Fig. 4

Effects of lurbinectedin, ecubectedin and PM54 on immunogenic cell deaths of MPM cells. (A) Workflow of experiment. Dendritic cells (DCs), generated in 4 days from healthy donors PBMC, were co-incubated 18 h with MPM cells, previously grown 24 h in drug-free medium (untreated), cisplatin + pemetrexed (Pt + PMX), lurbinectedin (L), ecubectedin and PM54 at their IC₅₀. An aliquot of DC-MPM co-cultures were collected to measure DC-mediated phagocytosis by flow cytometry. An aliquot of DCs that have been in contact with MPM cells was collected and incubated 10 days with CD8⁺T-lymphocytes, obtained from PBMC of the same donor. CD8⁺T-lymphocytes were then collected and co-incubated with the MPM cells of the same patient for 18 h. Finally, CD8⁺T-lymphocytes were collected and analyzed for the activation markers by flow cytometry, MPM cells were stained with Annexin V-FITC/PI to measure necro-apoptotic cells, as index of immune-killing mediated by CD8⁺T-lymphocytes. (B) The percentage of cells positive for surface calreticulin (CRT) was measured by flow-cytometry. (C) ATP release was measured by a chemiluminescent-based assay. (D) HMGB1 release was measured by ELISA. (E) Phagocytized MPM cells were counted by flow cytometry. (F) Percentage of CD8⁺CD107a⁺INFγ⁺ cells, as index of cytotoxic T-lymphocyte activation. (G) Percentage of annexin V-FITC⁺/PI⁺ MPM cells, as index of tumor cells immune killing by CD8⁺T-lymphocytes, measured by flow cytometry. In all panels, data are expressed as means ± SD of 12 MPM samples (n = 3 independent experiments, in duplicates). *p < 0.05, ***p < 0.001: vs. untreated cells; °°°p < 0.001: vs. Pt + PMX