Intravital Imaging of Glioblastoma Mouse Models

Glioblastoma multiforme (GBM) is a malignant form of brain tumor, which is characterized by extensive cellular and genetic heterogeneity, and poor prognosis. Calcium (Ca) signaling has been proposed to be directly involved in cancer proliferation and invasion, however there are no demonstrations in vivo of the correlation between GBM cell motility and calcium signaling. In light of this idea, we have produced a strain of mouse glioma cells (GL261) expressing a red fluorescent protein and a genetically encoded calcium sensor (GCaMP6s). Our findings demonstrate a heterogenous expression of Ca activity where most of the tumor volume is occupied by tightly packed spherical cells characterized by minimal motility and low Ca activity. Additionally, the dense tumoral area appear to have ensembles of cells displaying highly synchronized Ca waves propagating within cells and between distinct ensembles of cells based on their common activation. However, this core, is surrounded by sparse cells displaying a very polarized morphology, elevated migrational ability and active Ca signaling. We postulate that these regions represent the infiltrating component of the tumor. Next, in order to study GBM during development, we generated a spontaneous glioma model by overexpressing the active mutant of Ras (HRasV12) flanked by the consensus sequence for the piggyBac transposase. Using in utero electroporation technique, we transfected a small population of glia precursors with HRas, GCaMP6 and a red reporter. Two photon imaging demonstrated that, within 14 days from 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 active Ca activity. Finally, we explored effects of a potential combination of drug (patented, in collaboration with SISSA, Trieste) for the treatment of GBM on our GL261 mouse model.

The seminar is realized in the framework of th funded projects:

• Progetto DECODE-EE, Regione Toscana
• Telethon grant GGP 13187
• Progetto Gliomics, Regione Toscana
• PRIN 2015 and 2018
• Fondazione Pisa