COLLECTIVE MIGRATION STUDY OF BREAST CANCER CELLS IN 2D

Abstract

Cancer related deaths are mainly attributed to the metastatic spread of cancer cells to distant organs resulting in the formation of secondary tumors. Collective cell migration is one of the factors among many others that are strongly implicated in the growth and progression of cancers. Collective cell migration has been studied by various techniques in 2D and 3D, including wound healing assays which remain among the most commonly used techniques. To improve the information obtained from these methods, various modifications have been employed such as time-lapse microscopy and analysis of cell migration by cell tracking. The latter achieved considerable success with cell migration parameters derived from cell track data. However, these methods do not analyze collective migration in detail at the individual cell level, nor delineate the underlying mechanisms involved in the development of the collective behavior. Moreover, collective cell migration of breast cancers has not been studied in detail. We utilized a modification of the ring assays for the study of collective migration of breast cancer cell lines on 2D surfaces. We also improved this method by introducing kinematic and kymographic analysis including quantitative estimation of parameters for single cell motility mechanisms. Furthermore, with these assay methods and analytical technique, we have analyzed the migratory behavior of breast cancer collective cell migration on collagen IV coated surfaces. Results showed that collagen IV, a component of the basement membranes, increased the migration of the highly invasive MDA-MB-231 cells. Analyses also revealed that the abnormal collective cell migration in MCF-7 cells was due to altered lamellipod polarization, while the haphazard collective migratory patterns of the highly invasive MDA-MB-231 cells were the result of mesenchymal modes of migration that was facilitated by lack of intercellular adhesions and directional freedom of lamellipod polarization. The method of assay was also used to analyze two different protein knock down systems to evaluate the capabilities of this system. Finally, we tested the hypothesis that lamellipod polarization affects the collective cell migration in epithelial cell lines. In the course of these findings, it was also shown that the enzyme Rho Kinase was involved in the regulation of lamellipodial polarization in epithelial cells, and inhibition by the ROCK inhibitor resulted in disruption of collective cell migration. In conclusion, this study emphasizes the usefulness of 2D non-wounding assay methods in studying the collective migration of cells. The improved analysis methods also helped in revealing the details of collective migration of breast cancer cells as well as elucidating lamellipodial polarization as one of the crucial factors in the development of individual cell motility, and therefore collective cell migration.