Pre-clinical drug testing in 2D and 3D human in vitro models of glioblastoma incorporating non-neoplastic astrocytes: tunneling nano tubules and mitochondrial transfer modulates cell behavior and therapeutic respons

The role of astrocytes in the glioblastoma (GBM) microenvironment is poorly understood; particularly with regard to cell invasion and drug resistance. To assess this role of astrocytes in GBMs we established an all human 2D co-culture model and a 3D hyaluronic acid-gelatin based hydrogel model (HyStem™-HP) with different ratios of GBM cells to astrocytes. A contact co-culture of fluorescently labelled GBM cells and astrocytes showed that the latter promotes tumour growth and migration of GBM cells. Notably, the presence of non-neoplastic astrocytes in direct contact, even in low amounts in co-culture, elicited drug resistance in GBM. Recent studies showed that non-neoplastic cells can transfer mitochondria along tunneling nanotubes (TNT) and rescue damaged target cancer cells. In these studies, we explored TNT formation and mitochondrial transfer using 2D and 3D in vitro co-culture models of GBM and astrocytes. TNT formation occurs in glial fibrillary acidic protein (GFAP) positive "reactive" astrocytes after 48 h co-culture and the increase of TNT formations was greater in 3D hyaluronic acid-gelatin based hydrogel models. This study shows that human astrocytes in the tumour microenvironment, both in 2D and 3D in vitro co-culture models, could form TNT connections with GBM cells. We postulate that the association on TNT delivery non-neoplastic mitochondria via a TNT connection may be related to GBM drug response as well as proliferation and migration

A human co‐culture cell model incorporating microglia supports glioblastoma growth and migration, and confers resistance to cytotoxics

Despite the importance of the tumor microenvironment in regulating tumor progression, few in vitro models have been developed to understand the effects of non‐neoplastic cells and extracellular matrix (ECM) on drug resistance in glioblastoma (GBM) cells. Using CellTrace‐labeled human GBM and microglial (MG) cells, we established a 2D co‐culture including various ratios of the two cell types. Viability, proliferation, migration, and drug response assays were carried out to assess the role of MG. A 3D model was then established using a hyaluronic acid‐gelatin hydrogel to culture a mixture of GBM and MG and evaluate drug resistance. A contact co‐culture of fluorescently labeled GBM and MG demonstrated that MG cells modestly promoted tumor cell proliferation (17%‐30% increase) and greater migration of GBM cells (>1.5‐fold increase). Notably, the presence of MG elicited drug resistance even when in a low ratio (10%‐20%) relative to co‐cultured tumor cells. The protective effect of MG on GBM was greater in the 3D model (>100% survival of GBM when challenged with cytotoxics). This new 3D human model demonstrated the influence of non‐neoplastic cells and matrix on chemoresistance of GBM cells to three agents with different mechanisms of action suggesting that such sophisticated in vitro approaches may facilitate improved preclinical testing

Caveolin-1, a key mediator across multiple pathways in glioblastoma and an independent negative biomarker of patient survival

Glioblastoma (GB) remains an aggressive malignancy with an extremely poor prognosis. Discovering new candidate drug targets for GB remains an unmet medical need. Caveolin-1 (Cav-1) has been shown to act variously as both a tumour suppressor and tumour promoter in many cancers. The implications of Cav-1 expression in GB remains poorly understood. Using clinical and genomic databases we examined the relationship between tumour Cav-1 gene expression (including its spatial distribution) and clinical pathological parameters of the GB tumour and survival probability in a TCGA cohort (n=155) and CGGA cohort (n=220) of GB patients. High expression of Cav-1 represented a significant independent predictor of shortened survival (HR = 2.985, 5.1 vs 14.9 months) with a greater statistically significant impact in female patients and in the Proneural and Mesenchymal GB subtypes. High Cav-1 expression correlated with other factors associated with poor prognosis: IDH w/t status, high histological tumour grade and low KPS score. A total of 4879 differentially expressed genes (DEGs) in the GB tumour were found to correlate with Cav-1 expression (either positively or negatively). Pathway enrichment analysis highlighted an over-representation of these DEGs to certain biological pathways. Focusing on those that lie within a framework of epithelial to mesenchymal transition and tumour cell migration and invasion we identified 27 of these DEGs. We then examined the prognostic value of Cav-1 when used in combination with any of these 27 genes and identified a subset of combinations (with Cav-1) indicative of co-operative synergistic mechanisms of action. Overall, the work has confirmed Cav-1 can serve as an independent prognostic marker in GB, but also augment prognosis when used in combination with a panel of biomarkers or clinicopathologic parameters. Moreover, Cav-1 appears to be linked to many signalling entities within the GB tumour and as such this work begins to substantiate Cav-1 or its associated signalling partners as candidate target for GB new drug discovery

Modulating autophagy as a therapeutic strategy for the treatment of paediatric high‐grade glioma

Paediatric high grade glioma (pHGG) represent a therapeutically challenging group of tumours. Despite decades of research there has been a minimal improvement in treatment and the clinical prognosis remains poor. Autophagy, a highly conserved process for recycling metabolic substrates is upregulated in pHGG, promoting tumour progression and evading cell death. There is significant cross talk between autophagy and a plethora of critical cellular pathways, many of which Accepted Article This article is protected by copyright. All rights reserved. are dysregulated in pHGG. The following article will discuss our current understanding of autophagy signalling in pHGG and the potential modulation of this network as a therapeutic target

Caveolin-1, a driver of invasive phenotype in in-vitro 3D-spheroid assays comprised of high grade GBM cells association with an AKT-inhibited phenotype

INTRODUCTION Glioblastoma multiforme (GBM) cells display a highly invasive phenotype, a hallmark which counters effective surgical and radiotherapy strategies. Caveolin-1 (Cav-1) is the main structural and functional component of caveolae. The impact of the expression of Cav-1 within a range of tumour and tumour-associated stromal cells is variable with both oncogenic and tumour suppressive roles reported which appear to be both disease-specific and context-dependent. Our hypothesis is that Cav-1 serves as promoter of invasion of GBM cells. MATHERIALS AND METHODS To investigate our hypothesis we used a lentiviral shRNA approach to silence Cav-1 in three GBM cell lines (U87, UP007, UP029) derived from adult brain tumours. We employed an in-vitro 3D cell-sprouting invasion assay with GBM cell spheres embedded in Matrigel. Quantification of invasion was undertaken using a novel image analysis tool or 3D systems, INSIDIA (ImageJ Macro for High-throughput Spheroid Invasion Analysis). Parallel migration and invasion studies were performed using a Boyden Chamber approach, as well as cell-cell adhesion assays. Activation of signalling pathways in 2D and 3D cultures were performed by proteomic array and Western Blot analysis. RESULTS AND CONCLUSION GBM cells expressing Cav-1 (Cav-1 +ve) displayed a higher invasive capacity compared cells where Cav-1 had been silenced Cav-1 –ve), the latter also showing increased cell-cell adhesion. A significant finding from the signalling analysis was an inverse association between Cav-1 silencing and activation of AKT evidenced by increased phosphorylation at both Ser473 and Thr308 sites. Ongoing studies are exploring this signalling axis and its relationship to the invasive phenotype. CM and MG acknowledge Cancer Research Wales support. GP and HF acknowledge Brain Tumour Research support