Evidence for a secreted chemorepellent that directs glioma cell invasion

Abstract

Secreted chemotropic cues guide the migration of neuronal and glial cell precursors during neural development. It is not known if chemotropism contributes to directing the invasion of brain tissue by glioma cells. A model system has been developed that allows quantification of invasive behavior using gliomas spheroids embedded in collagen gels. Here we provide evidence that glioma spheroids secrete a chemorepellent factor(s) that directs cells away from the spheroid and into the collagen matrix. The relationship between total invasion, cell number, and implantation distance suggests that glioma cells respond to a gradient of the chemorepellent cue(s) that is well established at 48 h. C6 astrocytoma cells normally invade the collagen at an angle perpendicular to the spheroid edge. In contrast, an adjacent spheroid causes cells to turn away from their normal trajectory and slow their rate of invasion. Astrocytoma cells are repelled by an adjacent glioma spheroid but rapidly infiltrate astrocyte aggregates, indicating that astrocytes do not express the repellent cue. Uniform concentrations of repellent factor(s) in spheroid conditioned medium overwhelm endogenous gradients and render glioma cells less able to exhibit this chemotropic response. Concentration gradients of spheroid conditioned medium in cell migration assays also demonstrate the chemorepellent cue(s)'s tropic effect. Our findings indicate that glioma spheroids produce a secreted diffusible cue(s) that promotes glioma cell invasion. Identification of this factor(s) may advance current therapies that aim to limit tumor cell invasion.

Inhibition of medulloblastoma cell invasion by Slit

“…Human fetal astrocytes were obtained as described above, and hanging drop aggregates (45 000 cells/drop) were prepared as described (Ruffini et al, 2004;Werbowetski et al, 2004). UW3 and astrocyte aggregates were labeled with 0.075 mg/ml DiI and DiO solutions in DMEM, respectively (Molecular Probes, Eugene OR, USA).…”

Isolation of a Natural Inhibitor of Human Malignant Glial Cell Invasion: Inter α-Trypsin Inhibitor Heavy Chain 2

“…C6 (murine glioblastoma) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 125 units/mL penicillin G, 125 Ag/mL streptomycin sulfate, and 2.2 Ag/mL amphotericin B (Fungizone) and seeded into spinner culture flasks as described (5,6). Tumor spheroids were implanted into 48-well culture dishes containing 500-AL aliquots of a collagen type I solution (Vitrogen 100; COHESION, Palo Alto, CA) and cell invasion was assessed daily as previously described (5,6). All culture reagents were obtained from Life Technologies Invitrogen (Burlington, Ontario, Canada) unless otherwise stated.…”

“…There are many known positive regulators of malignant brain tumor cell proliferation and invasion; however, little is known about negative modulators of malignant glial cell invasion (1,(3)(4)(5).…”

“…Our laboratory has provided evidence for a secreted chemorepellent that directs glioma cell invasion (5). Conditioned medium from both U251 and C6 spheroids significantly inhibited invasion of both spheroid types when implanted into three-dimensional collagen type I gels, providing evidence for a potential inhibitory or kinetic effect as well (5).…”

“…Conditioned medium from both U251 and C6 spheroids significantly inhibited invasion of both spheroid types when implanted into three-dimensional collagen type I gels, providing evidence for a potential inhibitory or kinetic effect as well (5). Because this model can be used as a functional assay to identify endogenous and/or serum-derived inhibitors/repellents of glioma invasion, a purification strategy was designed and optimized to isolate these inhibitors/repellents from the conditioned medium of glioma spheroids.…”