Abstract PR008: Oligodendrocyte-secreted factor L-PGDS potentiates phagocytosis of tumor-associated myeloid cells in pediatric high-grade gliomas

Abstract

Pediatric brain tumors are the most common solid tumors in children. Pediatric high-grade gliomas (pHGGs) account for approximately 17% of brain tumors in children aged 0–14 years and remain largely incurable. Immunotherapies, including immune checkpoint blockade, have shown minimal benefit in pHGGs, largely due to immunosuppressive tumor-associated microglia/macrophages (TAMs), which can comprise up to 30% of the tumor mass. Our previous work showed that TAMs in pHGGs adopt predominantly immunosuppressive M0/M2-like states with impaired phagocytic activity. We hypothesized that restoring TAM phagocytosis would enhance their anti-tumor function by promoting tumor cell clearance, antigen processing, and antigen presentation. However, the mechanisms responsible for defective TAM phagocytosis remain poorly understood. To identify regulators of myeloid cell phagocytosis in pHGGs, we developed a CRISPRa gain-of-function screening platform. The system was first validated in THP-1 cells expressing CRISPRa machinery (pKVL2-U6gRNA_SAM[BbsI]-PGKpuroBFP-W) using a gRNA targeting CD206, demonstrating the suitability of the platform. We then screened a lentiviral CRISPRa library targeting the promoters of 6,213 genes encoding membrane-associated proteins. In parallel, we performed a complementary ligand-receptor screen using the 40 most abundant cytokines and secreted factors in the brain together with CRISPRa activation of their corresponding receptors. These complementary approaches identified lipocalin-type prostaglandin D2 synthase (L-PGDS/PTGDS) and its downstream signaling through interacting its receptor MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) as potent enhancers of myeloid phagocytosis. Single-cell RNA sequencing and immunofluorescence demonstrated that PTGDS is highly and selectively expressed by oligodendrocytes, revealing an unexpected paracrine function beyond myelin production. Although PTGDS knockout did not significantly affect adult myelination, it markedly impaired microglial phagocytosis of myelin debris in mouse brains, indicating that oligodendrocyte-derived L-PGDS primarily regulates microglial function rather than myelin maintenance. In vitro validation further confirmed these findings. Recombinant L-PGDS treatment or CRISPRa-mediated activation of MARCKS significantly enhanced phagocytosis of Fluorophore-conjugated beads, myelin debris, and apoptotic cells in THP-1 cells, while recombinant L-PGDS similarly increased phagocytic activity in immortalized mouse microglia. Collectively, our study identifies L-PGDS as a previously unrecognized oligodendrocyte-derived regulator of microglial phagocytosis and establishes a novel oligodendrocyte–microglia paracrine signaling axis that constrains pHGG growth. Because impaired phagocytosis is a hallmark of tumor-associated macrophages across many cancers, targeting this pathway may provide a broadly applicable strategy to reprogram immunosuppressive TAMs into tumoricidal cells and improve the efficacy of cancer immunotherapy in pHGGs.