Cancer metastasis is a multistep process that begins with the invasion of tumour cells into the stroma and migration towards the blood vessels. Tumour cells that have entered the bloodstream must then survive and leave by a process known as extravasation. Finally, extravasated cells proliferate and establish the secondary site in the metastatic cascade. Although extravasation encompasses key events during cancer cell invasion to aid in the development of effective treatments, an in vivo model that rapidly, reproducibly and economically recapitulates cancer cell extravasation is needed. Therefore, the objectives of my research were to 1) establish and validate an in vivo model of cancer cell extravasation, and 2) identify novel cellular and molecular events.
I used the chorioallantoic membrane of chicken embryos as a model system of extravasation as it provides an accessible and highly vascularized structure. The combination of the chorioallantoic membrane of chicken embryos, nanoscale flow cytometry, and confocal microscopy-based intravital imaging allowed me to observe that extravasating prostate cancer cells exhibited significant cell volume reduction. This reduction is suggestive of an invasive cell phenotype. However, cell volume reduction at certain threshold also decreased cancer cell extravasation efficiency. I also found that cancer cell released extravascular vesicles during extravasation, and an increase in extracellular vesicle release reduced cell volume. I then tested the hypothesis that extracellular vesicle release and extravasation may be linked to modes of cell death. Real-time imaging of extravasating cancer cells that released extracellular vesicles did not show activation of caspase-3. Activation of necroptosis, however, increased extracellular vesicle release and decreased cell extravasation and secondary colony formation. These results suggest that necroptosis may be targeted to induce extracellular vesicle release, decrease extravasation, and halt cancer metastasis.
Collectively, my work lays out the protocols for the use of the chorioallantoic membrane of chicken embryos as a model system to investigate cancer cell extravasation and invasion. Use of this model system allowed me to identify extracellular vesicle release during extravasation and discover that necroptosis may be a potential regulator of cancer metastasis
