In most solid malignant tumors, no metastases are present at diagnosis but still, up to 50% of patients suffer relapse, which is assumed to be due to cells shed either directly into the circulation or via the lymphatic system. Lymph nodes are no barrier to dissemination[15]. The key questions are when and how dissemination occurs, how disseminated cells survive, and what induces them to form metastases. In blood, tumor cells are not readily detectable. In fact, we have noted that in freshly drawn unfixed blood samples, circulating tumor cells become detectable only after the sample rests at room temperature overnight or is treated with a surfactant[16], indicating that their surface molecules initially are somehow masked.
A critical step for distant metastasis formation is survival of cancer cells in the bloodstream. The main threats to circulating tumor cells are shear stress and natural killer (NK) cytotoxic cells. It has been reported that platelets protect circulating tumor cells[17], which correlates with the tumor cells’ abilities to aggregate platelets[18]. Indeed, platelets are known to bind benign exfoliated epithelial cells that enter the circulation (e.g. upon wounding). Whereas normal epithelial cells may not be capable to proliferate, and seem to become eliminated after days or weeks[7] circulating tumor cells persist[13] when coated with platelets[19]. Surface coating of the tumor cells with platelets may serve as a shield against immune assault because the effect of anti-tumor attacks mediated by NK cells is primarily based on the direct interaction with circulating tumor cells[20].
Platelets that coat CETCs also prevent antibodies from accessing the tumor cell surface and it is likely the reason for the inability to detect these cells in freshly drawn blood. This evasion of circulating tumor cells from immune recognition may also be the reason why anti-EpCAM- antibodies have, so far, not been successful in treating epithelial tumors[21]. Drugs that theoretically would be effective may not be able to access the clogged cells, an important consideration in adjuvant or neoadjuvant systemic chemotherapy.
In our study, platelet inactivation on day 1 was likely due to "platelet storage lesion", a phenomenon resulting in the platelet’s disc-to-sphere transformation and known to be associated with reduced responsiveness to adenosine diphosphate for platelet aggregation[22]. We believe our observation was due to hypoxia, an effect of the sample resting on the bench. Platelets were released from the epithelial cells during overnight storage, making the CETCs accessible to the specific antibody. The subsequent cap formation may be a particular characteristic of epithelial cells[23] and the joint movement of EpCAM on the tumor cells with the platelets is suggestive of a preferential binding of the platelets to EpCAM. Activation of platelets did not lead to thrombus formation due to the coagulation-inhibitory environment in this artificial system.
Our in vitro observations may differ from what occurs in vivo, where an intact coagulation system is present. The association between abnormalities of haemostasis and cancer is described as recurrent migratory thrombophlebitis[24] with platelet-rich microthrombi in the vasculature. The link between cancer and risk of venous thromboembolism (VTE) has been well-documented[25]. Cancer patients on chemotherapy are 47 times more likely to experience VTE[26] and this correlates with the metastatic potential of cancer cells.
Circulating platelet/tumor cell complexes (like the ones visualized in this study) may arrest in thrombi or sites where the blood flow slows down, like the liver, lung or the bone marrow, contributing to metastasis formation in these organs. Under physiological-normal conditions, the endothelium prevents thrombosis by providing a surface that discourages the attachment of cells and clotting proteins. Upon vascular injury, initiation of clot formation occurs, with resultant activation of the endothelium[27]. Activated endothelial cells express a variety of molecules and receptors that increase platelet adhesion to the site of injury. If, accidentally, a platelet covered tumor cell is trapped in the clot, platelets detach from the tumor cells. At the same time they release growth factors such as platelet-derived growth factor, vasoactive agents and chemokines, and pro-angiogenic growth factors (VEGF). A small fraction of tumor cells subsequently clonally expand to form tumor metastases.