Glioblastomas (GBMs) are primary brain tumors endowed with inter- and intra-patient heterogeneity and extreme di↵usivity. As heterogeneity is studied with genomic and transcriptomic analysis, little is known on how it is reflected on cell migration, mechanoproperties and motility modes. Generally, the tumor cells invade the brain moving on brain vasculature or white matter tracks: Patient-Derived Xenograft (PDX) has been a standard to reproduce them in order to study GBM invasion. However, PDX presents many disadvantages, including time consumption, hard standardization, high cost and ethical concerns. The present PhD thesis report aims at summarizing the existing literature through an historical journey that gradually walks the reader towards the state of the art in the biological knowledge, therapeutic treatments, and bioengineering of GBM. It also reports the results of this PhD work. They include novel bioengineering tools for studying the mechanoproperties in GBM and the development of methods to dissect their migration and motility modes. Finally, a stand-alone assay aims at fostering a discussion on how the scientific mindset and science have evolved and are evolving to drive technological innovation in nowadays’ world. The main goal of this PhD work was to develop bioengineering tools to crack mechanoproperties and GBM motility. Initially, by utilizing clones of patient-derived GBM cells that were either highly proliferative or highly invasive, I co-studied their cellular architecture, migratory, and biophysical properties. One of the milestones of this PhD work consists in the link between that invasiveness and cellular fitness. The most invasive cells were sti↵er, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 supports the mechanical cohesion of the cytoskeleton and enhances cell’s mechanoproperties, leading to a higher motility and invasive phenotype. In order to scale up the motility screen to several GBM clones, I co-developed SP2G (SPheroid SPreading on Grids), the live imaging of GBM spheroids spreading on grid micropatterns mimicking the brain vasculature. To counteract the issues in PDX and rapidly identify the most invasive sub-populations hidden in heterogeneous GBMs, we developed an in vivo mimicry platform named SP2G (SPheroid SPreading on Grids). Live imaging of tumor-derived spheroids spreading on gridded micro patterns imitating the brain vasculature mimicked 3D motility features observed in brain or 3D matrices. Using patient-derived samples coupled with a semi-automated ImageJ/Fiji macro suite, SP2G easily characterized and sorted di↵erences in cell migration and motility modes through a set of 6 parameters (area expansion, di↵usivity, boundary speed, collective migration, directional persistence, hurdling). Moreover, SP2G exposed the hidden intra-patient heterogeneity in cell motility that correlated molecularly to specific integrins. Thus, SP2G is intended as a versatile and potentially pan-cancer workflow to identify the invasive tumor sub-populations in patient-derived specimens. SP2G represents an integrative tool, available as open-source Fiji macro suite, for therapeutic evaluations at single patient level.

MECHANOPROPERTIES, HETEROGENEITY AND CELL MIGRATION IN GLIOBLASTOMA / M. Crestani ; tutor: N. Gauthier, P. Monzo. - : . Dipartimento di Oncologia ed Emato-Oncologia, 2022. ((34. ciclo, Anno Accademico 2022.

MECHANOPROPERTIES, HETEROGENEITY AND CELL MIGRATION IN GLIOBLASTOMA

M. Crestani
2022

Abstract

Glioblastomas (GBMs) are primary brain tumors endowed with inter- and intra-patient heterogeneity and extreme di↵usivity. As heterogeneity is studied with genomic and transcriptomic analysis, little is known on how it is reflected on cell migration, mechanoproperties and motility modes. Generally, the tumor cells invade the brain moving on brain vasculature or white matter tracks: Patient-Derived Xenograft (PDX) has been a standard to reproduce them in order to study GBM invasion. However, PDX presents many disadvantages, including time consumption, hard standardization, high cost and ethical concerns. The present PhD thesis report aims at summarizing the existing literature through an historical journey that gradually walks the reader towards the state of the art in the biological knowledge, therapeutic treatments, and bioengineering of GBM. It also reports the results of this PhD work. They include novel bioengineering tools for studying the mechanoproperties in GBM and the development of methods to dissect their migration and motility modes. Finally, a stand-alone assay aims at fostering a discussion on how the scientific mindset and science have evolved and are evolving to drive technological innovation in nowadays’ world. The main goal of this PhD work was to develop bioengineering tools to crack mechanoproperties and GBM motility. Initially, by utilizing clones of patient-derived GBM cells that were either highly proliferative or highly invasive, I co-studied their cellular architecture, migratory, and biophysical properties. One of the milestones of this PhD work consists in the link between that invasiveness and cellular fitness. The most invasive cells were sti↵er, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 supports the mechanical cohesion of the cytoskeleton and enhances cell’s mechanoproperties, leading to a higher motility and invasive phenotype. In order to scale up the motility screen to several GBM clones, I co-developed SP2G (SPheroid SPreading on Grids), the live imaging of GBM spheroids spreading on grid micropatterns mimicking the brain vasculature. To counteract the issues in PDX and rapidly identify the most invasive sub-populations hidden in heterogeneous GBMs, we developed an in vivo mimicry platform named SP2G (SPheroid SPreading on Grids). Live imaging of tumor-derived spheroids spreading on gridded micro patterns imitating the brain vasculature mimicked 3D motility features observed in brain or 3D matrices. Using patient-derived samples coupled with a semi-automated ImageJ/Fiji macro suite, SP2G easily characterized and sorted di↵erences in cell migration and motility modes through a set of 6 parameters (area expansion, di↵usivity, boundary speed, collective migration, directional persistence, hurdling). Moreover, SP2G exposed the hidden intra-patient heterogeneity in cell motility that correlated molecularly to specific integrins. Thus, SP2G is intended as a versatile and potentially pan-cancer workflow to identify the invasive tumor sub-populations in patient-derived specimens. SP2G represents an integrative tool, available as open-source Fiji macro suite, for therapeutic evaluations at single patient level.
GAUTHIER, NILS
SCITA, GIORGIO
GLIOBLASTOMA ; MIGRATION ; HETEROGENEITY ; MICROSCOPY
Settore BIO/13 - Biologia Applicata
MECHANOPROPERTIES, HETEROGENEITY AND CELL MIGRATION IN GLIOBLASTOMA / M. Crestani ; tutor: N. Gauthier, P. Monzo. - : . Dipartimento di Oncologia ed Emato-Oncologia, 2022. ((34. ciclo, Anno Accademico 2022.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/946015
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