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. 2024 Sep 17;84(24):4214–4232. doi: 10.1158/0008-5472.CAN-24-0830

Figure 3.

Figure 3.

Autophagy-deficient cancer cells induce DC activation and T-cell priming via increased accumulation of intracellular antigens. A, Schematic outline of the coculture assay involving BM-derived DCs and KPC cells. B, Flow cytometry analysis of the expression of the activation and maturation markers, CD80, CD86, MHC I, MHC II on DCs cocultured with KPC1/2 shNC, KPC1/2 + 20 µmol/L CQ, or KPC1/2 shATG5#1, #2 cells. C, Schematic outline of the coculture assay involving DCs and OT-1 cells. D, DCs were pre-cocultured with KPC1 shNC, KPC1 + 20 µmol/L CQ, or KPC1 shATG7#1, #2 cells and then cocultured with OT-1 cells (extracted from the spleen of the OT-1 mouse). Flow cytometry was used to evaluate the proliferation of CFSE-labeled OT-1 cells. E, Schematic outline of the coculture assay involving autologous human PBMC–derived DCs and PDAC cells. F, Flow cytometry analysis of the activation and maturation markers on human DCs cocultured with autologous PDAC cells transfected with siNC; siATG5#1, #2; or siATG7#1, #2. G, KPC1-EGFP and KPC2-EGFP were treated with the autophagy inhibitors CQ (20 µmol/L) or 3-MA (2.0 mmol/L). Flow cytometry was used to assess the changes in EGFP expression. Representative histograms (top) and the corresponding quantification of EGFP expression in KPC1-EGFP and KPC2-EGFP (bottom) are shown. H, KPC1-OVA cells were treated with CQ (20 µmol/L) or 3-MA (2.0 mmol/L). IF analysis was performed to assess the accumulation of intracellular OVA. I, DCs were cocultured with KPC1 cells (no dye), KPC1-dye siNC, KPC1-dye, KPC1-dye + 20 µmol/L CQ, or KPC1-dye siATG7#1, #2. Flow cytometry was used to detect the signal of green dye in DCs to compare the amount of antigen captured by DCs. Representative dot plots are shown. J, Flow cytometry analysis of DCs indirect cocultured with KPC1/2 shNC or KPC1/2 shATG5#1, #2 cells. Error bars, mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001; analyzed using one-way ANOVA. FACS, flow cytometry analysis. (A,C, and E, Created with BioRender.com.)