Endocrine Abstracts

Background: Ferroptosis is an emerging form of regulated necrotic cell death characterized by excessive lipid peroxidation. Adrenocortical carcinoma (ACC) cells have been shown to be highly susceptible to ferroptosis caused by active steroid hormone synthesis. While therapeutic activation of ferroptosis has been proposed as a novel treatment strategy, its impact on the tumor immune microenvironment (TIME) remains elusive. Macrophages are an important TIME component and modulate inflammation through phagocytosis and the release of cytokines, however the relationship between tumor cell ferroptosis and macrophages is unclear.

Objectives: Characterization of the mediators and consequences of ferroptosis in ACC cell lines on macrophages.

Methods: Prostaglandin E2 (PGE2) secretion in cell culture supernatants was quantified by LC–MS/MS and ELISA. Human peripheral blood mononuclear cells were isolated with Lymphoprep™ gradient and macrophages differentiated with GM-CSF/M-CSF and polarizing factors. Macrophages were characterized by western blotting, immunofluorescence and metabolic phenotyping. Phagocytosis of ferroptotic ACC cells by macrophages was analyzed by flow cytometry.

Results: Treatment of the ACC cell line NCI-H295R with the ferroptosis inducer RSL3 resulted in increased secretion of the immune modulator PGE2. The release of PGE2 was completely blocked upon inhibition of both ferroptosis with the antioxidant Liproxstatin-1 and cyclooxygenase (COX) with celecoxib or diclofenac. Treatment of isolated macrophages with PGE2 lead to macrophage polarization towards an anti-inflammatory M2-like phenotype, which was characterized by high expression of CD163. Co-culturing of these macrophages with RSL3 treated NCI-H295R cells resulted in efficient clearance of ferroptotic ACC cells.

Conclusion: Ferroptosis induction with RSL3 leads to the release of PGE2, an immune suppressor involved in M2-like polarization. ACC cells undergoing ferroptosis are phagocytosed by M2-like macrophages, which is expected to further maintain an anti-inflammatory TIME.