Advanced technologies to study Glioblastoma microenvironment

Glioblastoma multiforme (GBM) is one of the most feared of all of human diseases for its near uniformly fatal prognosis and associated loss of cognitive function as part of the disease process. Despite advances in chemotherapy, radiotherapy (RT), and surgical resection, the residual cell population within brain parenchyma post-surgery causes inevitable recurrence.The moniker "multiforme" derives from the first histopathologic descriptions of the tumor's varied morphologic features and the presence of heterogeneous cell populations within a single tumor, in which lesions with a high degree of cellular and nuclear polymorphism and numerous giant cells coexist with areas of high cellular uniformity.Due to the heterogeneity of glioblastoma cells and the yet poorly understood mechanisms of the neoplastic cells infiltration into the brain parenchyma, it is not entirely clear to what extent histopathological characteristics are governed by inherent genetic properties of the tumor or are influenced by the local microenvironment. To better understand the role of tumor cell population in the complex heterogeneity of glioblastoma, we paired laser capture microdissection and Next generation seuquencing technology to study intra-tumoral differences into specific histological regions. We here applied LCM protocol to pick up astrocytes, neurons,vascular proliferations in different histological contest and determining a suitable RNASeq method for limited quantities of RNA. We initially validated our RNA seq data obteined from each cellular population with matching RNA-Seq experiement of BioProject database. Principal Component Analysis (PCA) provided a consensus list of 12950 genes with greater than 1-log-fold difference between group of tumor regions (pseudopalisading cell, tumor core, neoplastic astrocytes in satellitosis, vascolar microproliferation and normal vessel) when compared to control tissue. Consistent data emerged, in which multiple therapeutic targets significant to glioblastoma as PI3k-AKT-MTOR signaling, neoangiogenic process, remodeling process i.e metalloproteinase, integrins families and metabolic process according to others studies. Our results are promising and suggest as LCM, a microscopy-based technique to collect specific cells populations from FFPE, coupled to NGS technology may be attractive methods to study glioblastoma heterogeneity and also for prospective and retrospective studies where FF specimens are not available. GBM cells dynamically respond to their local tissue microenvironment, which, in turns, affects tumor migration and drug resistance. Currently little is known about the role of non-neoplastic cells, particularly, the microglia cells in GBM cell behavior and response to cytotoxic. In vitro drug testing is usually performed in 2D tumor cell cultures without the presence of other microenvironment features of a GBM. In order to address these limitations, we established a 3D oculture model comprising both human GBM cell lines and and microglia (MG) to investigated the influence of the in vitro tumour microenvironment on the growth, proliferation and resistance to cytotoxic telozolomide (TMZ).