Preclinical testing of a novel therapeutic approach to counteract Glioblastoma

Gliomas are brain tumors due to an excessive proliferation of glial cells. Among many types of gliomas, Glioblastoma (GB) is the most aggressive form and currently there is no effective treatment for this disease, whose average survival is of almost 8 months after diagnosis. Indeed, the elevated mortality, the short life expectancy, and an increasing number of cases confirm that GB represents a huge risk for public health. Its development is fast and destructive, resulting in severe and invalidating symptomatology that makes GB a significant social and economic burden for National healthcare systems. Thus, there is an urgent need to find effective therapeutic approaches aimed at targeting glioma progression, ameliorating patients’ quality of life, and increasing their life expectancy. In my PhD thesis, I evaluated the antineoplastic effect of a recombinant protein, CTX-CNF1, that was designed in the lab. CTX- CNF1 is composed of CNF1, a bacterial protein toxin effective in counteracting GB and protecting peritumoral tissue from neural dysfunctions, and Chlorotoxin (CTX), a protein already employed in the clinic for its capacity to cross the blood-brain barrier (BBB) and selectively recognize glioma cells. Preliminary data showed that i) CTX-CNF1 is effective in leading both murine and human glioma cells to death through the activation of a senescence process as CNF1, and that ii) a single systemic administration of CTX- CNF1 (80 nM) is capable of targeting glioma cells with high specificity as CTX, ultimately producing a significant increase in the survival of glioma-bearing animals. Thus, we designed a more clinical-like protocol, performing weekly systemic administrations of CTX-CNF1 (80 nM) for 3 weeks starting from MRI diagnosis of GB. This repetitive treatment was effective in i) ameliorating glioma- bearing mice motor deficits (seen with Grip Strength and Grid Walk tests), ii) progressively reducing their tumoral mass, and consistently iii) increasing their survival. This was seen on two GB murine syngeneic models, GL261 and CT-2A. Remarkably, 50% of GL261-bearing mice were still alive 6 months after tumor induction and no glioma mass was detectable with MRI at this time point. These survived-cured mice were rechallenged with another glioma in the contralateral hemisphere and resulted in being less susceptible to developing another GB when compared with same-age mice. This suggests the possibility of an existence of an immune memory in CTX-CNF1 treated animals. Importantly, the specific class of cytotoxic T lymphocytes (CD8+) was significantly upregulated and functionally activated in glioma-bearing mice treated with CTX-CNF1 48 hours after the treatment. This is in line with previous studies in which CNF1 was found to influence the functionality of the immune system. In addition, indirect evidence of CD8+ cells action in CTX-CNF1 treated mice was given by the fact that the survival of mice treated with both CTX-CNF1 and an anti-CD8 was similar to the one of vehicle glioma-bearing animals.