Fig. 4

NXS2 cells are susceptible to the cytotoxic payload DM4 and expose immunogenic cell death (ICD) markers after treatment with DM4, which may also induce tumor resistance in NXS2 cell lines by upregulation of PD-L1. (A) In vitro IC₅₀ curves assessed through cytotoxicity assays by exposing for 72 h NXS2 CTRL and hLGALS3BP cells to increasing doses of payload DM4 (left panel) or naked 1959-sss antibody (right panel). IC₅₀ values are reported for each cell line considered upon the different treatments. Values refer to two independent biological replicates. Surface levels of ICD markers (B) calreticulin, (C) HSP70, and (D) HSP90 expressed as mean fluorescence intensity (M.F.I.) signals evaluated through FACS analysis on NXS2 cells treated or not with DM4 5nM for 72 h or with Oxaliplatin (OXA) 60µM for 48 h. Asterisks indicate statistical significance through unpaired t-test compared to each experimental time control (PBS); *p < 0.05, **p < 0.01. Exact p-values are reported. Values refer to two or three independent biological replicates. (E) Surface levels of PD-L1 evaluated through FACS analysis expressed as fold change of mean fluorescence intensity (M.F.I.) signals of treated samples over controls of NXS2 control and hLGALS3BP cells treated with 3nM DM4 or with DMA (control) for 72 h. Asterisks indicate statistical significance through unpaired t-test compared to each experimental control (DMA); *p < 0.05. Exact p-values are reported. Values refer to three independent biological replicates