Assessment of Intratumoral Heterogeneity in Isolated Human Primary High-Grade Glioma: Cluster of Differentiation 133 and Cluster of Differentiation 15 Double Staining of Glioblastoma Subpopulations

Abstract

BACKGROUND: Gliomas are the most common primary brain tumors, representing 50–60% of malignant primary brain tumors. Gliomas are highly heterogeneous with marked inter- and intratumoral diversity. Gliomas heterogeneity is a challenging issue in the development of personalized treatment. The simplest method for studying heterogeneity is using ex vivo cell cultures; in our case, the cell lines were isolated from patient with glioblastomas.
 AIM: Here, we reported distinct cell subpopulations heterogeneity in glioblastoma cells.
 METHODS: Human glioblastoma cells isolation is conducted by enzymatic method with combination of collagenase I, hyaluronidase, and trypsin enzyme in proportional amount from patient. Immunostaining was performed to assess glial fibrillary acidic protein (GFAP), Ki-67, isocitrate dehydrogenase-1 (IDH-1) status, and program death ligand-1 (PD-L1) expression. Primary glioblastoma cell line was characterized by flow cytometry (fluorescence-activated cell sorting) analysis based on cluster of differentiation (CD) 133 and CD15 marker expression. U87MG and CGNH-89 cell lines were used as control. Distinct subpopulation analysis was performed by double staining of CD133 and CD15 in isolated primary glioblastoma cell line and its comparative control cells.
 RESULTS: Our isolated glioblastoma cells morphology was adherent cells which were able to form spheres depending on environment. Immunostaining confirmed GFAP, Ki-67, IDH-1 mutants, and PD-L1 expression. Our isolated glioblastoma cells expressed CD133 and CD15, coexpressed CD133/CD15 in different patterns. The highest subpopulation in primary glioblastoma was CD133+/CD15+.
 CONCLUSION: Glioblastoma cells can be isolated using enzymatic methods. Isolated glioblastoma cells consist of four different subpopulations distinguished by CD133/CD15 double staining. Intratumoral heterogeneity exists and directly or indirectly depends on their microenvironment.