Set up of different strategies to selectively target Glioblastoma Cells

Glioblastoma Multiforme (GBM) is the most common malignant brain tumor in adults, displaying fast growth, vascular proliferation, diffuse infiltrative nature into the brain parenchyma and necrosis areas. Despite optimal multimodal therapeutical treatment including surgical resection followed by radiotherapy and chemotherapy, the prognosis of patients remains still dismal, with a median survival of 14 months. One hypothesis seems to be related to the existence in the brain of the Cancer Stem Cells (CSCs). These cells are so-called Glioma Stem Cells (GSCs) and are described as a sub population of undifferentiated tumor-cells with stem-like properties, self-renewal, multi-lineage differentiation ability, maintained proliferation and highly tumorigenic potential in immune-deficient mice. GSCs seem to be play a key role in GBM initiation and progression, contributing to chemotherapy and radiotherapy resistance and tumor recurrence. Therefore, there is an urgent need to develop novel therapeutic approaches, to selectively target GSCs. In this study, we focus on two different therapeutical approaches: - the use of Mesenchymal Stem Cells (MSCs) as delivery vehicles for brain tumor therapy, exploiting their inherent tumor-tropism probably related to the effect of cytokines and chemokines, secreted by infiltrated tissues. - Virotherapy, employing the R3616 as neuroattenuated Herpes Simplex Virus type1. R3616 is a HSV1 mutant deleted for both copies of the gene γ34. 5, important for viral replication and neurovirulence. The effect of these deletions inhibits autophagy, blocks virus-induced host protein shutoff, and attenuate virus replication in GSCs, minimizing neurotoxicity. GSCs employed for this study have been isolated from GBM specimens obtained during surgical procedures of tumor removal and cultured in adhesion under normoxic and hypoxic conditions or in suspension as neurospheres under normoxic conditions. Neuropheres were phenotypically characterized for some of stemness markers, such as CD133, Nestin, Sox2 and CD44. In order to choose the best cell candidate for cell delivery experiments, we investigated between hMSCs isolated from human bone marrow of healthy donors (BM-MSCs) and glioma patients adipose tissue (AT-MSCs) obtained during GBM surgical resection. hMSCs from both sources were phenotypically characterized and cultured under hypoxic and normoxic conditions in order to mimic the peculiar and heterogeneous GBM microenvironment towards cells would be further delivered. The BM-MSCs retained: a. ) faster growth, b. ) a greater in vitro multilineage differentiative potential and c. ) increased migratory ability than AT-MSCs, especially in hypoxic conditions; thus suggesting to be the most suitable vehicles for our future analyses. Moreover, we evaluated BM-MSCs tropism to GBM derived cells modulated by oxygen levels or by the presence and absence of soluble factors shown to be able to stimulate astro-glial and neuronal differentiation, such as BMP2 and WNT3a respectively. Indeed, BMPs are members of the Transforming Growth Factor-β (TGF-β) Superfamily shown to stimulate both proliferation and mitotic arrest, to promote astro-glial differentiation reducing cell growth and increasing portion of GFAP+ cells. Wnt3a ligand mediates neuronal differentiation and proliferation inhibition of GBM cells and this phenomenon is enhanced under hypoxic conditions. hMSCs tropism was assessed both when primary GBM –derived cells were pre-treated alternatively with BMP2 or WNT3a and when such factors were delivered towards GBM cells. Our data demonstrated pro-differentiating molecules as enhancing agents of hMSCs migratory properties, especially in hypoxia. Experiments involving viruses were performed both on HSV1 wild type and R3616 to test the ability to replicate in and to kill GSCs. As expected for a neuroattenuated virus, R3616 replication in GSCs was widely impaired and the killing ability were decreased compared to the wild type. Both viruses were also employed to test their replication capacity and cytotoxicity in BM-MSCs. Compared to HSV1 wild type, R3616 retained impaired replication efficiency and attenuated killing ability in hMSCs. BM-MSCs inherent tropism was also investigated in GSCs infected with HSV1 wild type or R3616. Interestingly, we found that GSCs R3616 infected significantly increased the migratory properties of BM-MSCs. In conclusion, these findings support the use of BM-MSCs as a promising cell vehicle candidate for tumor-specific delivery and open the way to the development of engineered BMPs-expressing MSCs to further assays in GBM -derived cells. Additionally, the R3616 ability to kill GSCs encourages next efforts to combine both methods to obtain an efficient strategy to selectively target GSCs. Our preliminary data have already demonstrated that co-treatment of R3616 and BMP2 increases the differentiation markers expression. Accordingly, BM-MSCs retained a preferential inherent homing towards more differentiated cells.