Tumor cells can migrate in 3D matrices in either a mesenchymal-like or amoeboid mode. HT1080 fibrosarcoma cells cultured in 3D collagen gels change their morphology from mesenchymal-like (elongated) to amoeboid (round) following protease inhibitor (PI) treatment or active Rho or ROCK expression. In this study, we examined the role of LIM-kinase 1 (LIMK1) in the PI- or Rho/ROCK-induced cell morphological change. We showed that LIMK1 was activated after PI treatment of HT1080 cells in 3D collagen gels and this activation was blocked by a ROCK inhibitor. While overexpression of LIMK1 induced cell rounding, knockdown of LIMK1 or the expression of kinase-inactive LIMK1 suppressed PI- or Rho/ROCK-induced cell rounding. These results suggest that LIMK1 plays an essential role in the PI- or Rho/ROCK-induced mesenchymal-to-amoeboid cell morphological transition of HT1080 cells cultured in 3D collagen gels. Furthermore, LIMK1 knockdown suppressed the invasive activity of HT1080 cells in collagen gels with or without PIs, indicating that LIMK1 mediates both the mesenchymal and amoeboid modes of invasion of HT1080 cells. (C) 2010 Elsevier Inc. All rights reserved.