Fig 1: Low-dose gemcitabine treatment enhances exposure of immunogenic molecules in lung cancer in vivo. (A) C57BL/6 mice (6–8 weeks) were inoculated with 106 LLC cells. Mice were divided into four groups: PBS served as a vehicle control group. The other three groups were injected (i.p.) with gemcitabine (120, 60, or 30 mg/kg, four times every 3 days) plus cisplatin (3 mg/kg, twice every 6 days). (B,C) Calreticulin (CRT) membrane exposure and high-mobility group box 1 protein (HMGB1) release were detected by immunofluorescence in frozen sections of subcutaneous tumors. The low-dose treatment group showed stronger mean fluorescence intensity (MFI) per square centimeter of CRT and HMGB1 than medium- or high-dose groups. Scale bar = 50 μm. Data are representative of three independent experiments. The unpaired Student's t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: Low-dose gemcitabine treatment enhances exposure of immunogenic molecules in lung cancer in vitro. (A,B) Immunofluorescence analyses of calreticulin (CRT) exposure (red) after 24 h of chemotherapy and high-mobility group box 1 protein (HMGB1) exposure (red) after 48 h of chemotherapy on the murine Lewis lung carcinoma (LLC) cell line (A) and human lung cancer (A549) cell line (B). LLC cells were treated with vehicle control [dimethyl sulfoxide (DMSO)], gemcitabine (Gem; 5, 10, 50, 100, 500 nM), mitoxantrone (Mtx; 1 μM), or cisplatin (CDDP; 5 μM). Scale bar = 20 μm. (C,D) We counted the number of fluorescent cells showing CRT and HMGB1 expression. The number of cells per field of view and the percentage of positive cells per field of view are shown. Data are representative of three independent experiments. The unpaired Student's t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: PTT using aFn14-PBNP triggers thermal and immunogenic cell death in treated GBM tumor lines. (A–D) U87 and U251 cells undergo thermal (A) and immunogenic cell death (B–D) after PTT with aFn14-PBNP, as noted by a decrease in cell viability (A), decrease in intracellular ATP (B), increase in surface calreticulin expression (C), and decrease in intracellular HMGB1 (D) with increasing laser powers (0.75–2 W). (E–M) PTT-induced changes in immunophenotype in GBM tumor lines, including MHC expression (E,F), tumor specific antigen expression (G), immune checkpoint inhibitor expression (H,I), and T cell costimulatory markers (J–M). CTRL = untreated cells not irradiated; laser alone = untreated cells irradiated with laser; aFn14-PBNP = aFn14-PBNP-treated cells not irradiated; remaining conditions are aFn14-PBNP-treated cells irradiated at indicated laser powers. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.
Supplier Page from Abcam for Anti-HMGB1 antibody [EPR3507] (Alexa Fluor® 647)