Mechanical and transcriptional alterations during cancer cell transendothelial migration

Abstract

Cancer metastasis results in over ninety percent of cancer-related deaths. A critical aspect of metastasis is the extravasation of tumor cells through blood vessel endothelial cell junctions, thought to be smaller than the tumor cell nuclear diameter. The significant deformations required for extravasation change the chromatin spatial configuration along with the distribution of nuclear enzymes and transcription factors. However, it remains unknown whether tumor cells modulate their mechanical properties in order to facilitate this extravasation and whether the transcriptome is altered in this process to further downstream colonization. Improved understanding of how tumor cells undergo transendothelial migration and the resulting transcriptomic implications may help uncover new approaches to prevent cancer metastasis.

In this thesis, we utilized an in vitro 3D cell model for transendothelial migration, in which tumor cells cross an endothelial monolayer and travel into a collagen gel without interference of stiff substrates and fixed pores. We employed two non-perturbative optical methods, Brillouin confocal microscopy and confocal reflectance quantitative phase microscopy, to map the mechanical properties of live transmigrating tumor cells. First, we demonstrated agreement in the measurements from these two methods, by testing changes in tumor cell nuclear mechanical properties in response to doxorubicin, a common chemotherapeutic. Our subsequent studies of tumor cells during transendothelial migration revealed that they soften and that this softening persists (both in bulk internal and nuclear membrane measurements). These results are the first demonstration of tumor cell mechanical property modulation during transendothelial migration.

We also investigated transcriptomic alterations during transendothelial migration. We accomplished this by separating extravasated from non-extravasated tumor cells at different time points and performing RNAseq. We found that significant transcriptomic alterations occur during transendothelial migration, especially up-regulation of genes involved in the epithelial-to-mesenchymal transition and lamellipodia assembly, which support further migration into the stroma. Intriguingly, the lamin genes, which are a primary determinant of nuclear stiffness, were not differentially regulated. These experiments provide new insights into genetic alterations that occur in tumor cells during extravasation. In summary, these findings shed light on the modulation of tumor cell mechanics and transcriptome during metastasis across the blood vessel endothelium.