Introduction: Oxidative stress-based therapies exploiting elevated tumor reactive oxygen species (ROS) offer promise but are limited by insufficient selectivity and adaptive resistance. To overcome these barriers, we developed a combinatorial strategy that synergistically integrates a ROS-responsive prodrug with pharmacologic ascorbate, a tumor-selective pro-oxidant, to initiate a self-amplifying oxidative cycle specifically within the tumor microenvironment.

Methods: We evaluated the combination of pharmacologic ascorbate (P-AscH⁻) with a novel ROS-responsive prodrug across multiple cancer cell lines, including triple-negative breast cancer (TNBC) models (MDA-MB-231, MDA-MB-468). Cytotoxicity was assessed via MTT and clonogenic assays, while mechanisms were probed using flow cytometry for apoptosis (Annexin V/PI) and DNA damage (γH2AX). In vivo efficacy was evaluated in orthotopic and patient-derived xenograft (PDX) models of TNBC. Biosafety was assessed via serum chemistry and histopathology.

Results: The combination demonstrated strong synergistic cytotoxicity (combination index < 0.5) across all TNBC lines, with significantly attenuated effects in non-malignant fibroblasts. Mechanistically, ascorbate selectively amplified H₂O₂ flux in cancer cells, triggering prodrug activation and establishing a feed-forward oxidative loop, evidenced by a 4-fold increase in γH2AX foci and 70% apoptosis versus monotherapies. In vivo, the combination achieved complete and durable tumor regression in 80% of PDX-bearing mice, with no recurrence over 120 days and no significant off-target toxicity.

Conclusions: This redox-based combination strategy establishes a novel paradigm for precision cancer therapy by dynamically manipulating tumor redox biology. The self-amplifying oxidative mechanism enhances prodrug specificity and potency while repurposing ascorbate as a targeted synergistic agent. This approach is now being advanced toward translational studies.