Vascular endothelial growth factor A contributes to glioma-induced migration of human marrow stromal cells (hMSC)

https://doi.org/10.1016/j.expneurol.2005.11.027Get rights and content

Abstract

Objective

It has been demonstrated that murine neural stem cells (mNSCs) and human mesenchymal stroma cells migrate toward experimental gliomas, making stem cells a candidate for cellular carrier systems of anti-glioma therapy. However, few data are available on the factors involved in regulating stem cell migration. The aim of our study was to characterize the migratory and invasive behavior of adult human marrow stromal cells (hMSC) that interact with glioma cells, especially focusing on vascular endothelial growth factor A (VEGF-A)-mediated effects.

Methods

Human MSC were isolated from bone marrow biopsies carried out for hematological indications. The chemokinetic activity of hMSC in response to glioma-conditioned medium as well as VEGF-A was analyzed using a modified Boyden chamber assay. Invasion of hMSC and glioma spheroids was investigated using confrontational cultures. To provide analogous data from a well-described system, invasion of murine C17.2 neural stem cells was assessed. VEGF-A secretion by gliomas and the expression of VEGF-receptor 2 in hMSC were evaluated.

Results

Human MSC showed an extensive invasion into glioma spheroids. Glioma-conditioned medium significantly increased hMSC migration and also invasion, driven by chemotaxis. VEGF-A also showed significant pro-migratory and pro-invasive effects on hMSC, but in a reduced fashion compared to glioma-conditioned medium.

Conclusions

Human MSC show intensive migratory and invasive behavior in the presence of glioma cells and glioma-conditioned medium. Among others, VEGF-A seems to be one important factor in enhancing and directing stem cell motility.

Introduction

The treatment of malignant brain tumors, such as glioblastoma multiforme, invariantly fails despite extensive surgical excision and adjuvant radio- and chemotherapy. This treatment failure is due to diffuse infiltration of the surrounding neural tissue, often distinct from the primary tumor localization. These invasive tumor cells are inaccessible to current therapeutic strategies and are responsible for recurrent tumor growth. Promising gene therapeutic concepts resulted in limited distribution of the suicide gene after local administration using murine fibroblasts as cellular carriers for the viral gene therapy vectors (Rainov and Kramm, 2001). Murine embryonic neural stem cells (mNSC) have been demonstrated to show extensive tropism, which is promoted by a cerebral pathology, and to migrate expeditiously throughout a tumor mass. The surrounding of the invading tumor border was infiltrated by these cells as well as single cells tracked by mNSC (Aboody et al., 2000). The practical implication is that mNSC can actually seek out migrated tumor cells and foci at distinct sites of the primary tumor mass. Therefore, neural stem cells were considered potential cellular delivery systems for gene therapy of surgically inaccessible malignant gliomas.

However, several problems have to be faced when the stem cell approach is applied to a human adult environment. The mNSC described above are derived from an embryonic, immortalized, c-myc transfected murine cell line (Aboody et al., 2000). There is no human analogue of this particular cell line, which could be used as a cellular vector within the human brain. In addition, the use of embryonic stem cells incorporates ethical as well as several unsolved practical problems regarding their proliferative capacities when transplanted into an adult microenvironment. Therefore, a cell type has to be defined that meets the following criteria: (i) cells should be available from each glioma patient to create an autologous system without allogeneic immune responses. (ii) Within human brain parenchyma, cells should exhibit active motility directed toward glioma tissue.

The aim of this study was to determine the migratory and invasive capacity of human bone marrow stromal cells (hMSC), which can easily be obtained by bone marrow aspiration. Since recruitment of MSC by VEGF-A in other organ systems has been reported (Chen et al., 2004) and gliomas are highly VEGF-A expressing tumors, we focused on the potential role of VEGF-A in glioma-induced directional motility of hMSC. To provide an impression for the extent of VEGF-A-mediated effects on human MSC in relation to a well-established model system for stem cell–glioma interaction, analogous experiments were performed with the murine embryonic C17.2 cell line (Aboody et al., 2000).

Section snippets

Cell lines

The human glioma cell lines U-87, U-373, U-373GFP, U-251MG, and MZ-54GB were used. To allow identification of glioma cells during interaction with hMSC, U-373 cells have been stably transfected for green fluorescent protein (GFP) expression. Monolayer cultures of U-87 were grown in 75 cm2 tissue culture flasks (Nunc, Denmark) in a standard tissue culture incubator (5% CO2 in air, 100% humidity). The cells were grown in complete growth medium consisting of Dulbecco's Modified Eagle's Medium

hMSC antigen expression

FACS analysis showed a homogeneous population of bone marrow stromal cells according to size and granularity. Approximately 99% of the cells proved to be positive for CD 105 and 95% for CD 44. CD 11b, CD 14, CD 31, and CD 34 were not detected. These results are similar to those of previous investigations on the phenotype of hMSC (Conget and Minguell, 1999, De Ugarte et al., 2003, Majumdar et al., 2000, Reyes and Verfaillie, 2001). Immunohistochemistry of human VEGF-R2 detected a strong and

Discussion

The co-localization of glioma cells and C17.2 neural embryonic stem cells and the eradication of experimental gliomas by transfected stem cells support the hypothesis that gene therapy based on stem cell vectors could lead to a valuable improvement of glioma therapy (Aboody et al., 2000).

Neural stem cells have been described in the adult mammalian brain within the subventricular zone along the entire neuraxis (D'amour and Gage, 2003). It could be shown that migration of adult neural stem cells

Conclusion

Human MSC exhibit an intense migratory and invasive potential directed toward malignant gliomas in vitro. This property can be markedly enhanced by human malignant glioma cells via the secretion of soluble factors. VEGF-A seems to play an important role in this process. Since glioma-cell-conditioned medium shows much stronger effects on hMSC migration and invasion than VEGF-A alone, additional currently undefined cytokines have to be involved in this directional hMSC locomotion. Due to their

Acknowledgments

This work was supported by grants of the Friedrich-Baur-Stiftung, Burgkunstadt, Germany. We gratefully thank Mrs. Stefanie Lange, who provided helpful discussions and technical assistance.

References (28)

  • K. D'amour et al.

    Genetic and functional differences between multipotent neural and pluripotent embryonic stem cells

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • R.H. Goldbrunner et al.

    ECM dependent and integrin mediated tumor cell migration of human glioma and melanoma cell lines under serum-free conditions

    Anticancer Res.

    (1996)
  • R.H. Goldbrunner et al.

    Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms

    J. Neurosci. Res.

    (1999)
  • R.H. Goldbrunner et al.

    Vascular endothelial growth factor-driven glioma growth and vascularization in an orthotopic rat model monitored by magnetic resonance imaging

    Neurosurgery

    (2000)
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    Both authors contributed equally to this work.

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