Enteric glia have been recently recognized as key components of the colonic tumor microenvironment indicating their potential role in colorectal cancer pathogenesis. Although enteric glia modulate immune responses in other intestinal diseases, their interaction with the colorectal cancer immune cell compartment remains unclear. Through a combination of single-cell and bulk RNA-sequencing, both in murine models and patients, here we find that enteric glia acquire an immunomodulatory phenotype by bi-directional communication with tumor-infiltrating monocytes. The latter direct a reactive enteric glial cell phenotypic and functional switch via glial IL-1R signaling. In turn, tumor glia promote monocyte differentiation towards pro-tumorigenic SPP1⁺ tumor-associated macrophages by IL-6 release. Enteric glia cell abundancy correlates with worse disease outcomes in preclinical models and colorectal cancer patients. Thereby, our study reveals a neuroimmune interaction between enteric glia and tumor-associated macrophages in the colorectal tumor microenvironment, providing insights into colorectal cancer pathogenesis.

Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM‐associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient‐derived GBM stem cell cultures and combined single‐cell RNA sequencing of GAM‐GBM co‐cultures and real‐time in vivo monitoring of GAM‐GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM‐induced GAM polarization and the ability to attract and activate GAMs—features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock‐out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM‐GBM interactions can be studied in a clinically relevant way using co‐cultures and avatar models, while offering new opportunities to identify promising immune‐modulating targets.

Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs – features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.

Background: Immunotherapies have improved outcomes in many cancers but show limited efficacy in glioblastoma (GBM). This study aimed to determine whether immunotherapy could be tailored to GBM by functional subtyping of vascular-immune landscapes. Methods: We employed single-cell RNA sequencing, multiplex immunohistochemistry to characterize three distinct TIME subtypes in human and murine GBMs. We evaluated responses to combination of anti-angiogenic immunomodulating therapies (CD40 Ag, anti-PDL1, PI3Kγ/δ inhibition) in orthotopic syngeneic GBM mouse models. Results: We identified three distinct functional TIME subtypes with unique vascular-immune landscapes in human and murine GBM: TIME-low (immune-low/deserted, leaky vasculature), TIME-med (intermediate immune-infiltration, angiogenic), and TIME-high (heavily infiltrated with immunosuppressive myeloid cells and dysfunctional T cells). Representative mouse models of TIME-GBMs responded in subtype specific ways to anti-angiogenic immunomodulating therapies. TIME-low GBMs enhanced T-cell activity but relapsed due to emerging myeloid immunosuppression, concomitant with mesenchymal transition. TIME-med displayed the most immune-activated, yet angiogenic phenotype, and showed overall good responses to various anti-angiogenic immunomodulating therapies. TIME-high GBMs were mostly non-responsive but improved when the myeloid-cell PI3Kγ was targeted. However, CD40 agonist treatment, expected to enhance APC function, unexpectedly worsened survival by promoting angiogenesis and heightening immunosuppression, leading to dysfunctional T cells and reduced NK cell recruitment, and subsequent enhanced tumor propagation. Conclusions: Our study reveals three GBM TIME subtypes with distinct vascular-immune landscapes that require tailored therapies. TIME-med tumors are predicted to respond best to immunotherapies, TIME-low tumors show transient effects with anti-angiogenic immunomodulating therapies, while TIME-high tumors, due to their profound immunosuppression, can even have worse outcomes. Key points: Three TIME subtypes were identified in GBM with distinct vascular-immune landscapes TIME subtypes show divergent immunotherapy responses TIME classification supports personalized treatment strategy for GBM immunotherapy Importance of the Study: This study advances glioblastoma immunotherapy by providing the first comprehensive single-cell characterization of TIME subtypes, moving beyond bulk RNA-sequencing to reveal detailed functional states of immune cells. We establish clinically relevant murine models that recapitulate human TIME subtypes, enabling preclinical testing of TIME-targeted therapies. Our findings identify TIME-low GBM as immune deserted and TIME-med tumors as the most immunotherapy-responsive subtype that should be prioritized for clinical selection. We found that high immune infiltration correlates with non-responsiveness and even unexpected detrimental effects with CD40 agonist treatment in TIME-high tumors—critical information given ongoing clinical trials. Identifying distinct immunosuppressive mechanisms across TIME subtypes and differential treatment responses provides a framework for personalized immunotherapy selection. The immediate translational impact of this work highlights the importance of TIME classification for treatment stratification and the urgent need to consider TIME status in clinical trial design, potentially explaining variable patient responses in previous GBM immunotherapy trials.

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