Platelets and tumor cells: a new form of border control

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Just as countries need to protect their borders for the purposes of national security, so too must tissues maintain proper borders for normal function. During metastasis, tissue borders are breached as cancer cells move from their place of origin to distant organs, where their growth may lead to tissue dysfunction. Although most cancer patients die from metastases rather than their primary tumor, the molecular underpinnings of cancer spread have remained elusive. Addressing this knowledge gap has been challenging because each step in the so-called “metastatic cascade” – invasion, bloodstream entry, extravasation, and growth at a distant site – is rare and inefficient (Talmadge and Fidler, 2010). In this issue of Cancer Cell, a study describes a new molecular pathway by which platelets, the clot-forming cells of the blood, can serve as cellular “coyotes,” smuggling tumor cells across intact blood-tissue borders (Schumacher et al., 2013).

A relationship between cancer and clot formation was first recognized by French physician Armand Trousseau, who noted in 1865 that patients with internal malignancies often presented with thrombophlebitis (“phlegmasia alba dolens”) (Trousseau, 1865). Trousseau realized that the site of blood clotting could be far from the tumor and suspected that “a special alteration of the blood” was responsible. The clinical significance of this association was demonstrated shortly afterwards when Trousseau used it to diagnose his own terminal gastric cancer. However, nearly 150 years later, the mechanism(s) and biological importance of this special alteration of the blood remain complex and mysterious.

Platelets are small, anuclear cells that circulate in the blood in very high numbers. Circulating platelets are quiescent, but in the setting of a vessel injury they become activated by exposure to collagen, the coagulation protease thrombin, and other molecules not normally present in the blood. Activated platelets drive thrombus formation by creating shear-resistant aggregates, supporting coagulation factor activation and further thrombin generation, and by releasing vasoactive and prothrombotic agents from intracellular granules. Elevated platelet numbers have been associated with tumor metastasis and poor prognosis clinically for over a century (reviewed in (Erpenbeck and Schon, 2010)). A causal role for platelets in mediating cancer spread was first revealed when mice treated with neuraminidase exhibited a marked reduction in tumor metastasis due to an unexpected drop in their platelet counts (Gasic et al., 1968). More recent genetic studies have confirmed this observation, establishing that both reduced platelet numbers and reduced platelet activation can dramatically inhibit tumor metastasis in mice (Camerer et al., 2004; Palumbo et al., 2005). Thus platelet activation appears to be a key component of Trousseau's proposed “special alteration of the blood.”.

How do platelets support tumor metastasis? Since platelets do not leave the bloodstream, they must help tumor cells survive in the blood environment and/or exit the bloodstream. One idea is that platelets may act as protective “cloaks” for circulating tumor cells (CTCs), shielding them from immune destruction. During passage in the blood, tumor cells are exposed to natural killer (NK) cells that are capable of directly lysing them. Depletion of NK cells negates the protective effect of platelets during hematogenous metastasis in mice (Nieswandt et al., 1999). Further supporting this model, impairments in platelet activation due to loss of Gaq or impaired thrombus formation due to lack of fibrinogen lead to a reduced rate of metastasis only when NK cells are present (Palumbo et al., 2005). Platelet and endothelial cell adhesion proteins may also facilitate metastasis by augmenting tumor cell extravasation, a hypothesis supported by the observation that tumor metastases are reduced in mice lacking the vascular adhesion molecules P- and L-selectin (Wahrenbrock et al., 2003). More recently, genetic studies have demonstrated that platelet activation and release of TGF-β may enhance metastasis by inducing epithelial tumor cells to convert to a mesenchymal phenotype that facilitates tumor cell extravasation and tissue invasion (Labelle et al., 2011). These and other studies reveal a complex scenario in which activated platelets facilitate metastasis through numerous mechanisms.

In this issue of Cancer Cell, Schumacher et al. (2013) reveal a novel mechanism by which platelets facilitate tumor metastasis in a manner independent of their role in clotting. To discover platelet-derived agents that might facilitate tumor cell extravasation from the blood, the authors studied the effects of activated platelet supernatant on tumor cell migration through an endothelial monolayer. These studies identified platelet dense granule-derived ATP as a potent facilitator of tumor cell endothelial transmigration. To test the role of platelet-derived ATP in tumor metastasis, the authors employed animals lacking Munc 13-4, an exocytosis priming protein that is required for platelets to release their dense granules but is dispensable for all other platelet functions. Munc 13-4-deficient platelet supernatant was unable to facilitate tumor cell transmigration in vitro. Importantly, implantation of B16F10 melanoma and Lewis lung carcinoma cells into Munc 13-4-deficient mice resulted in a dramatic reduction in lung metastases while having no effect on primary tumor growth.

The authors then used their endothelial transmigration assay to identify P2Y2 as the ATP receptor most likely to mediate platelet-assisted tumor cell transmigration and confirmed this role in vivo using P2Y2-deficient mice. Although tissue-specific conditional deletion studies were not done, radiation chimeras confirmed that Munc 13-4 is required in circulating cells and P2Y2 in non-hematopoietic cells for these effects. These elegant studies support a molecularly straightforward model in which activated platelets associated with CTCs release ATP, which then acts on endothelial P2Y2 receptors to reduce endothelial barrier function and facilitate tumor cell movement out of the blood (Figure 1).

One of the most exciting aspects of this study is that it provides “actionable intelligence” in the form of a set of molecular targets that could potentially be used to tighten tissue border security in patients with cancer. While wholesale blockade of platelet activation and/or adhesion might reduce tumor metastasis, it would also increase the risk of bleeding. The study by Schumacher et al. suggests that the P2Y2 receptor might provide a means of attacking platelet-promoted metastasis without disabling normal platelet hemostatic function. P2Y2 receptors have diverse roles in vivo in many cell types, but the knockout mice are relatively healthy and the related P2Y12 purinergic receptor is the target of a number of successful drugs. These characteristics identify P2Y2 as an attractive drug target to block tumor metastasis, but going forward it will be important to determine whether the current findings apply solely to implantable melanoma and lung carcinoma cell lines, or whether Munc 13-4/P2Y2 inhibition also works more generally in genetically engineered models.

A larger question, not addressed by this study, is what patient population would benefit most from anti-metastatic therapy? If a tumor is detected prior to metastasis, then surgical removal of the tumor would be a preferable and a more definitive way of preventing spread. However, if metastasis has already occurred, even at the microscopic level, how will reducing further tumor cell extravasation at that point lead to an improved outcome? As with any issue of border control, there will be debate concerning the best approach. But before even considering such questions, it is necessary to know what makes borders porous and what can be done to plug the holes; the work of Schumacher et al. provide an important new piece of that basic knowledge.