Stem cell marker analysis of glioblastoma tumor cells and infection studies using Zika virus protein E pseudotyped lentiviral particles

Abstract

As part of this study, a novel cell culture system for glioblastoma (GBM) cells was developed. For this purpose, GBM tumor samples from areas adjacent to the subventricular zone (SVZ) were used. These areas contain many GBM stem cells (GSCs) and astrocyte-like stem cells. It was demonstrated that the cell cultures maintained their heterogeneity for at least three months when cultured in CSF-DF medium. Based on these findings, a system was developed that enables examination of primary GBM tumor cells under conditions that closely resemble the in vivo environment. In total, twelve primary cell cultures were successfully established (AKH-14 to AKH-24C). Immunological examination of the primary cell cultures revealed the presence of both stem cell- and GBM-specific markers, as well as the surface proteins AXL/Gas6 and integrin αvβ5, which act as entry receptors for the Zika virus (ZIKV). Expression of the stem cell markers OCT4, SOX2, NANOG, Nestin, integrin α6, CD44, and CD133 was detected in the cell cultures. The GBM markers EGFR and PD-1 were overexpressed in all primary cell cultures. Additionally, the isolation of astrocyte-like stem cells was demonstrated by the expression of glial fibrillary acidic protein (GFAP). Immunological analyses revealed that each cell culture exhibited a distinct biomarker expression pattern, suggesting the existence of many different GSC subpopulations. The two ZIKV receptors, AXL/Gas6 and integrin αvβ5, were successfully detected in all primary cell cultures. This study also involved the development of a pseudotype system in which the transmembrane and cytoplasmic (TMCY) region of ZIKV was replaced with that of vesicular stomatitis virus (VSV-G). This approach differs from methods previously established within the working group. The ZIKV E protein was cloned into two expression vectors, pCMV-VSV-G and pMD2.G, to produce the expression vectors pCMV-EG1 and pMD2-EG2. These constructs were used in combination with the packaging plasmid pNLgfpAM to produce the pseudotypes EG1-HIVgfp and EG2-HIVgfp. Infection experiments with both pseudotypes demonstrated successful infection of the primary cell cultures AKH-13, AKH-14, AKH-17, AKH-19, AKH-23, and AKH-24C.