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Updated: Jul 11, 2026
Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion
Published on: October 20, 2016
Glioma invasiveness responds variably to irradiation in a co-culture model
Jean L Nakamura1, Daphne A Haas-Kogan, Russell O Pieper
1Department of Radiation Oncology, University of California-San Francisco, San Francisco, CA 94143, USA. nakamura@radonc17.ucsf.edu
Purpose:
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 And Materials:
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.
