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Immunotherapeutic approaches to manage patients with advanced gastrointestinal malignancies are desired; however, mechanisms to incite tumor-specific immune responses remain to be elucidated. Rose bengal (RB) is toxic at low concentrations to malignant cells and may induce damage-associated molecular patterns; therefore, we investigated its potential as an immunomodulator in colon cancer. Murine and human colon cancer lines were treated with RB (10% in saline/PV-10) for cell cycle, cell death, and apoptosis assays. Damage-associated molecular patterns were assessed with western blot, ELISA, and flow cytometry. In an immunocompetent murine model of colon cancer, we demonstrate that tumors regress upon RB treatment, and that RB induces cell death in colon cancer cells through G2/M growth arrest and predominantly necrosis. RB-treated colon cancer cells expressed distinct hallmarks of immunogenic cell death (ICD), including enhanced expression of calreticulin and heat-shock protein 90 on the cell surface, a decrease in intracellular ATP, and the release of HMGB1. To confirm the ICD phenotype, we vaccinated immunocompetent animals with syngeneic colon cancer cells treated with RB. RB-treated tumors served as a vaccine against subsequent challenge with the same CT26 colon cancer tumor cells, and vaccination with in vitro RB-treated cells resulted in slower tumor growth following inoculation with colon cancer cells, but not with syngeneic non-CT26 cancer cells, suggesting a specific antitumor immune response. In conclusion, RB serves as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer.""Discussion:
...In the current study, we have provided several lines of evidence demonstrating that RB exhibited pronounced direct cytotoxicity in colorectal cancer cells both in vivo and in vitro. Our data also confirmed RB-induced prominent cell growth arrest at the G2/M phase and predominant necrotic cell death that was partially dependent on lysosome function. Furthermore, the findings from the current study identified that RB promoted expression of hallmarks related to ICD in colon cancer cell lines. Vaccination with RB-treated colon cancer cells and intralesional tumor injection resulted in retardation in tumor growth or prevention of subsequent tumor formation following challenge with the same tumor cells. These findings show that RB may serve as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer. Additional studies are warranted to elucidate the therapeutic potential of RB-induced ICD."