Abstract 188: Investigating the macrophage and microglia response to ferroptosis in glioblastoma

Abstract

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor. The median survival is less than two years. The mesenchymal (MES) subtype of GBM is particularly associated with treatment resistance, poor prognosis, and enriched infiltration of immune cells, including tumor-associated microglia and/or macrophages (TAMs). Despite promising results in preclinical studies, clinical trials targeting TAMs have been unsuccessful. Necrosis is a hallmark of GBM and correlates with unfavorable outcomes, but how necrosis results in poor prognosis is not well understood. We previously showed that neutrophils promote tumor necrosis and GBM progression by inducing ferroptosis, a form of regulated cell death due to iron-dependent lipid peroxidation. Understanding the effects of GBM ferroptosis on TAMs can provide further insights into how tumor necrosis ultimately leads to poor prognosis in GBM. We developed an orthotopic xenograft GBM mouse model harboring a constitutively active mutant of the Hippo pathway transcriptional coactivator with PDZ-binding motif (TAZ). Tumors of this model displayed MES characteristics and developed necrosis to an extent similar to patients with GBM. Using this model, confocal microscopy and flow cytometry studies revealed the presence of immune cells positive for markers of neutrophils and macrophages/microglia in the necrotic region of the tumor. Although we have yet to establish an in vitro model to study these cells, we found that conditioned media (CM) derived from ferroptotic GBM cells induced more macrophage differentiation from bone marrow cells when compared to live GBM CM. This led us to hypothesize that ferroptosis in GBM is immunogenic and orchestrates the immune response mounted by TAMs. We conducted an RNA sequencing (RNA-seq) study of bone marrow cells cultured in ferroptotic CM. Compared to live CM, ferroptotic CM upregulated several pro-inflammatory cytokine signaling pathways. We validated the mRNA expression levels of the identified cytokines by RT-qPCR. RNA-seq analysis also revealed the activation of phagosome formation pathway. Through phagocytosis assay, we showed that macrophages preferentially engulfed ferroptotic GBM cells over necrotic or live cells. To identify transcriptional changes in GBM cells undergoing ferroptosis, we carried out an RNA-seq study showing that ferroptotic GBM cells also upregulated several pro-inflammatory cytokine signaling pathways, which were validated by RT-qPCR. We also found that ferroptotic GBM cells released ATP extracellularly, where such release temporally coincided with the increase in pro-inflammatory cytokines. Our results suggest that ferroptosis in GBM is immunogenic and induces a pro-inflammatory response in macrophages/microglia. We are conducting further in vitro and in vivo studies to better understand how these macrophages/microglia modulate other immune cells and play a role in GBM progression. Citation Format: Stephen Y. Chih, Miaolu Tang, Soo Yeon Kim, Tong Lu, Rachel Harter, Jessica Thorpe, Wei Li. Investigating the macrophage and microglia response to ferroptosis in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 188.