Single cell spatial analysis reveals the topology of immunomodulatory purinergic signaling in glioblastoma

Abstract

How the glioma immune microenvironment fosters tumorigenesis remains incompletely defined. Here, we use single-cell RNA-sequencing and multiplexed tissue-imaging to characterize the composition, spatial organization, and clinical significance of extracellular purinergic signaling in glioma. We show that microglia are the predominant source of CD39, while tumor cells principally express CD73. In glioblastoma, CD73 is associated with EGFR amplification, astrocyte-like differentiation, and increased adenosine, and is linked to hypoxia. Glioblastomas enriched for CD73 exhibit inflammatory microenvironments, suggesting that purinergic signaling regulates immune adaptation. Spatially-resolved single-cell analyses demonstrate a strong spatial correlation between tumor-CD73 and microglial-CD39, with proximity associated with poor outcomes. Similar spatial organization is present in pediatric high-grade gliomas including H3K27M-mutant diffuse midline glioma. These data reveal that purinergic signaling in gliomas is shaped by genotype, lineage, and functional state, and that core enzymes expressed by tumor and myeloid cells are organized to promote adenosine-rich microenvironments potentially amenable to therapeutic targeting.

Exploring glioblastoma stem cell heterogeneity: Immune microenvironment modulation and therapeutic opportunities

“…Remodeling in response to hypoxic or low glucose environments occurs through various mechanisms including epigenetic modification ( 3 , 126 , 127 ), HIF-1α signaling ( 125 , 128 , 129 ), and altered expression of metabolic enzymes and transporters ( 49 , 130 ). In general, metabolic profiles in GBM cells are spatially distinct and attributed to TME interactions and restricted nutrient availability ( 122 , 125 , 131 , 132 ) with different GSC subsets having distinct metabolic preferences and dependencies ( 121 , 133 , 134 ).…”

“…Furthermore, Coy et al. identified increased levels of extracellular immune-suppressive adenosine in GBM attributed to high expression of CD73, which functions alongside CD39 to metabolize ATP into adenosine, in hypoxia-responsive GBM cells ( 131 ). Collectively, these findings demonstrate a connection between TME-driven metabolic reprogramming and GSC-mediated immune suppression, and prompt further investigation into therapeutically relevant metabolic vulnerabilities in these immune-suppressive cells.…”

“…While many of the above spatial technologies have yet to be applied in the context of GBM, RNAScope and MSI methods have been utilized with the former identifying expression-based prognostic markers from GBM tissues ( 214 , 215 ). On the other hand, MSI methods have investigated GBM heterogeneity based on metabolic and proteomic profiles and drug distribution in tumors ( 131 , 216 – 220 ).…”

SLC11A1 as a stratification indicator for immunotherapy or chemotherapy in patients with glioma

“…To deepen our understanding of SLC11A1’s function in glioma’s tumor immune microenvironment (TIME), we utilized glioma single-cell data derived from the GEO dataset (GSE131928) ( 9 ), unsupervised analyses of this data identified 7 cell states representing the stromal, immune, and neoplastic cells commonly observed in glioma. According to the marker genes in the study of Neftel et al, neoplastic cells were split across four pan-glioma cell states, AC-like (EGFR), MES-like (CHI3L1), NPC-like (ELAVL4) and OPC-like (PDGFRA) ( 9 ), that observed across many glioma single-cell studies ( 36 , 37 ). We also identified macrophages/monocytes by C1QB, C1QC and FCER1G ( 38 ), CD8 Tex cells by CD3D and IFITM1 ( 39 ), and oligodendrocyte by ERMN and KLK6 ( 40 ).…”

Chemical QuantArray: A Quantitative Tool for Mass Spectrometry Imaging

“…Immunohistochemical expression of CD73 was evaluated using a semiquantitative scoring system. 25 In brief, cases were scored according to the following system: Absent (0): No expression of CD73 by tumor cells, or weak membranous staining in <10% of tumor cells; Weak (1): Weak membranous staining in >10% of tumor cells; Moderate (2): Moderate membranous staining in >10% of tumor cells, or strong/ circumferential membranous staining in <10% of tumor cells; Strong (3): Strong/circumferential membranous staining in >10% of tumor cells. The overall percentage of tumor cells expressing CD73 in each specimen was also assessed (Table 1).…”