Implantation of C6 astrocytoma spheroid into collagen type I gels: invasive, proliferative, and enzymatic characterizations

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✓ 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.

Article Information

Address reprint requests to: Rolando F. Del Maestro, M.D., London Health Sciences Centre, Westminster Campus, Room 6109, 800 Commissioners Road, East, London, Ontario N6A 4G5, Canada. email: rdelmaes@julian.uwo.ca.

© AANS, except where prohibited by US copyright law.

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Figures

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    Photomicrographs showing a C6 spheroid and C6 astrocytoma cell invasion into collagen type I gel. A: The C6 astrocytoma cell invasion from a small spheroid after 24 hours. Original magnification × 88; bar = 100 µm. B: Some cells appear to display a bipolar morphology (straight arrow), whereas others are more rounded in appearance (curved arrow). Original magnification × 210; bar = 100 µm.

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    Graph displaying total invasion distance from C6 spheroids monitored for 12 days. Conditioned medium was replaced on Days 3, 6, and 9. The maximum invasion distance was approximately 2100 µm and the maximum daily invasion distance was seen on Day 1. The majority of invasive activity was seen from Day 1 through Day 5. Values represent the means ± standard deviations (SDs) for three experiments.

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    Bar graph showing the influence of three different growing conditions (on plastic, on type I gel, and in type I gel) on three different enzymatic activities from medium-sized spheroids (general proteolytic [GP], collagen type I degrading [Type I], and collagen type IV degrading [Type IV] activities). The means ± SDs of spheroid diameters used for each of these growing conditions were 469 ± 47, 469 ± 38, and 458 ± 38 µm, respectively. Enzymatic activities were assayed in the manner described in Materials and Methods. Values represent the means ± SDs for four experiments compared with activity on plastic. *p < 0.05. NS = not significant.

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    Graph showing the effect of the metalloprotease inhibitor 1,10 o-phenanthroline and TIMP-2 on C6 astrocytoma cell invasion. Values represent the means ± SDs for three experiments. *p < 0.05, **p < 0.01 compared with invasion distance of control on each day.

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    Bar graph depicting changes in labeling indices in the three microregions studied (spheroid surface, all invading cells, and invading edge) over 5 days. Spheroids were assessed on Days 1, 3, and 5. White bars on Days 1 and 5 show labeling indices of spheroid surface microregions cultured on plastic as controls. Values represent the means ± SDs for three experiments. *p < 0.05, **p < 0.01 compared with labeling indices of each area on Day 1. # p < 0.05 between two specific microregions.

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    Graph showing total invasion distances from three sizes of spheroid. The mean diameters of the spheroids used are shown. Total invasion distance was measured in the manner described in Materials and Methods. Values represent the means ± SDs for five experiments.

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    Bar graphs depicting changes in collagen type I degrading activity in the conditioned media from cultures of large and small spheroids on Days 1, 3, and 5. Each activity was measured in the manner described in Materials and Methods. Values represent the means ± SDs for four experiments. *p < 0.05 compared with activity in medium from culture of small spheroid on Day 1.

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    Bar graph showing the effects of various substrates mixed with collagen type I gel on total C6 astrocytoma cell invasion at 5 days. Values represent the means ± SDs for three experiments. Col IV = collagen type IV; FN = fibronectin; H = hyaluronic acid (µg/ml); LN = laminin. *p < 0.05, **p < 0.01 compared with the control value, which was standardized to 100%.

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