Abstract A011: Genome-wide CRISPR screening identifies novel regulators of macrophage phagocytosis against glioblastoma

Abstract Glioblastoma (GBM) is an aggressive brain cancer characterized by profound immunosuppression, largely driven by tumor-associated macrophages (TAMs), which account for 30-40% of the tumour bulk. In normal tissue homeostasis, macrophages detect, engulf, and clear stressed and cancerous cells through phagocytosis. TAMs, however, display markedly impaired ability to do so, and instead promote tumour growth. We sought to identify molecular pathways that regulate macrophage phagocytosis of GBM cells, with the goal of reprogramming TAMs into anti-tumor effectors. We performed a pooled genome-wide CRISPR knockout screen in BV2 macrophages. Mutagenized macrophages were co-cultured with murine glioma cells expressing ZsGreen, a fluorescent reporter. Upon phagocytosis, macrophages engulf ZsGreen and thus fluoresce, allowing for separation and collection of phagocytic and non-phagocytic macrophages by flow cytometry. Synthetic guide RNAs (sgRNAs) were recovered and sequenced to identify gene knockouts that enhanced phagocytosis. Analysis of sgRNA enrichment in ZsGreen+ macrophages revealed overrepresentation of gene knockouts involved in glycosaminoglycan biosynthesis, particularly the chondroitin sulfate pathway. Targeted disruption of CS biosynthesis genes in follow-up CRISPR knockout macrophage cell lines increased uptake of GBM cells in vitro, confirming CS synthesis loss enhances tumor cell phagocytosis. To contextualize these findings in GBM, we characterized O-glycan synthesis in patient tumors. We found CS synthesis gene programs are upregulated in GBM compared to normal brain tissue, and myeloid cells are the dominant producers of CS within the GBM niche. These data identify CS synthesis as a novel inhibitory pathway of macrophage phagocytic function in GBM. This work highlights glycosaminoglycan metabolism as a potential target for therapeutic reprogramming of TAMs to enhance innate immunity and immunotherapy efficacy in GBM. Citation Format: Shan Grewal, Vaseem Shaikh, Adrian Granda Farias, Kevin R. Brown, Lucas Asselstine, Mohamed Taleb, Chitra Venugopal, Patricia Mero, Jason Moffat, Sheila K. Singh. Genome-wide CRISPR screening identifies novel regulators of macrophage phagocytosis against glioblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr A011.