Abstract
Tumor cell migration toward and intravasation into capillaries is an early and key event in cancer metastasis, yet not all cancer cells are imbued with the same capability to do so. This heterogeneity within a tumor is a fundamental property of cancer. Tools to help us understand what molecular characteristics allow a certain subpopulation of cells to spread from the primary tumor are thus critical for overcoming metastasis. Conventional in vitro migration platforms treat populations in aggregate, which leads to a masking of intrinsic differences among cells. Some migration assays reported recently have single-cell resolution, but these platforms do not provide for selective retrieval of the distinct migrating and non-migrating cell populations for further analysis. Thus, to study the intrinsic differences in cells responsible for chemotactic heterogeneity, we developed a single-cell migration platform so that individual cells’ migration behavior can be studied and the heterogeneous population sorted based upon chemotactic phenotype. Furthermore, after migration, the highly chemotactic and non-chemotactic cells were retrieved and proved viable for later molecular analysis of their differences. Moreover, we modified the migration channel to resemble lymphatic capillaries to better understand how certain cancer cells are able to move through geometrically confining spaces.
Highlights
Cell migration is an essential process in angiogenesis, cancer metastasis, wound healing, inflammation, and embryogenesis
Different channel cross-sectional sizes and geometries have been used to study the effects of geometry on cell migration[15,17,18,19,20], while in others the migration channel was filled with hydrogel or extra-cellular matrix components in order to simulate the cancer invasion process through stroma[21,22]
Using p38γ short hairpin RNA (shRNA) knockdown and scrambled vector control breast cancer cells in our newly developed lymphatic-mimetic device, we show that the knockdown p38γ cells are unable to effectively traverse choke points at the critical size of 6 μ m × 10 μ m, which is the size of lymphatic vessels in vivo[26]
Summary
Cell migration is an essential process in angiogenesis, cancer metastasis, wound healing, inflammation, and embryogenesis. Recently one study has demonstrated the separation of a cancer cell line population based on chemotactic phenotype, it did not enrich for increased chemotaxis in the selected subpopulation as compared to the parent population and required the loading of thousands of cells[16] To overcome these and other limitations, we designed a single-cell migration platform that allows for the post-migration collection and analysis of differing chemotactic subpopulations of cells and that can be modified to geometrically mimic the tight spaces in the pre-lymphatic and lymphatic capillaries that a cancer cell must navigate on its way to metastasize[3,26,28]. After the migration assays, the highly chemotactic cells can be retrieved for further downstream molecular and phenotypic analysis in comparison to the non-chemotactic subpopulation
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