Fig 1: Demethylation of GSDME expression enhances IR700DX-6T-PDT sensitivity and improves the antitumor efficacy of IR700DX-6T-PDT in combination with PD-1 blockade. (A) SW620 cells was treated with 5 µM decitabine for 2 days and then GSDME mRNA (below) and protein (above) levels were assessed by qPCR and Western blot, respectively. (B) SW620 cells with low endogenous GSDME expression were treated with 5 µM decitabine and IR700DX-6T-PDT, and then analyzed for LDH release. (C) Expressions of GSDME mRNA (below) and protein (above) levels in 4T1 cells after having been treated with 1.25 µM decitabine for 5 days. (D) LDH release of 4T1 cells after indicated treatment. (E) Schematic illustration of the experimental timeline for tumor inoculation and combination treatment with decitabine, IR700DX-6T, and anti-PD-1 antibody. The 4T1 tumor-bearing mice were treated with PBS, decitabine, decitabine+anti-PD-1, IR700DX-6T+light, IR700DX-6T+light+decitabine, IR700DX-6T+light+decitabine+anti-PD-1, respectively (doses: decitabine, 80 µM, 3 times; anti-PD-1, 100 µg, 4 times; IR700DX-6T+light, 7.5 nM IR700DX-6T, light irradiation 18 J/cm2). (F) Growth curves of tumors after various treatments (n=5). (G) Tumor weight in a subcutaneous 4T1 murine model (n=5). (H) Representative flow cytometry analysis (left) and proportions (right) of CD8+ T cells and CD80+CD86+ cells within the tumor (n=3). (I) Multiplex IHC (CD3, CD4, CD8, and cleaved-CASP3) of tumor tissues after different treatments (n=16). CD3: orange; CD4: green; CD8: white; cleaved-CASP3: pink; DAPI: blue. Scale bar, 20 µm. (J) The proposed working model. IR700DX-6T-mediated PDT induces pyroptosis and boosts antitumor immunity. Data are presented as mean±SEM. **p<0.01. cleaved-CASP3, cleaved-caspase3; GSDME, gasdermin E; IHC, immunohistochemistry; LDH, lactate dehydrogenase; PDT, photodynamic therapy; PBS, phosphate buffered saline.