Glioma invasiveness responds variably to irradiation in a co-culture model.
OBJECTIVE
We developed a co-culture system to quantitate the growth and invasion of human malignant gliomas into a background of confluent normal human astrocytes, then used this assay to assess independently the effects of irradiating both cell types on glioma invasion.


METHODS
Enhanced green fluorescent protein (EGFP)-labeled immortalized human astrocytes, human malignant glioma cells, or transformed human astrocytes were focally plated onto a confluent layer of normal human astrocytes, and the invasiveness of EGFP-labeled cells was scored after 96 h. To address the consequences of irradiation on glioma invasion, the invasiveness of irradiated glioma cell lines and irradiated astrocytic backgrounds was assessed. Fluorescence-activated cell sorting was used to quantitate the total number of EGFP-labeled cells.


RESULTS
Growth in the co-culture assay consistently reflected transformation states of the plated cells. Immortalized, but untransformed human astrocytes failed even to establish growth on confluent normal human astrocytes. In contrast, all malignant human glioma cell lines and transformed human astrocytes demonstrated various degrees of infiltration into the astrocytic bed. Irradiation failed to alter the invasiveness of U87, A172, and U373. A 1-Gy dose slightly reduced the invasiveness of U251 MG by 75% (p < 0.05 by one-way analysis of variance and post hoc Neuman-Keuls), without reducing total cell numbers. Independently irradiating the human astrocytic bed did not alter the invasiveness of nonirradiated U251, whereas the matrix metalloproteinase (MMP) inhibitor GM6001 reduced U251 invasiveness in the co-culture assay.


CONCLUSIONS
Growth in the co-culture assay reflects the transformation status and provides a useful in vitro model for assessing invasiveness. Human glioma invasiveness in the co-culture model responds variably to single low-dose fractions. MMP activity promotes invasiveness in the co-culture model. Reduced invasiveness in irradiated U251 appears to be mediated by MMP-independent mechanisms.
Nakamura
Jean L
JL
Department of Radiation Oncology, University of California-San Francisco, San Francisco, CA 94143, USA. nakamura@radonc17.ucsf.edu
Haas-Kogan
Daphne A
DA
Pieper
Russell O
RO
D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D001932:Brain Neoplasms / Q000201:enzymology / Q000473:pathology / Q000532:radiotherapy*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D018920:Coculture Techniques / Q000379:methods; D004151:Dipeptides / Q000494:pharmacology; D005434:Flow Cytometry; D005910:Glioma / Q000201:enzymology / Q000473:pathology / Q000532:radiotherapy*; D049452:Green Fluorescent Proteins; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D009361:Neoplasm Invasiveness; D011480:Protease Inhibitors / Q000494:pharmacology
D004151:Dipeptides; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020778:Matrix Metalloproteinase 2