Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model

Object. Although it is known that malignant astrocytomas infiltrate diffuse ly into regions of normal brain, it is frequently difficult to identify une quivocally the solitary, invading astrocytoma cell in histopathological pre parations or experimental astrocytoma models. The authors describe an exper imental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion. Methods. Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane ( OA-mm pore size) that was bathed in medium supplied by the lower chamber. T wo astrocytoma cell lines. U-87 MG (U87) and U343 MG-A (U343), were selecte d because of their differing basal cell motilities in monolayer cultures. T he astrocytoma cells were stably transfected with vectors that expressed gr een fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid-mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of G FP expression were selected using the direct visualization provided by fluo rescence microscopy and fluorescence-activated cell-sorter analysis. The GF P-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was me asured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing as trocytoma cells could be readily tracked and followed in this model system. individual astrocytoma cells that exhibited green fluorescence could be re adily identified following their migration through the brain slices. The GF P-laheled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma c ells also infiltrated farther than the GFP-labeled astrocytoma cells themse lves. The expression of antisense RHAMM I virtually abrogated the invasion of the brain slices by both astrocytoma cell lines. Conclusions. The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion , net only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but a lso because the GFP tag enables tracking of highly migratory and invasive o strocytoma cells under direct vision.