Short-term high-dose gemcitabine induces PERK-mediated immunogenic cell death and potentiates antitumor immunity in bladder cancer

Background: Gemcitabine is a widely used intravesical chemotherapeutic agent for non-muscle-invasive bladder cancer (NMIBC), yet its efficacy remains suboptimal, with high recurrence rates. While traditionally considered a cytotoxic agent, emerging evidence suggests that certain chemotherapeutics may exert immunomodulatory effects. This study investigated whether gemcitabine induces immunogenic cell death (ICD) and explored the underlying mechanisms involving endoplasmic reticulum (ER) stress. Methods: A retrospective analysis was conducted to assess the association between gemcitabine-related complications and NMIBC recurrence. In vitro assays were used to evaluate ICD hallmarks (ATP, HMGB1, CALR) and pro-inflammatory cytokines. RNA sequencing and western blotting were performed to explore ER stress pathways. Functional assays included dendritic cell (DC) maturation, phagocytosis, and CD8+T cell proliferation and cytotoxicity. In vivo vaccination and T cell depletion models were used to evaluate antitumor immunity. Results: Clinical data revealed an inverse correlation between gemcitabine-related complications and tumor recurrence. Gemcitabine induced classical ICD markers in bladder cancer cells and elicited CD8+T cell-mediated antitumor immunity in vivo. Mechanistically, gemcitabine activated the PERK branch of the ER stress response, promoting CALR translocation and DAMP release. PERK overexpression enhanced DC maturation, phagocytosis, and stimulation of CD8+T cell proliferation and cytotoxicity, whereas PERK knockdown impaired these effects. Conclusions: This study demonstrates that short-term, high-dose gemcitabine induces PERK-dependent ICD, which enhances antitumor immune responses via DC and CD8+T cell activation. These findings provide a mechanistic rationale for combining gemcitabine with immunotherapy and suggest PERK as a potential biomarker and therapeutic target for ICD enhancement in bladder cancer.