Calcitriol and hyperthermia potentiate gemcitabine efficacy - A Multifactorial Preclinical Evaluation in Pancreatic and Breast Cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, characterized by chemoresistance and poor prognosis. Combination therapies that target multiple tumor vulnerabilities simultaneously are a promising strategy to overcome treatment limitations. This study aimed to evaluate the therapeutic potential and synergistic effects of a triple-modality treatment comprising gemcitabine (GEM), calcitriol (CAL), and hyperthermia (HT) in preclinical models of pancreatic and breast cancer. In vitro experiments were conducted using three cancer cell lines: human PDAC (PANC-1), murine PDAC (Panc02), and murine triple-negative breast cancer (4T1). Treatments included gemcitabine (GEM), calcitriol (CAL), and hyperthermia (HT). Cell viability, apoptosis, and metabolic activity were assessed via cell counting, MTT assays, real-time live cell imaging, and flow cytometry. Protein expression of VDR and HSP70 was analyzed by Western blotting. In vivo, Panc02 tumors were orthotopically implanted in C57BL/6J mice and treated with GEM, CAL and HT. Tumor growth was monitored by ultrasound, and survival was evaluated using Cox regression models. Triple therapy significantly reduced cell viability and metabolic activity across all models, with the strongest cytotoxic effects observed in 4T1 cells. Synergistic effects were observed at low GEM concentrations, especially in resistant PANC-1 cells. In vivo, triple therapy inhibited tumor growth, reduced peritoneal metastases, and improved survival (~ 85%), with limited systemic toxicity. The combination of GEM, CAL, and HT shows strong synergistic anti-cancer effects both in vitro and in vivo. This triple therapy enhances treatment outcomes in resistant tumors such as PDAC and represents a clinically relevant, low-toxicity approach for multimodal cancer treatment.