Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo

Abstract

Background: Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133⁺ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133⁺ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133⁺ GBM cells and their effects on tumor development, remains poorly defined.

Methods/results: We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133⁺ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133⁺ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells.

Conclusions: Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133⁺ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Keywords: CD133+ cells; Chemokines; Exosomes; Experimental model; MSCs; Tropism.

Fig. 1

a The establishment of human GBM primary cell culture. b Isolation of tumor neurospheres derived from GBM primary cell culture. d Purification of GBM cells from tumor subspheres using CD133 microbeads. Immunophenotypic characterization by using flow cytometry to evaluate the efficiency of magnetic cell separation for the antigenic marker, CD133 (76.3%). e–h CD133⁺GBM cells were able to further generate subspheres, compared with the absence of subspheres obtained from CD133⁻ fractions (c). e, f GBM subspheres visualized by inverted microscopy. g, h GBM subspheres visualized by fluorescence microscopy. i–k TEM of the GBM subspheres. l–q TEM of the CD133⁺ stem cells. n = nucleus, c = cytoplasm, mi = mitochondria, rer = rough endoplasmic reticulum, pv = pinocytic vesicles, v = vacuoles, arrow = electron-dense granules or magnetic beads. Scale: i–k 5.0 μm, l 2.0 μm, m–q 1.0 μm. r Fluorescence detection of Qdots (705 nm) labeling in the CD133⁺ GBM cells. s Fluorescence detection of Qdots (705 nm) labeling in the CD133⁺ GBM cells and DAPI. Magnification: ×400. All figures are representative ones from assays performed at least five times

Fig. 2

a, b Culture of MSCs with approximately 80–90% confluence. Magnification: ×100. c Induction of adipocyte-like phenotype by oil red stain. d Induction of osteogenic-like by Alizarin red stain. c, d′ Undifferentiated control for adipogenic and osteogenic differentiation, respectively. e, f Fluorescence detection of MION-Rh labeling in the MSCs. Magnification: a, b, d, e ×100; c, f ×400. g Graphs summarize FACS analysis of MSC expression of cell markers: 73.2% of MSCs reacted with the anti-CD73 antibody; 98.7% of MSCs reacted with the anti-CD44 antibody; 95.9% of MSCs reacted with the anti-CXCR4 and anti-CCR2 antibodies and there was low or no expression of CD31 and CD45 markers. H RT-PCR analysis of CXCR4 and CCR2 mRNA levels expressed in MSCs (triplicate samples). FACS and RT-PCR analysis are representative of all collected MSCs samples

Fig. 3

Representative figure of migration assays of MSCs, in transwell dishes, in different conditions placed in the lower chambers: a MSCs not labeled [control], b conditioned medium supplemented with specific neutralized antibodies (anti-MCP-1 and anti-SDF-1), c conditioned medium supplemented with specific neutralized antibodies (anti-MCP-1), d conditioned medium supplemented with specific neutralized antibodies (anti-SDF-1), e CD133⁺ cell culture supernatants (TBSCM), f chemokines MCP-1 and SDF-1, g graph summarized of mean number of migrated MSCs in relation to different conditions, h RT-PCR analysis of MCP-1/CCL2 mRNA levels expressed in CD133⁺ GBM cells (n = 5; GBM1 GBM2 GBM3 GBM4 GBM5). i RT-PCR analysis of SDF-1/CXCL12 mRNA levels expressed in CD133⁺ GBM cells (n = 5; GBM1 GBM2 GBM3 GBM4 GBM5). L50: Ladder 50 bp; LM low mass ladder; R positive control (human reference total RNA Clontech)

Fig. 4

Tumorigenesis study for stereotaxic implantation of the cells in different conditions: A 1 × 10⁴ MSCs labeled MION-Rh; B 1 × 10⁴ CD133⁺ GBM cells labeled Qdots(705 nm); C 1 × 10⁴ MSCs labeled MION-Rh added 1 × 10⁴ CD133⁺ GBM cells labeled Qdots(705 nm); D implantation of 1 × 10⁴ CD133⁺ GBM cells labeled Qdots (705 nm), after the establishment of the GBM (28 days), was made the infusion in caudal vein 1 × 10⁴ MSCs (MION-Rh); the development of tumor was followed for 20 days. A₁, C₁, D₁ MSCs labeled MION-Rh and visualized by fluorescence detection. B₁, C₂, D₂ CD133⁺ GBM cells labeled Qdot 705 nm and visualized by fluorescence detection. A₂, B₂, C₃, D₃ MSCs labeled MION-Rh and CD133⁺ GBM cells labeled Qdot 705 nm using combined fluorescence and X-ray detection. A₃, C₄ IHC analysis for Prussian blue staining of the MSCs labeled with MION-Rh. B₄, D₄, D₅ Hematoxylin and eosin staining. B₅, C₆, D₆ IHC analysis for GFAP. C₅ IHC analysis for Ki67; B₆, C₇, D₇ IHC analysis for VEGF. Red arrow: site of stereotaxic implantation of MSCs labeled MION-Rh. Green arrow: site of stereotaxic implantation of CD133⁺ GBM cells labeled Qdot (705 nm).Green circle evidenced proliferation: MSCs and CD133⁺ GBM cells. These images are representative of all collected MSCs and GBM samples

Fig. 5

Implantation of 1 × 10⁴ CD133⁺ GBM cells labeled Qdots (705 nm), after the establishment of the GBM (28 days), was made the infusion in caudal vein 1 × 10⁴ MSCs (MION-Rh); the development of tumor was followed for 20 days. a MSCs labeled MION-Rh and CD133⁺ GBM cells labeled Qdot 705 nm using combined fluorescence and X-ray detection. b CD133⁺ GBM cells labeled Qdot 705 nm and visualized by fluorescence detection. c MSCs labeled MION-Rh and visualized by fluorescence detection. d–h, i–l MRI (T₂*-weighted images) of animal brain monitoring of the process of migration of MSCs, which were able to cross the blood-brain barrier of the animal and migrated to the tumor region, promoting GBM cell proliferation. l MRI (T₂*-weighted images) of animal brain without stereotaxic implantation of cells (control group). Red circle showed migration assays of MSCs and green circle evidenced tumor propagation. m IHC analysis for Prussian blue staining of the MSCs labeled with MION-Rh. n, r, s, t Hematoxylin and eosin staining. u IHC analysis for GFAP. o IHC analysis for Ki67; v IHC analysis for p53; p IHC analysis for CD44 staining of the MSCs; q IHC analysis for CD73 staining of the MSCs; w, x IHC analysis for CD63 staining of the MSCs-derived exosomes; y–z′ IHC analysis for CD9 staining of the MSCs-derived exosomes. These images are representative of all collected MSCs and GBM samples

Fig. 6

Schematic representation demonstrating that chemokines mediate MSC migration toward CD133⁺ stem cell of GBM and scanning electron microscopy of exosome, secreted by MSCs, promoting tumor dissemination