Earlier studies have demonstrated that primary cancers begin shedding neoplastic cells into the circulation at an early stage of metastases formation. Others based on antibody approaches for cell isolation and detection showed that shed carcinoma cells were rare, apoptotic and less threatening to disease progression, except an extremely small population of the capillary-attached cells present in metastatic spots of the primary tumors and seeds of secondary tumors. To isolate and characterize such rare metastatic cells, a fibrous collagenous matrix scaffolding coated with blood-borne adhesion molecules, including human plasma fibronectin, laminin and vitronectin, collectively called cell adhesion matrix (CAM), which supports the attachment of cancer cells to the CAM, has been developed. This method allows for the first time the single-step isolation of viable cancer cells from over 106 fold of background hematopoietic cells in the blood. The cells captured by the matrix are assayed ex vivo as putative metastatic cells: (1) for measurement of their metastatic potential, i.e., assaying their ability to digest and internalize fluorescently matrix fragments, simultaneously, (2) for their viability by apoptosis and cytotoxicity assays, and (3) for their cell proliferation. In addition, conventional pathological methods for detecting cancer cells were used, including cell size, nuclear shape, and immunocytochemical reactivity against cell type markers, such as cytokeratins, and pan-epithelial antigen BerEP4 present on normal and neoplastic epithelial cells, as well as the CD45 leukocyte marker. Initial studies on the isolated carcinoma cells from the peripheral blood of over 200 patients with metastatic diseases showed the co-expression of epithelial (Epi+) and endothelial (Endo+) phenotypes and membrane proteases that represent the invasiveness of metastatic cells in vivo. In addition to being generally positive for epithelial antigens (Epi+), the cancer cell invasion in vitro assay system identified the cancer cells in blood with their capability of acquiring an endothelial phenotype, i.e., these cells are recognized by antibodies directed against endothelial markers, including van Willebrand factor, CD31, VEGFR, etc; these cells uptake acLDL and form cellular network in matrix gels. These Epi+/Endo+ cells can also degrade and ingest fluorescent collagen fragments (Co+), indicating that putative cancer cells are Epi+/Endo+/Co+; able to resist staining by Hoechst dye (Hs-), thus very low rates of apoptosis. CAM-affinity purified cancer cells are rare in the blood of most normal donors, patients with benign disease, and cancer patients undergoing chemotherapy. Cell suspension derived from microdissection and collagenase digestion of primary tumors could also be purified by CAM; however, only less than 1% of CAM-enriched viable Epi+ cells are Endo+ and Co+, suggesting that a small population of tumor cells has potential to metastasize. Furthermore, immunohistochemistry of invasive breast ductal carcinoma identified capillary-lined cells with the epithelial and endothelial characteristics described above. At these sites, the carcinoma cells with large nuclei that line the microvessel, express both seprase/DPPIV and epithelial/endothelial marker antigens, suggesting that these cells are of epithelial origin and express endothelial characters at "angiogenic center", a phenomenon analogous to "vasculogenic mimicry" in other human malignant tumors. These viable metastatic cells, thus, are important targets for control of cancer progression. |
