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 Response

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.

The Organoid Era Permits the Development of New Applications to Study Glioblastoma

“…Similarly, another study hypothesizes that astrocytes in TME behave like microglia by reducing cancer sensitivity to drugs and interactions between astrocytes and GB cells could be associated with increased growth and invasion of the tumor [96]. To confirm this, it was demonstrated that the direct contact between 3D co-culture of astrocytes and GB cells enhances glioblastoma formation [97]. More in detail, the authors of this study suggested that astrocytes may rescue the damaged target cancer cells transferring organelles along tunneling nanotubes.…”

Identification of a Metabolism-Related Risk Signature Associated With Clinical Prognosis in Glioblastoma Using Integrated Bioinformatic Analysis

“…Reaserches have further elucidated the potential mechanism of conventional antidepressants therapy for GBM. By targeting the respiratory chain complex III and changing membrane potential, the tricyclics antidepressants mentioned above induce mitochondria-mediated apoptosis of malignant glioma cells, activate the intrinsic pathway of cytochrome-C release and caspase-3 dependent apoptosis process, and finally results in glioma cell death ( 106 – 112 ). It is worth mentioning that previous studies have also indicated that the anticoagulant ticlopidine synergized with imipramine to induce the death of human glioma cell lines and primary mouse glioma cells ( 98 , 99 ).…”

Practical Review on Preclinical Human 3D Glioblastoma Models: Advances and Challenges for Clinical Translation

“…The most commonly used culture media for spheroids are Eagle’s minimal essential medium (EMEM), Dulbecco modified Eagle’s minimal essential medium (DMEM) and Roswell Park Memorial Institute medium (RPMI) supplemented with penicillin/streptomycin and fetal bovine serum (FBS). Human serum has also been used to better mimic the natural microenvironment of cancer cells [ 32 , 33 ]. The majority of the existing GBM cell lines (U-87MG, U-105MG, U-118MG, U-138MG, U-178MG, U-251MG, U-343MG, U-373MG, U-1231MG, BMG-1, T98G, A172, SNB-19, ACBT, M059J, LN-229, Hu197, and LN-18) are able to form spheroids except the U-343MG cell line [ 7 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ].…”