Directional migration of adult hematopoeitic progenitors to C6 glioma in vitro

Abstract

Multiform glioblastoma is the most common primary, highly invasive, malignant tumor of the central nervous system, with an extremely poor prognosis. The median survival of patients following surgical resection, radiation therapy and chemotherapy does not exceed 12-15 months and thus, novel approaches for the treatment of the disease are required. The phenomenon of the directed migration of stem cells in tumor tissue presents a novel approach for the development of technologies that facilitate the targeted delivery of drugs and other therapeutic agents to the tumor foci. Hematopoietic cluster of differentiation (CD)34+/CD133+ stem cells possess significant reparative potential and are inert with respect to normal neural tissue. The aim of the present study was to investigate the substantiation ability of adult hematopoietic progenitors to the directed migration of glioma cells. A C6 glioma cell line, a culture of hematopoietic CD34+/CD133+ stem cells and primary cultures of rat astrocytes and fibroblasts were used. The cells were co-cultured for five days. The results revealed the formation of cell shaft hematopoietic stem cells on the perimeter of the culture inserts containing the glioma culture. However, this was not observed in the wells with fibroblast and astrocyte cultures. The results indicated that hematopoietic stem cells exhibit a high potential for the directional migration of C6 glioma cells, which allows them to be considered as a promising cell line for the development of novel anticancer biomedical technologies and increases our understanding with regard to previously unclear aspects of glial tumor carcinogenesis.

Keywords: glioblastoma multiform; glioma C6; hematopoietic stem cells; neural stem cells.

Figures

Figure 1

General scheme of the experiment. (A) Culture plates comprising (1) C6 glioma cells, (2) astrocytes, (3) fibroblasts, (4) hematopoietic stem cells and (5) an empty insert. (B) A culture insert with (1) C6 glioma cells co-culturing with (2) hematopoietic stem cells.

Figure 2

Characteristics of hematopoietic stem cells and neoplastic C6 glioma line. (A) Phase contrast microscopy of hematopoietic stem cells in culture (magnification, ×20). (B) Fluorescence microscopy of hematopoietic stem cells stained with Vybrant® CFDA SE Cell Tracer directly prior to co-culture (magnification, ×10). (C) Inverted microscopy of neoplastic cells of rat C6 glioma. Cells of different shapes and sizes were observed actively flattened on the surface, forming numerous outgrowths on the third day of cultivation. Multinucleated cells were clearly visible (magnification, ×32). (D) Neoplastic C6 glioma cells stained using anti-glial fibrillary acidic protein monoclonal antibody, with nuclei counterstained using DAPI (magnification, ×100). (E) Rat brain preparation six days after the implantation of C6 glioma cells. (F) T2-weighted magnetic resonance imaging of the rat brain seven days after implantation of C6 glioma cells. Signs of compression of the ventricles of the brain edema and dislocation of medial brain structures were visible.

Figure 3

Characteristics of cell lines and control fibroblast cultures and astrocytes, and reaction of different cell lines to co-culturing with glioma cells in the experiment. (A) Rat fibroblasts (hematoxylin and eosin; magnification, ×40). (B) Rat fibroblasts. Phagocytosis of collagen immobilized on the surface of the fluorescent microparticles FluoSpheres® Collagen I-Labeled Microspheres (magnification, ×630). (C) Rat astrocytes stained with anti-GFAP Mab and nuclei counterstained with DAPI (magnification, ×100). (D) Rat astrocyte stained with anti-GFAP Mab (magnification, ×200). (E) Formation of fluorescent cell shaft on the perimeter of culture inserts containing glioma cells, by hematopoietic stem cells stained with Vybrant® CFDA SE Cell Tracer (magnification, ×10). (F) Co-culturing hematopoietic stem cells and rat fibroblasts. The formation of the cell shaft was absent (magnification, ×10). GFAP, glial fibrillary acidic protein; Mab, monoclonal antibody.

Figure 4

Distribution analysis showing the number of hematopoietic stem cells co-cultured with glioma between the first to the fifth days of the experiment.

Figure 5

Comparative dynamics of the number of hematopoietic stem cells in culture inserts containing glioma C6 cells, astrocytes and fibroblasts between the first and fifth days of the experiment.