Fig. 3
Thioridazine enhances oxaliplatin-induced immunogenic cell death in CT26 mouse CRC cells. A Protein levels of autophagy and ER stress markers were examined in murine CRC cells CT26 following treatment with 50 µM OXA or co-treatment of 50 µM OXA and 5 µM THD for 24 h. 5 µM thiostrepton (TST) was used as a positive control for autophagy and ICD induction. The level of extracellular ICD markers including (B) ATP, (C) HMGB1, and (D) calreticulin (CRT), were examined using ATP detection kit, ELISA, and flow cytometer, respectively, after 24 h of single or combined drug treatment. *: statistical significance compared to vehicle group; #: statistical significance compared to OXA treated group. *: p < 0.05; **: p < 0.01; ***: p < 0.001. E Schematic of ICD vaccination assay in Balb/c mice for 33 days. Balb/c mice were subcutaneously injected into the right flank with 1 × 106 CT26 cells treated with OXA (50 μM) alone or in combination with THD (5 μM) for 24 h. One week later, each mouse was transplanted with 1 × 106 naïve CT26 cells into the left flank as a challenge to examine the efficacy of vaccination. F Images of mice that were sacrificed, and tumors harvested from the challenge site after 30 days are shown. The mice were implanted with pre-treated CT26 cells in the prime site, which received pre-treatment with OXA alone or in combination with either TST or THD. In the vehicle group, three mice developed tumors on the challenge site. In the groups treated with OXA alone or OXA in combination with TST, two mice in each group grew tumors. However, when OXA and THD were administered together, only one mouse developed a tumor. G Tumor volume was calculated using the equation: 0.5 × tumor length × (tumor width).2 after sacrifice. Tumor growth and body weight were measured every 3 days with an electronic caliper and an electronic weight scale, respectively, until sacrifice. Data are mean ± SEM (N = 3). Statistical analysis was performed using one-way ANOVA with Fisher’s LSD post hoc test. *: p < 0.05; **: p < 0.01; ***: p < 0.001