Supplementary Material for: Selective Tumor Cytotoxicity via Singlet Oxygen Generation: Investigating Eosinophil Peroxidase and Myeloperoxidase in Cancer Therapy

Abstract
Introduction: Eosinophil peroxidase (EPO) and myeloperoxidase (MPO) are large, cationic enzymes secreted by granulocytes that bind preferentially to negatively charged cancer cell membranes produced by the Warburg effect. In the presence of halide cofactors and hydrogen peroxide (H₂O₂), and under acidic conditions that potentiate catalysis, they generate singlet oxygen (¹O₂*), a metastable oxygen state whose microsecond lifetime defines its diffusion radius and therefore its cytotoxic target. This confines reactivity to enzyme-enriched cancer cell surfaces, producing a spatially restricted therapeutic effect [Supplementary Note 1].
Methods: Human bladder cancer cell lines (5637, T24) and normal urothelial cells (SV-HUC1) were treated with porcine EPO (pEPO) or porcine MPO (pMPO) aggregate formulations in acidic medium (pH 5.3). Activation occurred upon addition of 10 mM H₂O₂ immediately prior to cell contact. Viability was assessed by MTS assay, and IC₅₀ values were determined by nonlinear regression. Mixed cultures of GFP⁺ SV-HUC1 and mCherry⁺ malignant cells were analyzed by fluorescence microscopy and flow cytometry. MPO localization was assessed by immunofluorescence, and DNA damage was evaluated by Western blotting for γH2AX and phospho-ATM. Independent toxicity of enzymes, cofactors, and peroxide was also tested.
Results: Aggregate formulations selectively eliminated bladder cancer cells while sparing SV-HUC1. IC₅₀ values were in the nanomolar range for malignant cells, with SV-HUC1 remaining viable at concentrations up to 200 nM. In mixed cultures, malignant cells were preferentially eliminated while GFP⁺ SV-HUC1 remained intact. Immunofluorescence confirmed MPO binding to malignant membranes, and DNA damage markers were induced only in cancer cells. Component testing showed no cytotoxicity from enzymes, cofactors, or 10 mM H₂O₂ alone; only higher peroxide concentrations produced injury.
Conclusion: Selective cytotoxicity arises from concurrent enzyme binding and ¹O₂* generation under acidic conditions, not from direct peroxide toxicity. Haloperoxidase therapy therefore offers a precise, contact-driven approach for post–bulk tumor treatment in non-muscle invasive bladder cancer. [Graphical Abstract]