Tumor cell death by ferroptosis contributes to an immunosuppressive tumor microenvironment in syngeneic murine models of cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by profound metabolic rewiring and a strongly immunosuppressive tumor microenvironment, both of which contribute to poor therapeutic responses. Immunogenic cell death (ICD) represents a potential strategy to overcome immune suppression by coupling tumor cell death to anti-tumor immune activation. Here, we investigated whether targeting amino acid metabolism in PDAC can induce ICD and promote tumor immunity. Through a focused metabolic screen in a panel of syngeneic mouse cancer cell lines, we identified cysteine restriction as a robust inducer of multiple damage-associated molecular patterns (DAMPs) in vitro, hallmark features of ICD. In addition to driving DAMPs, cystine-deprived tumor cells also promoted dendritic cell phagocytosis, maturation, and proinflammatory cytokine production in vitro. Because cysteine deprivation is a known trigger of ferroptosis, we further demonstrated that pharmacologic inhibition of glutathione peroxidase 4 (GPX4) similarly elicited ICD-associated features, which were reversible by the ferroptosis inhibitor Ferrostatin-1 (Fer-1). To define additional immune-modulatory signals associated with ferroptosis, we performed metabolomic and lipidomic profiling of cells undergoing, but not yet committed to, ferroptotic death. These analyses revealed selective release of immunosuppressive metabolites and oxidized phospholipids. Consistent with this, conditioned media from ferroptotic cells impaired CD8⁺ T cell proliferation and cytotoxicity in vitro. Thus, together our results indicated that the induction of ferroptotic immunogenic cell death led to the release of both pro- and anti-inflammatory signals. Subsequent analysis in vivo revealed that ferroptotic tumor cells predominantly contributed to a tumor-protective environment. In particular, tumors inoculated with ferroptotic cells were enriched with immunosuppressive myeloid cells and exhibited reduced populations of tumor-infiltrating CD8+ T cells. Further investigation using immune compromised mice suggested that ferroptotic cells may suppress both adaptive and innate immune responses. Collectively, these results underscore the complex and highly context-dependent effects of ferroptosis on tumor immunity, highlighting the critical importance of in vivo models to determine true immunogenic potential within the tumor microenvironment.