A three-dimensional model has been developed in which C6 astrocytoma spheroids of defined sizes are embedded into collagen type I gels. The authors have monitored cell invasive behavior; obtained quantitative data on cell invasion, proliferation, and enzymatic activity; assessed cell-cell interactions by altering the spheroid size used; and studied cell-matrix interactions by modifying the matrix components. Their results show that C6 astrocytoma cells detach from the spheroid surface and invade the gel as single cells by means of a system that appears to be dependent on metalloprotease function. These invasive cells have a low proliferative index. Larger spheroids with central hypoxic microregions possess cells that invade the gel at faster rates; this could be correlated with the release of increased collagen type I degrading activity. Extracellular matrix proteins, such as laminin, fibronectin, and collagen type IV have no significant influence on invasive activity, whereas hyaluronic acid decreases and human central nervous system myelin increases invasion. New strategies directed at the treatment of malignant gliomas must take into account the subpopulation of malignant cells located long distances from the major tumor mass. The spheroid invasion model may provide specific insights into the behavior of these invasive cells.

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An experimental model of malignant glioma growth involving implantation of spheroids into a gel matrix of collagen type I has been developed. This model has been used to characterize changes in glioma cell invasion in response to single dose and fractionated radiation treatment. Suspensions of C6 astrocytoma cells were grown in spinner culture flasks to yield spheroids of varying size (300-1000 microm). Implantation of spheroids into a gel matrix of collagen type I was associated with measurable invasion of the surrounding gel by individual tumor cells. Changes in the distance of invasion in response to single dose and fractionated radiation were measured. Changes in apoptosis and proliferative indices in different regions of the spheroids in response to radiation were also assessed. In unirradiated gels, maximum depth of invasion, 1300-1750 microm, was achieved by 5 days after implantation. A radiation dose-dependent inhibition of invasion was noted and was most profound for larger spheroids. Fractionation of the radiation dose was associated with a partial recovery of invasion. Changes in apoptotic and proliferative indices in response to radiation depended on the region of the spheroid examined. Increases in apoptosis were noted for cells at the surface of the spheroid and invading cells while cells at the centre of the spheroid demonstrated virtually no increase in apoptosis. Likewise, a dose-dependent decrease in proliferative indices following radiation was noted among the invading cells and cells at the surface of the spheroid but not at the centre of the spheroid. We have described a model of malignant glioma invasion which possesses many of the qualities of in vivo malignant gliomas. Within this model, invasion appeared to be inhibited by radiation in a dose- and fractionation-dependent fashion. Measurement of apoptotic and cell proliferation indices favour a direct cytotoxic effect on the invading cells as the most likely mechanism for this phenomenon.

Tumor growth is dependent on the ability of neoplastic cells to induce angiogenesis. Remodelling of blood vessels requires reconstruction of the collagen (type IV) and non-fibrous protein components of basement membrane. This study assessed the general protease and collagenase (IV) activities of C6 astrocytoma cells in monolayer and spheroid culture and C6 astrocytoma spheroids growing in vivo. Extracellular release of non-specific proteases and collagenase IV was maximal during early exponential cell growth. Increased spheroid size resulted in enhanced extracellular activity of both enzyme groups assessed. The size of the implanted spheroid influenced the activity measured in vivo. General proteolytic activity was significantly greater in tumor tissue at all spheroid sizes while only the implantation of 750 microns spheroids resulted in significantly increased collagenase type IV activity. The growth of C6 astrocytoma cells in monolayer and spheroid culture in vitro and in vivo is associated with distinct alterations in intracellular and extracellular activity of the proteolytic enzymes assessed. Increased extracellular release of these enzymes may play important roles in tumor-associated angiogenesis, tumor invasiveness, tumor induced hemorrhage and tumor-associated edema.