Motility factor produced by malignant glioma cells: role in tumor invasion

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✓ To better understand the cellular mechanism of tumor invasion, the production of a cell motility-stimulating factor by malignant glioma cells was studied in vitro. Serum-free conditioned media from cultures of rat C6 and human T98G cell lines contained a factor that stimulated the locomotion of the producer cells. This factor was termed the “glioma-derived motility factor.” The glioma-derived motility factor is a heat-labile protein with a molecular weight greater than 10 kD and has relative stability to acid. The factor showed not only chemotactic activity but also chemokinetic (stimulated random locomotion) activity in the two types of glioma cells studied. Although glioma-derived motility factors in conditioned media obtained from two different cell origins are likely to be the same, chemokinetic migration of T98G cells to their conditioned medium was much stronger than that of C6 cells to theirs.

Coincubation of cells with cytochalasin B, which disrupts the assembly of cellular actin microfilaments, almost completely inhibited the cell migration stimulated by glioma-derived motility factor. Cytochalasin B also induced marked alterations in cell morphology, including cell retraction and arborization, while the drug did not affect cell attachment to culture dishes. These results indicate that glioma cells produce a motility factor which may play a role particularly when tumor cells are detached and migrate away from the original tumor mass, thus promoting tumor invasion. Also, glioma cell migration stimulated by the motility factor requires the normal organization of cytoskeletons such as actin microfilaments.

Article Information

Address reprint requests to: Takanori Ohnishi, M.D., Department of Neurosurgery, Osaka University Medical School, 1-1-50 Fukushima, Fukushima, Osaka 553, Japan.

© AANS, except where prohibited by US copyright law.

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    Photomicrographs showing glioma cells that have migrated to the lower side of a Nucleopore filter with 8-µm pores. Upper Pair: Migration of C6 glioma cells. Conditioned medium of cultured C6 glioma cells (left) or minimal essential medium (right) was used as a chemoattractant in the lower wells of the microchambers. Lower Pair: Migration of T98G cells. Conditioned medium of T98G cells (left) or minimal essential medium (right) was used as a chemoattractant. Diff-Quik stain, × 200.

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    Dose-dependent stimulation of migration of both types of glioma cells by serum-free conditioned medium obtained from each cell source. The C6 glioma cells were placed in the upper wells and the indicated dilutions of C6 conditioned medium were dispensed in the lower wells of the microchambers. The T98G cells were placed in the upper wells and the indicated dilutions of T98G conditioned medium in the lower wells. Circles and bars represent means ± standard error of the means for six experiments.

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    Checkerboard analysis of the migratory responses of C6 glioma cells (left) and T98G cells (right) to that cell type's conditioned medium. Various concentrations of serum-free conditioned medium were dispensed into the lower wells, and a cell suspension containing various concentrations of the conditioned medium of the same origin was added to the upper wells. The values shown indicate the cell number per sq mm that had migrated to the lower side of the filter, expressed as means ± standard error of the means for triplicate experiments. The far left vertical column indicates the migratory response to maximal positive gradient. Values on the uppermost line represent the migratory response to a maximal negative gradient. Values along the diagonal represent stimulated random migration that occurs in the absence of an established gradient.

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    Chemotactic responses of C6 glioma cells and T98G cells to the conditioned media obtained from both cell origins. The concentration of each conditioned medium (CM) used as a chemoattractant for both glioma cell lines was matched in terms of the number of cells counted during the preparation of those conditioned media. Minimum essential medium (MEM) was used as a control attractant. Blocks and bars represent means ± standard error of the means for six experiments.

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    Phase-contrast micrographs demonstrating morphological changes in T98G cells treated with cytochalasin B. The T98G cells were incubated with cytochalasin B (10 µg/ml) for 6 hours. Left: Untreated preparation. Right: Preparation treated with cytochalasin B. × 200.

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    Effects of cytochalasin B on glioma cell migration stimulated by the conditioned medium. The C6 cells in the upper wells of the microchambers were incubated with indicated concentrations of cytochalasin B for 4 hours and the number of cells having migrated to the lower side of the filter was counted. Circles and bars represent means ± standard error of the means for six experiments. Asterisks show data significantly different (t-test) from untreated group results: * = p < 0.01, and ** = p < 0.001.

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    Effects of anti-fibronectin antibody on the cell migration stimulated by glioma-derived motility factor. The C6 conditioned medium (CM) was added to the lower wells in the presence or absence of affinity-purified anti-fibronectin (anti-FN, 10 or 20 µg/ml) antibodies. After incubation for 4 hours, the number of C6 cells that had migrated to the lower side of the filter was determined. Minimum essential medium (MEM) was used as a control substance. Blocks and bars represent means ± standard error of the means for six experiments.

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