Effects of neural precursors-derived extracellular vesicles on glioblastoma stem-like cells biology: enlightening the role of cellular prion protein in brain tumor cells interactions.

Author(s)

João Pedro Alves de Araujo

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2024-06-27

Examining board members

Marilene Hohmuth Lopes; Luciana Biagini Lopes; Fernanda Ortis; Ana Cláudia Trócoli Torrecilhas

• Advisor: Marilene Hohmuth Lopes

Abstract

Glioblastoma (GBM) is an aggressive brain tumor that contains a subpopulation of tumor stem cells called glioblastoma stem-like cells (GSCs). GSCs are involved in therapy resistance and high rates of GBM relapse, with cellular prion protein (PrPC) being an important factor for their maintenance. In this context, neural stem cells (NSCs) have been pointed out as a probable origin of GSCs, and the crosstalk between NSC and GSCs, mostly mediated by extracellular vesicles (EVs), may contribute to increasing GBM aggressiveness, and data on NSCs in GBM are still scarce. We aimed to evaluate the impacts of this intercellular crosstalk on GSCs biology, which could help to better understand the role NSCs-derived EVs can play in this devastating brain tumor. NSCs were generated from induced pluripotent stem cells (hiPSCs), and characterized for the expression of specific markers for each cell type. Once characterized, the NSCs medium was collected to obtain EVs, which were molecularly characterized by western blotting for the expression of CD63, Alix, HSP90, and by NanoSight (NTA), which identified vesicles of size between 100 and 200nm. Both NSCs and GSCs were explored for their ultrastructure by electron microscopy, enlightening biogenesis processes (such as multivesicular bodies and their intraluminal vesicles), vesicle uptake (clathrin-mediated endocytosis), and cell-cell adhesion. Notably, PrPC knockout GSCs (PrPC-KO) seem to present dysfunction in cell adhesion under neurosphere conditions. We also found that PrPC silencing promotes CD63 downregulation in GBM cells lines and in patient-derived GSCs, leading to a reduction in the total number of EVs released by GSC cells. This data suggests a role for PrPC in modulating the tumor environment, as this molecule was also to be secreted in EVs. By treating GSCs with NSCs-derived EVs, we observed modulation in cell-adhesion properties through changes in the expression of proteins such as integrin α-V and NCAM. Functional assays of cell dissociation revealed that treatment with NSCs-derived EVs reduced the dissociation rates of neurospheres. Our data shed light on the role of PrPC in cell communication, and the influence of NSC-derived EVs on the adhesion properties of GSCs. (AU)

• FAPESP's process: 22/08106-1 - The role of extracellular vesicles of neural precursors in the PDX cells sensitivity to temozolomide
• Grantee: João Pedro Alves de Araújo
• Support Opportunities: Scholarships in Brazil - Master