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By detecting the alterations of gene expression during C6 astrocytoma cell invasion in a three-dimensional model using cDNA microarray, we found genetic evidence to postulate the mechanism of glioma cell invasion. There may be different kinds of subpopulations of cells in the spheroid core, including those arresting proliferation, those undergoing apoptosis, those still proliferating, and those initiating invasion. These subpopulations may arise from physically stressful conditions caused by excessive cell growth in spheroid core. In invading cells, there appears to be a subpopulation of cells that may reenter the cell cycle, when suitable microenvironments are encountered after enough invasion. Moreover, we also detected many molecules related to cell migration and invasion. The alerted genes involved in these processes may have potential as effective therapeutic targets for anti-invasive therapy.