Fig. 9

Doxorubicin-triggered immunogenic cell death is dependent either on C/EBP-β LIP or calreticulin. JC TetON LIP cells, transduced with a non-targeting (scrambled, scr) vector or with a knocking-out calreticulin (KOCRT) vector, were cultured in the absence (− doxy) or presence (+ doxy) of doxycycline (1 μg/ml) for 24 h, to induce C/EBP-β LIP. When indicated, 5 μM doxorubicin (D) was added for 24 h further. a. Whole cell lysates were probed with the indicated antibodies. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. Surface CRT was detected by flow cytometry. The histograms represent the results obtained from 1 out of 3 experiments. c. Tumor cells were stained with PKH2-FITC, DC were stained with an anti-HLA-DR-PE antibody. Tumor cells were co-incubated with DC for 24 h. Double-stained cells were counted by flow cytometry. Data are presented as means±SD (n = 3). *p < 0.001: “+ doxy” cells vs “- doxy” cells; °p < 0.001: “KOCRT” cells vs “scr” cells. d. T-lymphocytes were co-cultured with DC after phagocytosis, then incubated with JC cells. The percentage of CD8⁺CD107⁺T-cells was measured by flow cytometry. Data are presented as means±SD (n = 3). *p < 0.001: “+ doxy” cells vs “- doxy” cells; °p < 0.001: “KOCRT” cells vs “scr” cells. e. Cells were implanted orthotopically into 6 week-old female balb/C mice. When indicated, animals received 1 mg/ml doxycycline in the drinking water (+ doxy) to induce the intratumor LIP expression. Mice were randomized into 8 groups (n = 8 animals/group) treated on days 1, 6, 12 after randomization: 4 groups received 0.1 ml saline solution i.p. (left panel), 4 groups received 5 mg/kg doxorubicin (D) i.p. (right panel). Tumor growth was monitored daily by caliper measurement. Data are presented as means±SD. *p < 0.001: “+ doxy” cells vs “- doxy” cells; °p < 0.001: “KOCRT” cells vs “scr” cells. f. Photographs of representative tumors of each group