Fig. 4: Gemcitabine-cisplatin chemotherapy promotes an overwhelmingly high release of inhibitory DAMP to attenuate DC maturation.

a Western blot analysis of COX-2 expression in G69 cell lysates treated with either vehicle or GC for 48 h. GAPDH expression was used as loading control (n = 3 biologically independent experiments). b ELISA for PGE₂ release in the culture medium of G69 cells treated with either vehicle control or GC for 48 h (n = 4 independent biological samples). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test **p = 0.0016). c COX-2 expression in G69-tumor tissue lysates treated with either vehicle or GC for two chemotherapy cycles. GAPDH expression was used as a loading control (n = 3 biologically independent experiments). d ELISA for PGE2 release in the serum of G69-tumor-bearing mice treated with either vehicle or GC for 2 cycles (n = 5 and n = 4 biologically independent samples for vehicle and GC, respectively). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test ****p < 0.0001). e Immunofluorescence analysis of COX-2 expression in G69-tumor tissues treated with either vehicle or GC for two chemotherapy cycles (Scale bar: 100 μm; Images representative of n = 3 independent experiments). f Schematic representation of BMDC treatment with conditioned media in the presence or absence of iDAMP blockade. g Flow cytometric analysis of CD40, CD86, and MHCI expression in BMDCs treated with G69-conditioned media with or without iDAMP blockade. h Geometric mean fluorescence intensity (gMFI) of CD40, CD86, and MHCI molecule expression in BMDCs upon treatment with condition media as in g (n = 2 biologically independent samples). Data are presented as mean values. i Schematic representation of footpad vaccination assay. j Flow cytometric analysis of CD40, CD86 and MHCI expression in vaccination-draining lymph node CD11c + MHC2 + DCs upon injection of G69 cells treated with GC in the presence or absence of iDAMP blockade (n = 5 biologically independent samples). Non-vaccination-draining lymph node DCs were used as control (n = 3 biologically independent samples). Data are presented as mean values ± SEM. (One-way ANOVA-Tukey’s multiple comparisons test. CD40: GC vs GC+ Celex, p = 0.045; GC vs GC+ aPGE2, p = 0.036; CD86: GC vs GC+ Celex, p = 0.046; GC vs GC+ aPGE2, p = 0.0007; H2-K: GC vs GC+ Celex, p = 0.0004; GC vs GC+ aPGE2, p = 0.005). Source data are provided as a Source data file. ns not significant.