The Evaluation of Glioblastoma Cell Dissociation and Its Influence on Its Behavior.

Veronika Skarkova,Vera Kralova,Adam Skarka,Barbora Vitovcova,Petra Kasparova,Emil Rudolf,Marketa Krupova,Petr Krupa

doi:10.3390/ijms20184630

Abstract

Purpose: Primary cell lines are a valuable tool for evaluation of tumor behavior or sensitivity to anticancer treatment and appropriate dissociation of cells could preserve genomic profile of the original tissue. The main aim of our study was to compare the influence of two methods of glioblastoma multiforme (GBM) cell derivation (mechanic—MD; enzymatic—ED) on basic biological properties of thus derived cells and correlate them to the ones obtained from stabilized GBM cell line A-172. Methods: Cell proliferation and migration (xCELLigence Real-Time Cell Analysis), expression of microRNAs and protein markers (RT-PCR and Western blotting), morphology (phase contrast and fluorescent microscopy), and accumulation of temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide (AIC) inside the cells (LC-MS analysis) were carried out in five different samples of GBM (GBM1, GBM2, GBM32, GBM33, GBM34), with each of them processed by MD and ED types of isolations. The same analyses were done in the A-172 cell line too. Results: Primary GBM cells obtained by ED or MD approaches significantly differ in biological behavior and properties of these cells. Unlike in primary MD GBM cells, higher proliferation, as well as migration, was observed in primary ED GBM cells, which were also associated with the acquired mesenchymal phenotype and higher sensitivity to TMZ. Finally, the same analyses of stabilized GBM cell line A-172 revealed several important differences in measured parameters. Conclusions: GBM cells obtained by MD and ED dissociation show considerable heterogeneity, but based on our results, MD approach should be the preferred method of primary GBM cell isolation

Highlights

Glioblastoma multiforme (GBM) represents the most aggressive form of glial tumors, characterized by extensive genetic, as well as epigenetic, alterations leading to multiple changes in biological behavior of malignant cells [1,2]
Our results showed that both ED and mechanic dissociation (MD) approaches enable successful establishment of primary GBM cell lines, which could be maintained under standard laboratory conditions and subjected to various tests and assays
Our other obtained results show that A-172 cells are highly dissimilar in their morphology to the primary GBM cells; i.e., they are smaller and lack mesenchymal markers, despite the fact that their proliferation and migration are considerable. These results suggest that both ED and MD approaches may be used for establishment of primary GBM cell lines, they generate GBM cell populations differing significantly in their phenotype and biological behavior

Summary

Introduction

Glioblastoma multiforme (GBM) represents the most aggressive form of glial tumors, characterized by extensive genetic, as well as epigenetic, alterations leading to multiple changes in biological behavior of malignant cells [1,2]. Besides the above-mentioned limitations of the standard care strategy of GBM, there are additional therapy-related concerns, which include for instance the diffuse infiltrative nature of the tumor based, at least in part, on invasiveness of GBM cells. These cells typically invade up to several centimeters away from main tumor mass and can even cross into the contralateral hemisphere. GBM contains a subset of glioma stem cells (GSCs), which may have an increased invasive potential and are thought to be more resistant to radiotherapy and chemotherapy compared to non-stem tumor cells. Gliomas seem to prefer single-cell migration and invade over longer distances than other tumors that metastasize in the brain [8]

Objectives

Methods

Results

Discussion

Conclusion