Mitomycin C Induces Immunogenic Cell Death Biomarkers In Human Bladder Tumors

Abstract

IntroductionStandard‐of‐care for non‐muscle invasive bladder cancer is complete transurethral tumor resection followed by intravesical therapy with BCG, mitomycin C (MMC), gemcitabine, or other agents. Tumor recurrence is common and occurs in 40% to 80% of patients, with 10–20% progressing to muscle‐invasive disease. Earlier studies have shown selected cytotoxic agents induce immunogenic cell death (ICD) that promotes tumor response to immunotherapy and thereby offers the opportunity of developing synergistic combinations of ICD‐inducing cytotoxics with immune checkpoint blockers. The current qualifications for an ICD‐inducing cytotoxic include induction of DAMPs (danger‐associated molecular patterns) in vitro and vaccination with cytotoxic‐treated tumor cells in immunocompetent mice. Under these criteria, MMC is considered a poor ICD inducer. The present study tested the hypothesis that MMC can induce ICD in a more clinically relevant model, i.e., 3D histocultures of bladder tumors obtained from patients.MethodsWe evaluated the ability of MMC to induce two ICD biomarkers (i.e., ecto‐CRT (calreticulin) and release of HMGB1 from nucleus) and/or dendritic cell (DC) maturation in 3 bladder cancer cell lines with different differentiation status (RT4, T24, and TCCSUP), and 21 MMC‐treated human bladder tumor histocultures including 11 archived paraffin‐embedded tumors and 10 freshly obtained surgical tumor specimens. The methods included immunocytochemical staining flow/immune‐cytometry, immunoblotting, and immunohistochemical staining and quantitative imaging. Co‐cultures of human peripheral blood DC with MMC‐treated tumor cells (48 h) were used to detect DC maturation markers (CD83 and HLA‐DR).ResultsOver 85% of human bladder tumors (7/10 fresh tumors and 11/11 archived tumors) showed ecto‐CRT and/or HMGB1 release upon treatments with MMC at clinically achievable concentrations (1 and 3 μM). Induction of ICD markers by MMC was time‐dependent (peaked at 18 h) and increased with tumor grade (TCCSUP (grade 4) >T24 (grade 3)>RT4 (grade 1) (3 experiments, P<0.01, 2‐way ANOVA). HMGB1 release was increased in three cell lines with a dose‐dependent manner after 48 h MMC treatment (2 experiments, p value<0.05, 2‐way ANOVA). MMC further induced DC maturation in TCCSUP cells but not RT4 cells.ConclusionOur results indicate MMC induced ICD biomarkers and/or DC maturation in cultured human tumor cells and histocultures of human bladder tumors; the MMC effects were dependent on time, tumor grade and culture conditions.Support or Funding InformationSupported in part by Mosier Endowed Chair in Pharmaceutical Sciences at University of Oklahoma, Albert Charitable Trust, 1R01CA163015 from National Cancer Institute, and University of Kansas Cancer Center P30‐CA168524‐03.