We have studied tumor metastasis in pFN-deficient mice and show that pFN supports the retention of tumor cells in the lungs. The pro-metastatic effect of pFN is linked to clotting activity and mediated by tumor cell integrin αvβ3 in vivo. We attributed this novel function of pFN to the capacity of FibFN to activate integrin αvβ3 and, thus, to promote tumor cell adhesion to and invasion into clotted plasma in vitro.
Deposition of fibronectin into the extracellular matrix has been shown to support tumor cell proliferation and angiogenesis, which are crucial steps in the completion of metastasis (25
). Our results show that pFN supports lung metastasis at an earlier stage of metastasis when tumor cell fate is determined by the ability of circulating tumor cells to survive in and extravasate from the pulmonary vasculature. Tumor cell survival in the lung vasculature is largely dependent on clot formation, which protects tumor cells from the cytotoxic activity of natural killer cells (4
). In addition, clotting has been shown to promote tumor cell adhesion and extravasation (3
). The pivotal role of clotting has been demonstrated in several models of spontaneous and experimental lung metastasis (6
). Blood clots contain large amounts of pFN, which is cross-linked to fibrin (7
). Using the thrombin antagonist hirudin to inhibit clot formation, we found that the prometastatic function of clots depends at least in part on the presence of pFN. In agreement with previous studies, this cooperation between pFN and clotting was important for lung metastasis but was not relevant for metastasis to the liver, where circulating tumor cells have direct access to fibronectin and other extracellular matrix components expressed in the endothelial basement membrane of liver sinusoidal blood vessels (30
). The initial tumor cell arrest in the lung vasculature, in contrast, is mostly initiated by adhesive interactions with the endothelium suggesting that basement membrane proteins critical for tumor cell survival and extravasation are not immediately accessible (1
). In this environment, tumor cells appear to be unable to compensate for the lack of pFN or the inability to form clots.
The association of pFN with fibrin has been shown to promote cell adhesion to clotted plasma (21
). Despite this proadhesive function, pFN did not affect the initial tumor cell arrest. This result was not unexpected because soluble pFN is inactive and clots form only after tumor cells adhere to the lung vasculature (3
). One of the effects of blood clots is to sustain tumor cell adhesion, which involves integrin activation as tumor cells begin to spread alongside the lung vasculature (3
). Our results indicate that adhesive interactions of tumor cells with clotted plasma are uniquely mediated by integrin αvβ3, thus providing a possible explanation for the prometastatic activity of this adhesion receptor. Integrin αvβ3 recognizes a plethora of ligands including fibrin, which is the predominant adhesion protein in clotted plasma (35
). Yet, the efficacy of αvβ3 to mediate tumor cell adhesion, invasion and metastasis decreased significantly when pFN was absent from clotted plasma. Based on this finding, we concluded that incorporation of pFN into blood clots promotes metastasis by enhancing the adhesive function of αvβ3 towards clotted plasma. Moreover, we found that FibFN is important for tumor cells as it leads to activation of αvβ3. Activated integrins exhibit a conformational state of increased affinity for their ligands (23
). The interaction of integrin αvβ3 with its ligands promotes survival, and can also induce proliferation of metastatic tumor cells (37
). However, FibFN had no effect on tumor cell growth suggesting the primary function of clotted plasma is to prepare tumor cells for extravasation. Blood clots have been shown to promote endothelial retraction and, thus, to provide access to the pulmonary basement membrane (27
). In order to reach the basement membrane, tumor cells have to invade the surrounding layer of clot. Our results demonstrate that the invasion of blood clots requires FibFN-mediated activation of integrin αvβ3 and the proteolytic activity of MT1-MMP.
In solution, pFN has a compact conformation that limits accessibility to cryptic binding sites for integrins and cell surface proteoglycans buried within the molecule (38
). Our results suggest that cross-linking pFN to fibrin via coagulation factor XIIIa gives access to a binding site in pFN that activates integrin αvβ3. It is unlikely that integrin activation is the result of a pFN-binding protein because pFN remained inactive in absence of FXIIIa, which itself had no effect on αvβ3 (Supplementary Figure 3
). Moreover, we found that FibFN was unique in its ability to activate αvβ3 when compared to a number of known integrin activators or FN-binding proteins (39
). FibFN-induced cell adhesion and metastasis did not depend on crosstalk with the fibronectin-binding integrins α4β1 and α5β1. However, it is conceivable that FibFN could activate αvβ3 through ligation of the fibronectin receptors CD26 and CD44 or as a result of cryptic protein disulfide isomerase activity near the C-terminus of fibronectin (45
). Identifying the molecular mechanism of FibFN binding to tumor cells will expand our understanding of metastasis and may provide a novel target for the development of anti-metastatic treatments.