Immunogenic ferroptosis protects against the development of tumour growth
Authors: Saviuk М. О. , Turubanova V. D. , Efimova Yu. V. , Mishchenko Т. А. , Mishchenko Т. А. , Vedunova М. V. , Krysko D. V. 
Published: May 2024

Immunotherapy is a proven and effective anti-tumor strategy, which can be used alongside surgery, radiation therapy, and chemotherapy [1]. Immunogenic cell death (ICD) was identified as a critical factor determining the effectiveness of cancer treatment [2]. The concept of ICD combines the capacity to destroy cancer cells effectively, with the activation of a cancer cell-specific immune response, leading to potent and long-lasting anti-cancer immunity. ICD-inducing agents activate a perilous pathway that triggers the release of ICD mediators called damage-associated molecular patterns (DAMPs). DAMPs encompass a group of naturally occurring molecules that gain immunostimulatory qualities upon exposure to the outer cell membrane or when liberated into the extracellular matrix in a specific spatiotemporal fashion. ATP, the nuclear protein HMGB1, calreticulin (CRT), and type I interferons (IFNs) are among the identified factors [8].
The concept of ICD was initially described for cancer cells undergoing apoptosis, but it was expanded to encompass additional forms of cell death, such as necroptosis, pyrroptosis, ferroptosis, nontosis, etc. [11]. Ferroptosis is a regulated iron-dependent type of cell death that is characterized by the buildup of reactive oxygen species in the cell.
In this study, the immunogenicity of ferroptotic cancer cells in vitro was assessed and their potential as an alternative approach to cancer immunotherapy was tested.
Glioma GL261 and fibrosarcoma MCA205 cells were induced with one of the well-known inducers of ferroptosis, RSL3 (RAS-Selective Lethal 3). After 24 hours of RSL3 stimulation, 80% of GL261 cells and 90% of MCA205 cells showed positivity to Annexin V/Sytox Blue, indicating they were in the late stage of ferroptosis. Similarly, after 3 hours of RSL3 stimulation, 50% of GL261 cells and 45% of MCA205 cells were double positive with Annexin V/Sytox Blue indicating their late-stage ferroptotic state. We evaluated the immunogenic features of early and late ferroptotic cells in vitro, specifically at 3 or 24 hours after RSL3 stimulation. To achieve this, we compared the phenotype of dendritic cells (BMDCs) exposed to late ferroptotic cells with BMDCs exposed to viable cancer cells. Furthermore, immunogenic apoptosis was induced with MTX as a positive control and LPS as a secondary positive control. Late ferroptotic MCA 205 cells surprisingly did not induce phenotypic BMDC maturation, as indicated by the lack of surface activation of costimulatory molecules CD86, CD80, and MHCII. In contrast, a less pronounced phenotypic response compared to MCA205 cells was induced by early ferroptotic glioma GL261 cells. Nonetheless, a decrease in the ability to activate dendritic cells was observed for late ferroptotic glioma cells as well.
The study used the standard tumor prophylactic vaccination model on immunocompetent C57BL/6 J mice to assess the adaptive immune system activation by early ferroptotic cancer cells. Mice were immunized with early or late ferroptosis MCA205 cells. As a negative control, we used PBS or cells that underwent spontaneous necrosis. The mice that were immunized were later confronted with viable MCA205 tumor cells. Protection against tumor growth at the site of infection was deemed indicative of successful activation of the adaptive immune system. Remarkably, mice that received immunization with late ferroptotic MCA205 cells, induced with RSL3 for 24 hours, exhibited conspicuous tumor growth at the infection site, signifying that late ferroptotic cells are not immunogenic in vivo, as per our preliminary observations in vitro.