Invasive breast carcinoma cells from patients exhibit Mena ^INV - and macrophage-dependent transendothelial migration

Abstract

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 MenaINV, 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 MenaINV or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that MenaINV and TMEM frequency are correlated prognostic markers and CSF-1 and MenaINV may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.

Signatures of breast cancer metastasis at a glance

“…These AUC values, where 0.8 is good, 0.7 is fair and 0.6 is poor discrimination (Metz, 1978), indicate that TMEM composite is better at predicting metastasis than these other prognostics (Harney et al, 2015;Oktay and Jones, 2015;Pignatelli et al, 2014;Robinson et al, 2009;Rohan et al, 2014;Roussos et al, 2011c;Wyckoff et al, 2007). Previous reports on cancer angiogenesis and vasculogenesis have collectively demonstrated that newly constructed endothelia are composed of myeloid precursors expressing the receptor tyrosine kinase Tie2 (also known as TEK) (Riabov et al, 2014;Squadrito and De Palma, 2011), and, more recently, the mannose-receptor c-type 1 (MRC1) (Hughes et al, 2015a).…”

“…Mena Calc is predictive of metastatic relapse and survival in breast cancer patients (Agarwal et al, 2012;Forse et al, 2015). The relative expression of Mena INV to Mena 11a is directly related to TMEM assembly because tumor cells that both assemble and use TMEM for dissemination require the expression pattern Mena INV-high and Mena11a low (Pignatelli et al, 2014) (see poster). TMEM sites are the sites of intravasation in polyoma middle T oncoprotein (PyMT)-induced mouse mammary tumors and patientderived xenograft (PDX) mammary tumors, and are therefore necessary for the dissemination step of the metastatic process (Harney et al, 2015).…”

“…The detailed mechanism of TMEM function and the involvement of Mena isoforms in intravasation at TMEM sites have been described (Harney et al, 2015;Pignatelli et al, 2014;Roh-Johnson et al, 2014) and it has been shown that activation of the Tie2 signaling pathway in the perivascular macrophage stimulates the release of macrophage vascular endothelial growth factor (VEGF). VEGF in turn, promotes reversible local loss of endothelial adherens junctions at TMEM sites (Harney et al, 2015).…”

“…VEGF in turn, promotes reversible local loss of endothelial adherens junctions at TMEM sites (Harney et al, 2015). The TMEM macrophage also initiates Mena-dependent invadopodium assembly in the TMEM tumor cell, with which it is in direct contact, thereby completing a required invasive step for transendothelial migration at TMEM sites (Pignatelli et al, 2014;Roh-Johnson et al, 2014). The resulting reversible, local permeability of the endothelium is visualized in real time by multiphoton microscopy as a dramatic release of luminal blood vessel components into the subluminal space (termed 'bursting').…”

Endosialin-Expressing Pericytes Promote Metastatic Dissemination

“…ns, nonsignificant. (48,49); however, analysis of tumors growing in WT or EN KO mice did not yield any significant differences in macrophage numbers, indicating that macrophages are not the driving force for endosialin-mediated tumor cell intravasation (C. K€ onig and H.G. Augustin, unpublished data).…”