This model represents a reproducible approach to the preparation of implantable spheroids with invasive potential that compares with those produced using spinner culture. The use of hanging drops broadens the applicability of three-dimensional in vitro assays examining brain tumor invasiveness.

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.

Recent studies using molecular and cellular techniques of the factors regulating the invasion process have revealed a crucial role for a number of growth factors and cytokines. Their function lies on the one hand in the autocrine stimulation of the tumor cells themselves, resulting in the stimulation of protease expression and an enhancement of migratory potential. On the other hand, the growth factors and cytokines seem to play a major role in the paracrine activation of the tumor surrounding stroma. Through stimulation of the strong angiogenic response that is characteristic for gliomas and also of the expression of proteases in the stromal cells, they contribute critically to the generation of a stromal environment that is permissive or even inductive for tumor cell invasion. Understanding of the mechanisms by which soluble factors modulate glioma cell invasion therefore will help to determine targets for the modification of existing therapies and lead to the development of novel therapeutic strategies in the management of gliomas.