A major mechanism for carcinoma cell dissemination from the primary tumor to distant organs is via hematogenous spread. Multiphoton-based intravital imaging has demonstrated that invasive carcinoma cells in mouse and rat mammary tumors co-migrate and intravasate when associated with peri-vascular macrophages. Specifically, intravasation occurs at sites where a macrophage, tumor cell, and endothelial cell are in direct contact. The tumor cell that co-migrates and interacts with macrophages at intravasation sites is detected by the high expression of multiple pro-invasive isoforms of Mena, an Ena/VASP protein family member that is a key actin polymerization regulatory protein. We have identified this intravasation microenvironment in human breast cancer samples using a triple immunostain for paraffin-embedded tissue that simultaneously labels macrophages, endothelial cells, and invasive macrophage-associated carcinoma cells. The macrophage-associated carcinoma cells are recognized by high expression of pro-invasive isoforms of Mena using a pan-Mena antibody that stains all isoforms of Mena. We call the structure composed of these three cell types (perivascular macrophages, endothelial cells, invasive Mena over-expressing tumor cells) in direct contact with each other “TMEM”, for Tumor MicroEnvironment of Metastasis. The mechanistic role of Mena suggests that measurement of the invasive isoform in tumor tissue could be valuable for prediction of the risk of metastasis. We have described a multiplexed quantitative immufluorescence-based method (MQIF) in which the fraction of Mena protein with invasive isoforms is assessed by subtraction of the non-invasive isoform from the total Mena present in tumors. We call this biomarker Menacalc. Both of these mechanism-based markers – TMEM and Menacalc – have been utilized in clinical studies, the results of which provide evidence for their utility as prognostic markers in breast cancer.