Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity

Abstract

BackgroundImmunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term survival of patients. Induction of immunogenic apoptosis or necroptosis in cancer cells does activate antitumor immunity, but resistance to these cell death modalities is common. Therefore, it is of great importance to find other ways to kill tumor cells. Recently, ferroptosis has been identified as a novel, iron-dependent form of regulated cell death but whether ferroptotic cancer cells are immunogenic is unknown.MethodsFerroptotic cell death in murine fibrosarcoma MCA205 or glioma GL261 cells was induced by RAS-selective lethal 3 and ferroptosis was analyzed by flow cytometry, atomic force and confocal microscopy. ATP and high-mobility group box 1 (HMGB1) release were detected by luminescence and ELISA assays, respectively. Immunogenicity in vitro was analyzed by coculturing of ferroptotic cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and activation/maturation of BMDCs (CD11c+CD86+, CD11c+CD40+, CD11c+MHCII+, IL-6, RNAseq analysis). The tumor prophylactic vaccination model in immune-competent and immune compromised (Rag-2−/−) mice was used to analyze ferroptosis immunogenicity.ResultsFerroptosis can be induced in cancer cells by inhibition of glutathione peroxidase 4, as evidenced by confocal and atomic force microscopy and inhibitors’ analysis. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2−/− mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells.ConclusionsThese results pave the way for the development of new therapeutic strategies for cancers based on induction of ferroptosis, and thus broadens the current concept of immunogenic cell death and opens the door for the development of new strategies in cancer immunotherapy.

Ferroptosis becomes immunogenic: implications for anticancer treatments

“…In a recent issue of the Journal for Immunotherapy of Cancer , Efimova and colleagues described a novel approach for the induction of antitumor immunity by triggering ferroptosis-dependent ICD in preclinical models. 9 In a first step, the authors confirmed that the GPX4 inhibitor RSL3 induced cell death in mouse fibrosarcoma MCA205 cells via ferroptosis, rather than apoptosis and necroptosis. 9 The authors compared the impact of MCA205 cells treated with RSL3 for 1 h (“early ferroptotic cancer cells”) or 24 h (“late ferroptotic cancer cells”) on the maturation of mouse bone-marrow derived dendritic cells (BMDCs) using phagocytosis assays combined with the flow cytometric detection of DC maturation markers (e.g., MHC Class II, CD80, and CD86).…”

“… 9 In a first step, the authors confirmed that the GPX4 inhibitor RSL3 induced cell death in mouse fibrosarcoma MCA205 cells via ferroptosis, rather than apoptosis and necroptosis. 9 The authors compared the impact of MCA205 cells treated with RSL3 for 1 h (“early ferroptotic cancer cells”) or 24 h (“late ferroptotic cancer cells”) on the maturation of mouse bone-marrow derived dendritic cells (BMDCs) using phagocytosis assays combined with the flow cytometric detection of DC maturation markers (e.g., MHC Class II, CD80, and CD86). These co-culture assays led to the conclusion that early (but not late) ferroptotic cancer cells induced BMDC maturation, whereas late (but not early) ferroptotic cells were eliminated by BMDC-mediated phagocytosis.…”

“…Rechallenge of these “vaccinated” mice with live MCA205 cells revealed that only early (but not late) ferroptotic cancer cells could induce a protective immune response against this fibrosarcoma. 9 Finally, the authors proved that extracellular HMGB1 and adenosine triphosphate (ATP) were required for ICD caused by early ferroptotic cells. In particular, the pharmacological blockade of the ATP receptor (P2X7) using oxiATP reversed the tumor-protective effects of vaccination with early ferroptotic cancer cells.…”

“…In summary, Efimova et al reported that RSL3 stimulates ferroptosis that results in ICD, which occurs in an ATP- and P2X7-dependent manner. 9 It will be interesting to investigate whether this type of ICD can be induced by other ferroptosis activators, especially SLC7A11 inhibitors. Beyond this pharmacological question, a few general issues arise.…”

The dark side of ferroptosis in pancreatic cancer

“…In addition to causing inflammation-related immunosuppression, 3 several ferroptosis agents (e.g., RSL3) can provoke immunogenic cell death to improve cytotoxic T cell responses against tumors. 5 Similar, in addition to the chronic activation of the STING1 pathway that mediates genomic instability-induced tumorigenesis and metastasis, 6 the robust activation of the STING1 pathway by reagents (e.g., MSA-2, DMXAA, ADU-S100, and zalcitabine) or radiation therapy is an approach to enhance antitumor immunity or direct killing cancer cells in mouse models or clinical trials. [7][8][9] Acute activation of STING1-mediated T cell apoptosis may weaken anti-tumor immunity, 10 further arguing the dual role of STING1 in tumor therapy.…”