Local Delivery of a Ferroptosis-inducing Drug Causes Immunogenic Cell Death and Activates an Effective Immune Response in Glioma

Introduction: Novel immunotherapeutic strategies have the potential to impact poor prognosis in glioblastoma (GBM). Ferroptosis, a programmed form of cell death based on lipid peroxidation, has the potential added benefit of causing immunogenic cell death (ICD). We investigated the therapeutical potential of a ferroptosis-inducing drug (RSL3) to effectively kill glioma cells via ICD which subsequently activates microglia in the tumor microenvironment with increased phagocytosis, antigen presentation and expression of cytokines involved in T-cell recruitment.

Methods: The acute effects on the tumor microenvironment of a ferroptosis-inducing drug, RSL3, were investigated in patient-derived organotypic slice cultures generated from surgical samples of GBM by flow-cytometry, immunohistochemistry (IHC), and single-cell RNA sequencing (scRNAseq). The effects of RSL3 on phagocytosis were measured in glioma-myeloid co-cultures. To examine in vivo effects, RSL3 was infused intratumorally for 7 days via a micro-osmotic implantable pump in a murine glioma model. Animals were sacrificed and tumors were analyzed by IHC, flow-cytometry and scRNAseq.

Results: RSL3-driven ferroptosis caused ICD in glioma cells with induction of cellular stress and translocation of calreticulin from the endoplasmic reticulum to the cellular membrane which led to significantly increased phagocytosis of glioma cells and myeloid cell activation. In patient-derived slice cultures, RSL3 treatment reprogrammed local myeloid populations to promote phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3 in a murine glioma model polarized myeloid cells to a phagocytic phenotype and increased intratumoral levels of CD4+ and CD8+ T- cells.

Conclusion : Drug induced ferroptosis in glioma promotes endoplasmic reticulum-stress driven ICD, phagocytosis and antigen presentation with activation of intratumoral myeloid populations and induction of an adaptive immune response. The dual effects of direct tumor cell cytotoxicity and induction of an anti-tumor immune response make local delivery of RSL3 an attractive novel strategy in a clinical trial for glioma.