CO + EO assembloids are established as an original human model of GBM plasticity at the neurovascular interface and identify CELF2 as a master regulator of GSC tropism and AHNAK as a mediator of vascular affinity, revealing a molecular axis that governs GBM invasion and providing potential avenues for future therapeutic intervention.
Abstract
Glioblastoma (GBM) remains one of the most aggressive human cancers, driven by cellular plasticity and dynamic interactions with the neurovascular niche. Yet, further refinement of existing preclinical models is needed to improve the reproduction of the human brain–vascular interface and facilitate the study of tumor-host crosstalk and invasion. To address this limitation, we developed a human induced pluripotent stem cell (hiPSC)-derived cortico-endothelial (CO + EO) assembloid model by fusing cortical and endothelial organoids. These assembloids spontaneously form branched vascular networks enriched in tight junction proteins (CLDN5, OCLN, ZO-1), thereby recapitulating key BBB-like features and providing a physiologically relevant human platform to investigate GBM–neurovascular interactions. Using this system, we uncovered a previously unrecognized CELF2-dependent glioma stem cell (GSC) tropism. CELF2-expressing GSCs preferentially infiltrate neural regions, where they induce neuronal apoptosis and disrupt endothelial tight junction structure, supporting an aggressive phenotype. In contrast, CELF2-deficient GSCs lose neurotropism, acquire mesenchymal features, and are redirected toward vascular compartments. This endothelial affinity depends on the scaffold protein AHNAK, strongly expressed at the plasma membrane of CELF2-deficient cells and enriched at tumor-endothelial interfaces. AHNAK knockdown partially abolishes endothelial infiltration, demonstrating its role in GBM vascular tropism. Moreover, analysis of patient GBM specimens confirmed that CELF2-positive tumor cells are enriched in poorly vascularized, mitotically active regions and are largely excluded from vessel-rich zones, closely resembling the behavior observed in assembloids. Transcriptomic profiling further revealed that CELF2 promotes a neuronal progenitor-like program while repressing mesenchymal and vascular-associated gene signatures, thereby shaping tumor identity, invasive behavior, and tissue preference. Collectively, this study establishes CO + EO assembloids as an original human model of GBM plasticity at the neurovascular interface. We identify CELF2 as a master regulator of GSC tropism and AHNAK as a mediator of vascular affinity, revealing a molecular axis that governs GBM invasion and providing potential avenues for future therapeutic intervention.
This human iPSC-derived tumor–brain organoid platform provides a reliable and scalable system for studying complex tumor-neural interactions and exploring therapeutic approaches that aim to eliminate the tumor while preserving neural function.
Ewa Grassin, H. Chintalapudi, Xianjun Dong et al.· bioRxiv· 0 citations
BACKGROUND
Glioblastoma (GBM) is the most common adult primary brain malignancy. Recent studies demonstrate that temozolomide (TMZ) facilitates the persistence of quiescent glioma stem cells (GSCs), which are responsible for GBM recurrence. An ideal therapy should eradicate both proliferating cells and GSCs. Abexinostat (Abx), a histone deacetylase inhibitor, was identified through connectivity mapping to target the specific GBM signature. Here, we demonstrate the anti-proliferative effect of Abx on both differentiated cells and GSCs.
METHODS
Using patient-derived tumor cultures (PDCs) to test Abx in vitro, ATAC-seq identified chromatin accessibility. Single-spheroid and alkaline phosphatase staining assays were used to test stem cell self-renewal. Aldehyde dehydrogenase activity distinguished mesenchymal GSCs. The efficacy of Abx with TMZ was evaluated in GSC-expressing CK9751 PDC and mesenchymal patient-derived xenografts (PDXs).
RESULTS
In PDCs (CK9495 and CK9751), Abx decreased the DNA repair machinery (RAD51, CHK1, Ku70, and MGMT) and induced apoptosis. Focused ATAC-seq analysis for promoters of DNA repair (RAD51, Ku70, CHK1, and BRCA1) and stemness (CD44, KLF4, c-Myc, and BMI1) revealed Abx decreased chromatin accessibility. Abx decreased stem cell self-renewal and reduced the mesenchymal stem cell signature (CD44, ALDH1A3 expression, and ALDH1 activity) in vitro GBM models. Abx reduced tumor growth and stemness markers in CK9751 PDC and mesenchymal PDXs.
CONCLUSION
Abx reduced both DNA repair machinery and GSC markers by decreasing chromatin accessibility. Abx reduced tumor growth and mesenchymal GSCs in vitro and in vivo in GBM PDC and PDX models, supporting Abx's potential to prevent GSC-mediated therapy resistance and improve patient survival.
Cancer-associated fibroblasts (CAFs) are central orchestrators of tumor progression, stromal remodeling, and immune evasion in glioblastoma. While tumor-derived secretomes can induce CAF-like phenotypes, it remains unknown whether therapy resistance reprograms the secretome to generate CAFs with amplified immunosuppressive and immune-shaping functions.
Induced CAFs (i-CAFs) were generated from normal fibroblasts using conditioned media derived from therapy-resistant (R) and therapy-responsive(NR) glioblastoma cells. CAF induction was confirmed by morphological transformation, increased proliferation and migration, and upregulation of ACTA2 (>4-fold) and TGFB1. Secretome-driven mechanisms were interrogated using integrated proteomic profiling and pathway-level bioinformatic analysis. CAFs were incorporated into 3D tumor organoids (30%) using both glioma cell lines and patient-derived samples, and immune modulation was assessed using co-culture systems with primary human immune cells.
Secretome exposure induced a stable CAF phenotype with enhanced proliferation, migration, and clonogenicity. Proteomic and pathway analysis identified coordinated activation of metabolic and stromal programs, including glycolysis and lipid metabolism (PGK1, FASN, CPT1A), lactate transport (SLC16A3), and ECM remodeling/contractility networks (LAMA5, PALLD, MYH10). In parallel, stress-response and redox regulators (NQO1, PRDX6, HSPB1) and interferon-associated signaling (STAT1, ISG15, IFITM family) were enriched, indicating a metabolically active and immune-modulatory state. Functionally, CAF incorporation increased tumoroid size, structural integrity, and reduced heterogeneity. Importantly, resistant-derived CAFs exhibited significantly higher monocyte chemotaxis compared to NR-derived CAFs and normal fibroblasts (P < 0.05). CAF-containing systems promoted macrophage polarization toward an immunosuppressive CD206+ phenotype, while targeting stress-associated pathways partially restored CD80+ polarization, with a more pronounced effect in resistant CAF contexts. These findings were recapitulated in patient-derived tumoroids, confirming translational relevance.
Glioblastoma secretomes drive fibroblast reprogramming into metabolically reprogrammed, contractile, and immunomodulatory CAFs. Therapy resistance amplifies these effects, enhancing immune recruitment and immunosuppressive polarization. Targeting CAF metabolic and stress-response pathways may represent a strategy to reprogram the tumor microenvironment and restore anti-tumor immunity.
Mahdieh Shokrollahi Barough, Maryam sadat Mirlohi, Amir Seyfoori, Meitham Amereh, Patrick B. Walter, Ibrahim Numanagic, Mohsen Akbari. Paracrine remodeling of normal fibroblasts into glioblastoma-associated states: An immune-enriched stromal model of GBM [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A043.
M. S. Barough, M. Mirlohi, Amir Seyfoori et al.· Cancer Research· 0 citations
Key features and heterogeneity of ccRCC vascular architecture are defined and ponds are highlighted as candidate contributors to treatment failure and targets for future interventions.
Noémie Brassard-Jollive, Y. Atlas, C. Compere et al.· Angiogenesis· 0 citations
Background Blood–brain barrier (BBB) disruption is a hallmark of acute traumatic brain injury (TBI), yet the immune–vascular mechanisms underlying endothelial dysfunction remain incompletely understood. Perivascular macrophages (PVMs) are strategically positioned to modulate cerebrovascular homeostasis, but their role in acute post-traumatic endothelial activation has not been systematically characterized. Methods We re-analyzed a publicly available mouse single-cell RNA sequencing (scRNA-seq) dataset (GSE290150, 24 h post-TBI) to characterize the landscape of immune and vascular cell populations and to infer intercellular communication. Bone marrow–derived macrophages (BMDMs) served as a PVM surrogate model, and bEnd.3 cerebral endothelial cells were used for in vitro validation of key signaling pathways. Results Single-cell analysis revealed concurrent expansion of PVM-like cells and activated endothelial cells (Activated ECs) in the TBI brain, with CellChat-predicted enhancement of MIF–ACKR3 signaling from PVM-like cells toward Activated ECs. Inflammatory stimulation of BMDMs significantly increased MIF expression and secretion in a STAT3-dependent manner. Macrophage-conditioned medium induced endothelial activation–associated molecular changes, including upregulation of Angpt2 and Adm and downregulation of tight junction genes Claudin5 and Tjp1; these effects were attenuated by MIF inhibitor ISO-1 or ACKR3 antagonist CCX771. Recombinant MIF dose-dependently reproduced these changes, which were substantially abrogated by ACKR3 pharmacological blockade or siRNA knockdown. STAT3 inhibition further suppressed rMIF-induced endothelial transcriptional responses. Conclusion These findings provide transcriptomic and in vitro evidence supporting a PVM-derived MIF–ACKR3–STAT3 signaling axis associated with endothelial activation–related molecular changes in acute TBI. Direct functional effects on BBB integrity and in vivo relevance require further validation. This candidate axis highlights perivascular immune–vascular crosstalk as a potential avenue for therapeutic investigation.
Yanya Lin, Cheng-Yu Lin, Jianhui Chen et al.· Frontiers in Cellular Neuros...· 0 citations
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