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Participation of EGFR mutations in the formation of glioma-derived pericytes.

Association between the levels of angiogenic molecules and the aggressiveness and the genetic alterations of gliomas.

Correlation of SOX9 expression with EGFR mutations in gliomas.

List of glioma samples

Supplementary Materials and Methods

The extraordinary plasticity of glioma cells allows them to contribute to different cellular compartments in tumor vessels, reinforcing the vascular architecture. It was recently revealed that targeting glioma-derived pericytes, which represent a big percentage of the mural cell population in aggressive tumors, increases the permeability of the vessels and improves the efficiency of chemotherapy. However, the molecular determinants of this transdifferentiation process have not been elucidated. Here we show that mutations in EGFR stimulate the capacity of glioma cells to function as pericytes in a BMX- (bone marrow and X-linked) and SOX9-dependent manner. Subsequent activation of platelet-derived growth factor receptor beta in the vessel walls of EGFR-mutant gliomas stabilized the vasculature and facilitated the recruitment of immune cells. These changes in the tumor microenvironment conferred a growth advantage to the tumors but also rendered them sensitive to pericyte-targeting molecules such as ibrutinib or sunitinib. In the absence of EGFR mutations, high-grade gliomas were enriched in blood vessels, but showed a highly disrupted blood–brain barrier due to the decreased BMX/SOX9 activation and pericyte coverage, which led to poor oxygenation, necrosis, and hypoxia. Overall, these findings identify EGFR mutations as key regulators of the glioma-to-pericyte transdifferentiation, highlighting the intricate relationship between the tumor cells and their vascular and immune milieu. Our results lay the foundations for a vascular-dependent stratification of gliomas and suggest different therapeutic vulnerabilities determined by the genetic status of EGFR.Significance:This study identifies the EGFR-related mechanisms that govern the capacity of glioma cells to transdifferentiate into pericytes, regulating the vascular and immune phenotypes of the tumors.

List of GBM cell lines

Primers

Analysis of EGFR-IDH alterations and vascular molecules in human samples

Association of EGFR alterations with BBB leakage and definition of a hypoxia signature in gliomas.

Antibodies

The blood-brain barrier (BBB) defines the physiological function of the brain. Its disruption is common in multiple neuropathological diseases. Accumulating evidence suggest that BBB alterations occur in early stages of neurodegenerative diseases, being associated with neuronal loss processes. The vascular abnormalities and BBB leakage characterize the progression of gliomas, however it is unclear their correlation with the neurodegeneration observed in such tumors. Methods: To establish the different gene signatures we have performed an in-silico analysis using glioma data sets and we have validated the results in a cohort of 26 gliomas (high and low grade) and 7 controls from non-tumor donors. We have also used patient-derived xenografts (PDXs) to correlate the degree of BBB disruption (measured by IgG extravasation immunofluorescence staining) with the neuronal loss (evaluated by NeuN immunofluorescence staining) and with the motor impairment (assessed by a rotarod test). Results: We measured the expression of a series of signatures associated with different biological processes, relevant for glioma development. The “Synapse” and the “BBB dysfunction” signatures were inversely correlated during glioma development. Thus, the most aggressive tumors were associated with BBB breakdown and neuronal dysfunction. These phenotypes correlate with the mesenchymal subtype of gliomas and with a shorter survival. The vascular normalization induced by the isocitrate dehydrogenase 1 (IDH1) mutations rescued the neuronal loss, reducing the motor impairment and increasing the overall survival of PDX-bearing mice. Conclusions: Our data demonstrate that vascular normalization can revert the neuronal loss and the aggressiveness in those tumors, which is linked to the appearance of motor dysfunction symptoms. Overall, we propose that disruption of the BBB is associated with neurodegeneration in glioma patients. These results could help us to understand the progression of this pathology as well as other neurodegenerative diseases and therefore to improve their therapeutic approaches. Citation Format: Pablo Mata-Martínez, Berta Segura-Collar, Maria Garranzo-Asensio, Juan M. Sepúlveda-Sánchez, Aurelio Hernández-Lain, Pilar Sánchez-Gómez, Ricardo Gargini. Blood-brain barrier dysfunction leads to neuronal loss in glioma pathology [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO013.