show Abstracthide AbstractPhenotypic heterogeneity of cancer cells is caused not only by genetic and epigenetic alterations, but also by stochastic variation of intracellular signaling molecules. Using cells that stably express F?rster resonance energy transfer (FRET) biosensors, we here show correlation of Rac1 activity fluctuation with invasive property of C6 glioma cells. By long-term time-lapse imaging Rac1 activity in C6 glioma cells was found to fluctuate with a timescale significantly longer than the replication cycle. Because the level of Rac1 activity in each cell was robust to suspension-adhesion procedure, C6 glioma cells were sorted by Rac1 activity, yielding Rac1high and Rac1low cells. The Rac1high cells invaded more efficiently than did Rac1low cells in the Matrigel invasion assay. Among the top 14 membrane-related genes enriched in Rac1high cells, four genes were associated with glioma invasion and Rac1 activity as examined by siRNA knockdown experiments. Among transcription factors enriched in Rac1high cells, Egr2 was found to positively regulate expression of the four membrane-related invasion-associated genes. The identified signaling network may cause the slow fluctuation of Rac1 activity and heterogeneity in invading capacity of glioma cells.