Invasive breast carcinoma cells from patients exhibit Mena[superscript INV]- and macrophage-dependent transendothelial migration

Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)–responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding Mena INV , an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down Mena INV or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that Mena INV and TMEM frequency are correlated prognostic markers and CSF-1 and Mena INV may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes. intravital imaging of rodent mammary tumors, have also been detected in human invasive ductal carcinomas (IDCs) by triple immunohistochemistry (IHC) (22). A TMEM site is defined as a MENA -expressing tumor cell that is in direct contact with an endothelial cell and a perivascular macrophage. Case-control studies show that the number of TMEM sites is associated with increased risk of developing distant metastases in patients with IDC of the breast (22, 23). IDC sampled by fine-needle aspiration (FNA) biopsies indicates that the relative abundance of Mena INV correlates with TMEM frequency, stability, and/or function in patients (14). Increased Mena INV abundance increases transendothelial migration (TEM) in cultured cells and promotes cell migration, streaming, intravasation, and formation of spontaneous lung metastases in orthotopic xenografts in mice (6). Other studies show that Mena knockout mice bred with PyMT (polyoma virus middle T antigen) transgenic mice develop tumors with reduced intravasation and metastasis and have prolonged survival (24). Thus, Mena classic and/or Mena INV may promote metastasis by inducing TEM activity during intravasation. Here, we tested the hypothesis that the Mena INV isoform in particular is linked to TMEM score and the associated TEM at these proposed sites of intravasation (14, 25) by measuring the TEM activity of primary tumor cells from patients using new in vitro assays.