Role of metformin and AKT axis modulation in the reversion of hypoxia induced TMZ-resistance in glioma cells

Hypoxia is a key driver of tumor adaptation promoting tumor progression and resistance to therapy. Hypoxia related pathways might represent attractive targets for the treatment of Glioblastoma Multiforme (GBM), that up to date is characterized by a poor prognosis. Primary aim of this study was to investigate the role of hypoxia and hypoxia-related modifications in the effect of temozolomide (TMZ) given alone or in association with the antidiabetic agent Metformin (MET) or the PI3K/mTOR blocker, BEZ235. The study was conducted in the TMZ responsive U251 and resistant T98 GBM cells. Our results showed that during hypoxia, TMZ plus MET reduced viability of U251 cells affecting also CD133 and CD90 expressing cells. This effect was associated with a reduction of HIF-1\u3b1 activity, VEGF release and AKT activation. In T98 TMZ-resistant cells, TMZ plus MET exerted similar effects on HIF-1\u3b1. However, in this cell line, TMZ plus MET failed to reduce CD133 positive cells and AKT phosphorylation. Nevertheless, the administration of the dual PI3K/mTOR inhibitor BEZ235 potentiated the effect of TMZ plus MET on cell viability, inducing a pro-apoptotic phenotype during hypoxic condition also in T98 cells, suggesting the block of the PI3K/AKT/mTOR pathway as a complementary target to further overcome GBM resistance during hypoxia. In conclusion, we proposed TMZ plus MET as suitable treatment to revert TMZ-resistance also during hypoxia, an effect potentiated by the inhibition of PI3K/mTOR axis

Validation of an Engineered Cell Model for In Vitro and In Vivo HIF-1 alpha Evaluation by Different Imaging Modalities

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Metformin and temozolomide, a synergic option to overcome resistance in glioblastoma multiforme models

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor survival. Cytoreduction in association with radiotherapy and temozolomide (TMZ) is the standard therapy, but response is heterogeneous and life expectancy is limited. The combined use of chemotherapeutic agents with drugs targeting cell metabolism is becoming an interesting therapeutic option for cancer treatment. Here, we found that metformin (MET) enhances TMZ effect on TMZ-sensitive cell line (U251) and overcomes TMZ-resistance in T98G GBM cell line. In particular, combined-treatment modulated apoptosis by increasing Bax/Bcl-2 ratio, and reduced Reactive Oxygen Species (ROS) production. We also observed that MET associated with TMZ was able to reduce the expression of glioma stem cells (GSC) marker CD90 particularly in T98G cells but not that of CD133. In vivo experiments showed that combined treatment with TMZ and MET significantly slowed down growth of TMZ-resistant tumors but did not affect overall survival of TMZ-sensitive tumor bearing mice. In conclusion, our results showed that metformin is able to enhance TMZ effect in TMZ-resistant cell line suggesting its potential use in TMZ refractory GBM patients. However, the lack of effect on a GBM malignancy marker like CD133 requires further evaluation since it might influence response duration

Specific V-ATPase expression sub-classifies IDHwt lower-grade gliomas and impacts glioma growth in vivo

Background: Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. Methods: V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients\u2019 cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. Findings: GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0\ub703, by Cox proportional-hazards model). In vivo, V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. Interpretation: Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. Fund: This work was supported by Fondazione Cariplo (2014\u20131148 to VV), Fondazione IRCCS Ca\u2019 Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV)

A robust coregistration method for in vivo studies using a first generation simultaneous PET/MR scanner

Purpose: Hybrid positron emission tomography (PET)/magnetic resonance (MR) imaging systems have recently been built that allow functional and anatomical information obtained from PET and MR to be acquired simultaneously. The authors have developed a robust coregistration scheme for a first generation small animal PET/MR imaging system and illustrated the potential of this system to study intratumoral heterogeneity in a mouse model. Methods: An alignment strategy to fuse simultaneously acquired PET and MR data, using the MR imaging gradient coordinate system as the reference basis, was developed. The fidelity of the alignment was evaluated over multiple study sessions. In order to explore its robustness in vivo, the alignment strategy was applied to explore the heterogeneity of glucose metabolism in a xenograft tumor model, using ^(18)F-FDG-PET to guide the acquisition of localized ^1H MR spectra within a single imaging session. Results: The alignment method consistently fused the PET/MR data sets with subvoxel accuracy (registration error mean=0.55 voxels, <0.28 mm); this was independent of location within the field of view. When the system was used to study intratumoral heterogeneity within xenograft tumors, a correlation of high ^(18)F-FDG-PET signal with high choline/creatine ratio was observed. Conclusions: The authors present an implementation of an efficient and robust coregistration scheme for multimodal noninvasive imaging using PET and MR. This setup allows time-sensitive, multimodal studies of physiology to be conducted in an efficient manner

A GBM-like V-ATPase signature directs cell-cell tumor signaling and reprogramming via large oncosomes

Background: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF)

INSERT: A Novel Clinical Scanner for Simultaneous SPECT/MRI Brain Studies

A clinical SPECT insert for a commercial MRI scanner has been developed within the INSERT project, allowing simultaneous SPECT/MRI studies of the human brain. Here we present preliminary experimental results. The reconstructed resolution was 6-11 mm and the sensitivity 280-440 s-1/MBq. The INSERT is the first clinical SPECT prototype for simultaneous SPECT/MRI