title: Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model.
abstract: OBJECTIVE
  Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental 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 (0.4-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines, U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green 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 GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. Individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-labeled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM 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, not only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive astrocytoma cells under direct vision.
authors.affiliation: The Arthur and Sonia Labatt Brain Tumour Research Centre, Division of Neurosurgery, The Hospital for Sick Children, The University of Toronto, Ontario, Canada
authors.forename: S
authors.initials: S
authors.lastname: Jung
authors.forename: C
authors.initials: C
authors.lastname: Ackerley
authors.forename: S
authors.initials: S
authors.lastname: Ivanchuk
authors.forename: S
authors.initials: S
authors.lastname: Mondal
authors.forename: L E
authors.initials: LE
authors.lastname: Becker
authors.forename: J T
authors.initials: JT
authors.lastname: Rutka
mesh_terms: D001254:Astrocytoma / Q000473:pathology*; D001921:Brain / Q000473:pathology*; D001932:Brain Neoplasms / Q000473:pathology*; D016326:Extracellular Matrix Proteins; D049452:Green Fluorescent Proteins; D006801:Humans; D018960:Hyaluronan Receptors; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008854:Microscopy, Electron; D009361:Neoplasm Invasiveness / Q000473:pathology; D009924:Organ Culture Techniques; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C098946:hyaluronan-mediated motility receptor; D049452:Green Fluorescent Proteins