Modeling the role of urokinase plasminogen activator, uPA, and circulating Cancer-Associated Fibroblasts (cCAFS) in breast cancer cell extravasation

Circulating Cancer-Associated Fibroblasts (cCAFs) have been discovered in circulating tumor cell clusters from all stages of disease progression in breast Cancer patients. We have shown that CAFs promote lung metastases in the mouse tail vein model when they are clustered with triple negative breast Cancer (TNBC) MDA-MB231 cells. Following on this observation, we saw that MDA-MB231-luciferase labeled cells persist at higher levels when present in CAF23/MDA-MB231 co-clusters compared to MDA-MB231 mono-clusters within the first 3 days after tail vein injection. This prompted us to investigate whether CAFs aid Cancer cell extravasation from capillary venules into the lung parenchyma, which would impart better survival and faster seeding of metastases. Ex vivo lung extravasation assays showed that within the first 8-24 hrs after tail vein injection, more cells from CAF23/MDA-MB231 co-clusters extravasated than cells from MDA-MB231 mono-clusters. Using in vitro endothelial binding assays, we determined that CAF/TNBC co-clusters bind to HUVEC endothelial cells better than TNBC mono-clusters. Single Cell RNA-seq identified several genes in the fibrinolysis pathway whose expression increases in TNBC cells when they are clustered with CAFs. One of these genes is PLAU, which encodes the urokinase-type plasminogen activator, uPA. siRNA knockdown of PLAU decreased in vitro TNBC-endothelial cell interactions and ex vivo extravasation of MDA-MB231 mono-clusters, revealing a role for uPA/PLAU in breast Cancer cell extravasation. Our data helps to define the role of CAFs in breast Cancer extravasation and highlights the importance of our previous work showing that CAFs promote tumor cell dissemination and metastasis.

breast cancer; cancer-associated fibroblasts; circulating tumor cell clusters; extravasation; metastasis; urokinase-type plasminogen activator.