Abstract A053: Tumor-localized myeloperoxidase enhances tumor chemoresistance, migration, and invason.

Abstract Neutrophils frequently infiltrate solid tumors, where they can have pro-tumor effects that significantly worsen clinical outcomes for many types of cancer. However, our understanding of how neutrophils benefit tumors is far from complete. In the tumor microenvironment, neutrophils encounter myriad signals that stimulate the release of myeloperoxidase (MPO), a peroxidase that has commonly been thought to be cytotoxic due to its catalytic generation of the highly reactive oxygen species, hypochlorous acid. However, in this study we show that exogenous MPO, such as that released by neutrophils into the tumor microenvironment, acts as a tumor-promoter. Using fluorescent microscopy of cultured tumor cells, we show that MPO treatment significantly enhances chemoresistance of cells from every tumor model tested, including breast, ovarian, prostate, and bladder cancers, among others. Cells treated with both MPO and doxorubicin have a more than 2-fold increase in EC50 compared to cells treated with doxorubicin alone. Many cell types also show significant resistance to other chemotherapies, such as docetaxel, cisplatin, and ribociclib. Using transwell migration and Matrigel invasion assays, we showed that MPO-treated tumor cells have increased migration and invasion, indicating that MPO may also function to increase tumor metastasis. To determine whether MPO’s enzymatic activity is necessary for its pro-tumor effects, we generated an enzymatically dead mutant, MPO(M409V). We found that MPO(M409V) did not enhance chemoresistance. Additionally, cells treated with MPO(M409V) did not show increases in migration or invasion above untreated cells. We also wanted to understand how MPO’s localization affected tumor cells in these assays. We showed that wild type MPO binds and is rapidly internalized into tumor cells, and, by 14 hours, localizes exclusively to the nucleus. Previous studies have suggested that MPO internalization is due to a dense patch of basic residues on the surface of MPO. Therefore, we generated a charge-mutant MPO (MPO(R559E/R603A/R622E/K652E/R653P)), where 5 residues in the “basic patch” were changed to acidic or neutral amino acids. Immunocytochemistry with the charge-mutant MPO showed decreased intracellular localization, with no nuclear staining. When we tested the effects of the charge-mutant MPO on chemoresistance, migration, and invasion, we found no significant change compared to cells with MPO(M409V) or no MPO treatments. Together, these data show that MPO-treatment of cells increases chemoresistance, migration, and invasion in a manner that is dependent on MPO catalytic activity, electrostatic charge, and subcellular localization. Finally, we used immunocytochemistry on human tumor cells extracted by fine needle aspiration to show that MPO is found in human tumor cells, indicating that MPO could be a clinically-relevant tumor promoter. Citation Format: Rita P Loudermilk, James A Wells. Tumor-localized myeloperoxidase enhances tumor chemoresistance, migration, and invason. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A053.