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Digital archive of theses discussed at the University of Pisa


Thesis etd-12102018-074025

Thesis type
Tesi di dottorato di ricerca
Thesis title
Advanced technologies to study Glioblastoma microenvironment
Academic discipline
Course of study
tutor Prof. Naccarato, Antonio Giuseppe
  • Glioblastoma
  • LCM
  • Microenvironment
  • NGS
Graduation session start date
Release date
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).