) were isolated from syngeneic mice and differentiated in vitro. After co-culture, the percentages of phagocytized cells were determined by flow cytometry. B Murine NPM1-ALK⁺ R80 cells were treated with 0.2 μM of crizotinib (CRIZ) or ceritinib (CER), 2 μM of BKM120 (BKM), 1 μM of mitoxantrone (MTX) for 16–18 h or subjected to one cycle of freeze–thawing (F/T) and co-cultured with BMDCs for 4 h. Co-culture was stained using a fluorescent anti-CD11c antibody. The percentages of CMFDA⁺ cells within CD11c⁺ BMDCs reflect phagocytosis. The representative gating strategy is shown in B. C Means ± SD of three independent experiments each performed in triplicates (n = 9); ****p < 0.0001 (Student’s t test) (C). Scheme of vaccination experiment (D). Murine tumor cells were treated in vitro to reach 50–70% of mortality before being injected into the left flank of syngeneic mice. Two weeks later, mice were challenged with live cells into the opposite flank and tumor appearance and growth were monitored. Five million of NPM1-ALK⁺ R80 cells, previously treated for 24 h with 0.5 μM of CER, were injected into the left flank of C57BL/6 mice. After two weeks, mice were challenged with 1 × 10⁶ of live R80 cells into the opposite flank. Tumor surface in function of time is represented as mean ± SEM of mice belonging to the same group or for each mouse (E). Survival is also represented in E. Vehicle n = 9 and CER n = 10. Comparison between tumor growth curves was assessed using a type II ANOVA test, while the statistical significance of survival data was calculated using the Log-rank test. **p < 0.01, ***p < 0.001.