Abstract 358: Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment
Authors: Rocha Murilo R. , Chhabra Yash , Huser Laura , Harper Elizabeth Irene , Dixit Agrani , Wang Vania , Carey Alexis E. , Palm Cheyenne , Huang Fan , Weeraratna Ashani T. 
Published: March 2024

Abstract
Aging is an independent prognostic factor for melanoma, the most aggressive form of skin cancer. Although immune therapy has shown a dramatic improvement in melanoma treatment, resistance to it still occurs in some patients. Higher oxidative damage in tumor cells as well as lipid and iron accumulation are singular characteristics of the aged tumor microenvironment (TME). Here, we investigate the impact of these characteristics of the aged TME in the promotion of ferroptosis, a caspase independent iron mediated type of cell death, and as possible modulators of the response to checkpoint blockade therapy. Lipid peroxidation levels were assessed through immunohistochemical staining for the marker 4-HNE in tumors of young and aged mice injected intradermally with Yumm1.7 cells.Correlation analysis between transcriptomic data (TCGA-SKCM) was performed to identify common denominators among proteins associated with the aged TME, ferroptosis pathway and IFN-y signaling. Our in vitro experiments consisted of viability assays with combinations of IFN-y, young and aged skin fibroblast conditioned media (CM), arachidonic acid (AA) or ferroptosis inducers such as RSL-3. Protein levels of ferroptosis related markers in melanoma cells treated with fibroblast conditioned media, and in Wnt5a high/low cell lines were analyzed through western blot (WB). Tumors from aged mice showed stronger staining with 4-HNE antibody when compared to young. Wnt5a (present in the aged TME) showed a strong correlation with several ferroptosis related transcripts. WB analysis of Wnt5a high/low lines indicate differences in GPX4, FTL1, SLC3A2, GCH1 and DHODH. Wnt5a levels show an inverse correlation with ferroptosis resistance and anti-ferroptotic proteins. Our data indicate that aged TME favors ferroptosis death in melanoma cells. To understand how ferroptosis impacts tumor growth in young and aged mice, we used a syngeneic tumor model injecting Yumm1.7 cells intra-dermally in mice and treated with a lipid peroxidation inhibitor, Liproxstatin, or an ferroptosis inducer, IKE. Liproxstatin lead to reduced tumor growth only in young mice followed by changes in immune cell populations such as monocytes and macrophages. IFNy induction of these events might be associated with changes in proteins of the pathway or alterations in fatty acid uptake as suggested by lipid accumulation and AA potentiation of its cytotoxic effects. Further exploration of ferroptosis will impact our understanding on how aged TME modulates immune cell killing and provide evidence to target or revert resistance to checkpoint blockade therapy associated with an IFN-y signature and new strategies for treatment of older patients.
Citation Format: Murilo R. Rocha, Yash Chhabra, Laura Huser, Elizabeth Irene Harper, Agrani Dixit, Vania Wang, Alexis E. Carey, Cheyenne Palm, Fan Huang, Ashani T. Weeraratna. Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 358.