METFORMIN, CLIC1 AND TRANSCRANIAL STIMULATION: THE BASIS OF A NEW TARGETED THERAPY AGAINST GLIOBLASTOMA RELAPSE

Glioblastoma (GBM) is the most prevalent type of brain tumor and has a very poor prognosis. The current standard of care includes surgery to remove the mass of the tumor, followed by chemoradiation therapy. However, this technique is neither effective nor specific, and tumor recurrence occurs frequently, often fatally. As a result, new techniques for preventing glioblastoma relapse and increasing patients' life expectancy should be developed. GBM stem-like cells (GSCs) are primarily responsible for the high level of aggressiveness and recurrence. GSCs drive tumorigenesis and self-renewal, confer resistance to the tumor and guide tumor relapse. Recent publications proposed the transmembrane CLIC1 protein (tmCLIC1) as a potential pharmacological target since it is crucial for GSCs proliferation. tmCLIC1 inhibition shows tumor growth impairment both in vitro and in vivo. Recently, tmCLIC1 has been proposed to be one of metformin’s targets. In this work, we show molecular and functional evidence of the direct metformin-CLIC1 interaction, by NMR, MicroScale Thermophoresis, and single-channel patch-clamp experiments. However, due to the blood brain barrier, the high concentration of metformin (10mM) required to inhibit tumor progression, which has been beneficial in other types of tumors, cannot be reached in the brain. As a result, the primary goal of this study is to improve metformin antitumoral activity on glioblastoma by lowering its operative concentration to a level that allows it to enter the brain. Our strategy is to induce repetitive membrane depolarizations to the tumor to promote metformin-tmCLIC1 binding. In fact, interaction between the two 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. The application of EMF stimulation resulted in a 10-fold decrease of metformin concentration (1mM) needed to have the same antiproliferative effect in vitro on GSCs cultures and spheroids. To see if the impact was sustained in vivo, zebrafish embryos were orthotopically injected with GSCs, and the tumor mass was measured after 72 hours in absence or presence of 1mM metformin diluted in embryos’ water and of EMF stimulation. GSCs were orthotopically implanted into zebrafish embryos, and tumor mass was assessed after 72 hours in the absence or presence of 1mM metformin diluted in embryo water and EMF stimulation. The findings were comparable with those obtained in vitro, demonstrating that combining 1mM metformin and EMF decreases tumor growth in zebrafish embryos to the same level as metformin 10mM alone. In addition, we provide data showing that stimulation increases tmCLIC1 open probability by both imaging techniques and electrophysiological experiments. GSCs were transfected to encode a green-fluorescent chloride sensor that can be monitored in living cells under fluorescent microscope. We found that chloride flux increases upon EMF stimulation in wild-type GSCs while the increase is way lower in cells Clic1-/-, suggesting that CLIC1 activity is effectively stimulated by EMF application. For the same purpose, we performed single channel recordings of tmCLIC1 current in the same GSCs in control condition and consecutively switching on EMF stimulation. The outcome was that tmCLIC1 open probability increased significantly with stimulation. The long-term goal of the project is to combine transcranial stimulation and metformin administration to patients as adjuvant therapy to target chemotherapy-resistant cells that drive tumor relapse.