Heterogeneity Matters: Different Regions of Glioblastoma Are Characterized by Distinctive Tumor-Supporting Pathways

SIMPLE SUMMARY: 5-ALA Fluorescence Guided Surgery aims at extending the boundaries of glioblastoma (GBM) resection. It is based on the use of a fluorescent dye, 5-aminolevulinic acid (5-ALA). Depending on the fluorescence levels, it is possible to distinguish the core of the tumor, the infiltrating borders and the healthy tissue. Since GBM progression is supported by tumor cells and their interaction with the surrounding microenvironment, we hypothesized that 5-ALA intensity could identify microenvironments with different tumor supporting properties. Taking advantage of glioma-associated stem cells; a human in vitro model of the glioma microenvironment, we demonstrate that all regions of the tumor support the tumor growth, but through different pathways. This study highlights the importance of understanding the TME to obtain key information on GBM biology and develop new therapeutic approaches. ABSTRACT: The glioblastoma microenvironment plays a substantial role in glioma biology. However, few studies have investigated its spatial heterogeneity. Exploiting 5-ALA Fluorescence Guided Surgery (FGS), we were able to distinguish between the tumor core (ALA+), infiltrating area (ALA-PALE) and healthy tissue (ALA−) of the glioblastoma, based on the level of accumulated fluorescence. The aim of this study was to investigate the properties of the microenvironments associated with these regions. For this purpose, we isolated glioma-associated stem cells (GASC), resident in the glioma microenvironment, from ALA+, ALA-PALE and ALA− samples and compared them in terms of growth kinetic, phenotype and for the expression of 84 genes associated with cancer inflammation and immunity. Differentially expressed genes were correlated with transcriptomic datasets from TCGA/GTEX. Our results show that GASC derived from the three distinct regions, despite a similar phenotype, were characterized by different transcriptomic profiles. Moreover, we identified a GASC-based genetic signature predictive of overall survival and disease-free survival. This signature, highly expressed in ALA+ GASC, was also well represented in ALA PALE GASC. 5-ALA FGS allowed to underline the heterogeneity of the glioma microenvironments. Deepening knowledge of these differences can contribute to develop new adjuvant therapies targeting the crosstalk between tumor and its supporting microenvironment.