Intravital two photon clcium imaging of glioblastoma mouse models / Pillai, Vinoshene; relatore: RATTO, GIAN MICHELE; Scuola Normale Superiore, ciclo 31, 13-Oct-2021.

Intravital two photon clcium imaging of glioblastoma mouse models

PILLAI, Vinoshene

Settore BIO/09 - Fisiologia
Scienze biofisiche
Glioma is a brain tumor that derives from glial cells and it represents about 40% of all diagnosed central nervous system tumors. The most common type of glioma is glioblastoma multiforme (GBM), which is one of the most malignant tumors that can affect humans, with a median life expectancy of only 14 months. Its invasive properties and the presence of the blood-brain barrier makes GBM very hard to treat. Furthermore, GBM has a high degree of cellular and genetic heterogeneity, and the study of its genome is a very active field of research. As such, it complicates the diagnosis, treatment and prognosis. Recently, researchers are focusing on alterations in cellular signaling processes related to central nervous system (CNS) pathologies and diseases. Crosstalk among neurons, glial cells and/or between neurons and glial cells is important in controlling cellular processes, including cell proliferation, cell division and programmed cell death. Calcium signaling has been proposed to be directly involved in GBM proliferation, motility and invasion, but there are no demonstrations in vivo of the correlation between GBM cell motility and calcium signaling. In light of this idea, we have modified the mouse glioma cell strain (GL261) to express a red fluorescent protein and a genetically encoded calcium sensor (GCaMP6s). Two photon imaging data that we acquired demonstrate that most of the tumor volume, i.e. the core, is occupied by tightly packed spherical cells characterized by reduced cellular motility and low intracellular calcium ((Ca2+)i) activity. This core is surrounded by sparse cells displaying a very polarized morphology that migrate at a higher rate. These cells in the peripheral region are characterized by very active (Ca2+)i signaling. Additionally, we observed a novel phenomenon where groups of cells display synchronized (Ca2+)i waves propagating within the ensemble based on their common activation. Cells in these clusters/ensembles appear to have a direction-biased cellular motility. We postulate that tumor cells from peripheral regions and belonging to active clusters represent the infiltrating component of the tumor. Finally, by means of in utero electroporation, we transfected a small population of glial precursors with the active mutant of Ras (HRasV12), GCaMP6s and a red reporter, that subsequently generates a spontaneous glioma model. Two photon imaging demonstrated that, within 10 days from the electroporation, the brain is gradually invaded by hypertrophic cells characterized by elevated motility and infiltrative potential. Similarly to the GL261 model, the infiltration is organized in cell streams endowed by very elevated (Ca2+)i activity.
Scuola Normale Superiore
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Open Access dal 13/10/2022

Tipologia: Tesi PhD
Licenza: Solo Lettura
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