Humanized glioblastoma patient-derived orthotopic xenografts recreate a locally immunosuppressed human immune ecosystem amenable to immunotherapeutic modulation

ABSTRACT Immune-based strategies have so far failed to demonstrate clinical benefit in glioblastoma (GBM), largely due to the profound immunosuppressive tumor microenvironment (TME). To achieve more predictive preclinical insights, advanced in vivo models that faithfully recapitulate the human brain immune landscape are urgently needed. Here, we established GBM patient-derived orthotopic xenografts (PDOXs) across diverse mouse strains, including humanized models. Humanization was achieved through transplantation of CD34 + hematopoietic stem cells (HU-CD34 + ) or peripheral blood mononuclear cells (HU-PBMC). Both models successfully reconstituted human T-cells systemically, with stronger engraftment in HU-CD34 + mice. We observed selective infiltration and spatial organization to intracranial GBM tumors, including exhausted, memory-like, and regulatory CD4 + T-cell phenotypes, TIM-3 + immunosuppressive-like myeloid cells and intratumoral B cells. Mouse microglia-derived tumor-associated macrophages (TAMs) remained the dominant immunosuppressive immune population. Anti-PD-1 therapy, but not anti-GITR, modestly modulated the infiltration dynamics, demonstrating the susceptibility of the reconstructed adaptive immunity to immunotherapeutic intervention. These findings position humanized GBM PDOXs as a relevant preclinical platform to interrogate tumor-immune interactions and evaluate immunotherapeutic strategies in a human context. Key points GBM PDOXs developed in HU-CD34 + and HU-PBMC mice faithfully reconstitute systemic and local human adaptive immunity. Human immune components undergo selective infiltration, spatial organization and transition towards exhausted CD4 + T-cells and immunosuppressive CD11c + myeloid cells. Anti-PD-1, but not anti-GITR, locally promote human immune infiltration into intracranial GBM tumors, while sparing systemic compartments. Humanized GBM PDOXs provide a powerful preclinical platform to test novel immunotherapeutic strategies. Study importance Immune checkpoint blockade has shown limited efficacy in GBM, reflecting the highly immunosuppressive and lymphocyte-poor nature of the TME. Conventional syngeneic and GEMM models fail to recapitulate these features, contributing to the translational disconnect between preclinical success and clinical failure. Humanized mice provide a solution to interrogate human-specific immunity in vivo , but their use in GBM has remained limited. Here, we provide the first comparison of GBM PDOX modeling in two complementary modes of humanization based on CD34 + HSCs and PBMCs. We systematically profile systemic and intratumoral compartments, showing that these models faithfully reconstitute human adaptive immunity and capture the interplay with the murine brain TME. Furthermore, we demonstrate clinically-relevant responses upon treatment with checkpoint antibodies targeting PD-1 and GITR, showing modulation of human immune subsets without altering murine TAM immunosuppression, underscoring the translational value of the system. This study establishes humanized GBM PDOXs as a versatile platform for dissecting tumor-immune interactions in the brain and for preclinical evaluation and development of novel immunotherapies. Graphical abstract