P17.17.A Metformin, tmCLIC1 and transcranial stimulation: the basis of a new targeted therapy against glioblastoma relapse
Authors: Cannavale G , Verduci I , Cianci F , Balboni B , Palloni L , Ranucci M , Castiglione S , Martelli C , Tacconi C , Girotto S , Ottobrini L , Fantin A , Mazzanti M 
Published: September 2022

Abstract

Background
Glioblastoma (GBM) is the most common brain tumor with an extremely poor prognosis. Standard therapy relies on surgery, chemo- and radiotherapy but tumor relapse occurs inevitably with fatal outcome. Hence, new approaches should be designed to increase patients’ life expectancy. The high grade of aggressiveness and recurrence is attributed to GBM stem-like cells (GSCs). Recent publications propose the transmembrane Chloride Intracellular Channel 1 protein (tmCLIC1) as a potential pharmacological target since it is crucial for GSCs proliferation both in vitro and in vivo. Recently, we proposed tmCLIC1 to be one of metformin’s targets. Here, we show molecular evidence of direct metformin-tmCLIC1 interaction. However, the high concentration of metformin needed to impair tumor progression cannot be reached in the brain due to the blood brain barrier. Hence, the main aim of this work is to decrease metformin’s operative concentration to the level that can access the brain. Our strategy is to induce repetitive membrane depolarizations to the tumoral area to promote metformin-tmCLIC1 interaction, that only occurs when tmCLIC1 is in the open state. Pulsed depolarizing electromagnetic field (EMF) stimulation should increase tmCLIC1 close-to-open transitions and, consequently, the availability of metformin binding sites.

Material and Methods
We generated CLIC1-knockout of several patient-derived GSCs using CRISPR-Cas9 technology to assess the role of CLIC1 in metformin’s antiproliferative effect. In the first set of experiments, we used 10mM metformin. When applying concomitant EMF stimulation, we dropped to 1mM metformin. NMR experiments and single-channel electrophysiological recordings were provided to study metformin-tmCLIC1 interaction. Metformin-EMF system was applied in vitro on GSCs cultures and spheroids, and in vivo on zebrafish embryos and mice.

Results
NMR and electrophysiology demonstrate the direct binding between metformin and tmCLIC1, that figures as the main metformin receptor in GBM. The application of EMF stimulation resulted in a 10-fold decrease of metformin concentration to get the same antiproliferative effect in vitro. To assess whether the effect is maintained in vivo, zebrafish embryos were orthotopically injected with GSCs and the tumor mass was measured after 72 hours in absence or presence of metformin in embryos’ water and of EMF stimulation. Consistently, metformin coupled to EMF reduces tumor progression in zebrafish embryos to the same extent of metformin 10mM alone. Experiments on mice are currently on-going.

Conclusion
Our research has revealed a new interactor of metformin. The combination of metformin and EMF results in an effective strategy to impair GBM progression both in vitro and in vivo. The long-term goal is to combine transcranial stimulation and metformin administration to patients as an adjuvant therapy. AIRC#24758