Abstract
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.
Highlights
Glioblastoma (GBM) is the most common and most aggressive form of primary brain tumour in adults, carrying a dismal prognosis
By exploring the significance of astrocytes in the tumour microenvironment (TME), we demonstrated that astrocytes physically interact with GBM cells reducing drug sensitivity to TMZ, vincristine (VCR), and clomipramine (CLM) by developing tunneling nanotubes (TNT) connections
Two GBM cell lines (U-87 MG, UP-007) were stained using Cell Trace carboxyfluorescein succinimidyl ester (CFSE), (Figure 1A,B; Figure S1) and the percentage of CFSE-positive cells remained >95% during 7 days of culture demonstrating low toxicity even using a high concentration of dyes (10 μM)
Summary
Glioblastoma (GBM) is the most common and most aggressive form of primary brain tumour in adults, carrying a dismal prognosis. The GBM tumour microenvironment (TME) is characterized by complex networks of cancer cells and stromal cells such as astrocytes, microglia, and endothelial cells and an extensive release of soluble factors [6], which interact with the tumour and generate permissive conditions for tumour growth [7]. Astrocytes, the most numerous non-neuronal cells in the central nervous system (CNS), comprise approximately 50% of the human brain volume [17]. They dynamically respond to changes in the brain environment. In addition to the physiological roles that astrocytes play in the healthy CNS, during pathological conditions, such as trauma, ischemia, or stroke, astrocytes become activated, upregulating glial fibrillary acidic protein (GFAP) expression and showing a characteristic polarization with the formation of long processes [22]
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