Many experimental studies using in vitro assays and in vivo metastatic animal models have demonstrated a mechanistic link between tumor cell dissemination and platelet activation. Direct contact between platelets and tumor cells has been observed in the primary tumor microenvironment. Platelet involvement in primary tumor growth and invasiveness has not well been recognized. The process of metastasis initiation includes detachment of tumor cells from the primary site and migration to and intravasation into the blood vessel.
Several angiogenic molecules, including angiopoietins, VEGF, and TGF-β, are abundant in platelets and may affect the tumor microenvironment [1, 2, 22]. As a Tie2-antagonist, Ang-2 mediates angiogenic sprouting and vascular regression. We found that platelet-depleted tumors exhibited an increase in Ang-2 levels, leading to a delay in vessel maturation and diminished pericyte recruitment to blood vessels that were highly permeable and hemorrhagic. This finding is supported by the recent observation that platelet depletion displayed a significantly lower vessel density and poor vascular maturation in a tumor implantation model and in hindlimb ischemia animal models .
Furthermore, it is well known that VEGF plays an important role in the initiation of tumor angiogenesis. Platelet-derived TGF-β is known as an important growth factor involved in circulating dissemination . In fact, TGF-β also provides proliferative signals to tumor cells, which might contribute to the ECM breakdown that is required for vessel invasion to occur . We used microdialysis to examine the extracellular VEGF and TGF-β levels in solid B16/F10 tumors, and the results showed that platelet-depleted mice exhibited a decreased secretion of both VEGF and TGF-β compared to control mice. It should be noted that most serum VEGF is derived from platelets, which are activated upon coagulation . Further studies are required to clarify the role of platelets in the storage of VEGF released from the tumors.
Tumor progression and metastasis are strongly related to blood vessel maturation and stabilization in the tumor microenvironment. Platelets are involved in vessel maturation through multiple mechanisms, including releasing platelet-derived factors and cytokines and regulating bone marrow-derived cell recruitment [5, 25, 26]. VE-cadherin is crucial for vessel assembly and integrity during angiogenesis [18, 27, 28]. Likely, increased intratumoral VE-cadherin expression might contribute to vessel lumen development. VE-cadherin also promotes tumor progression not only by contributing to tumor angiogenesis but also by enhancing tumor cell proliferation via the TGF-β1 signaling pathway in breast cancer . Interestingly, we found a significant decrease in VE-cadherin expression in platelet-depleted tumors, suggesting that high VE-cadherin in tumors may lead to an enlarged vessel lumen and is linked to tumor progression in the presence of platelets.
Invasion through the ECM is an important step in tumor metastasis. Cancer cells initiate invasion by adhering to and spreading along blood vessel walls. Proteolytic enzymes, such as MMP, degrade ECM surrounding the blood vessels to allow cancer cells to invade. Alternatively, it is important to note that tumor metastasis is associated with blood vessel maturation and stabilization in the primary tumor. Intravasation of cancer cells does not occur solely though the vessel wall but also through the ECM (basement membrane). TGF-β1 is a crucial factor in inducing tumor growth and metastasis through up-regulating MMP-2, 9. Intratumoral TGF-β1 is constitutively secreted by B16/F10 tumor cells, as well as by direct platelet-tumor cell . We found a significant reduction of TGF-β1 in blood, extracellular space and intracellular tumors from platelet-depleted tumor-bearing mice. Circulating platelet-derived TGF-β1 has been reported to promote metastasis work by activating activate the TGFβ/Smad and NF-kB pathways in cancer cells .
Our data demonstrated that platelet depletion reduced metastasis and was further associated with decreased ECM degradation and reduced expression of MMP-2, 9 and PAI-1. The ECM surrounding blood vessels plays a critical role in the limitation of extravasation and intravasation in the tumor microenvironment. Thus, it is possible that platelet-promoted primary tumor metastasis is mainly associated with the integrity of the ECM in the tumor microenvironment as a part of vessel maturation.
Our data demonstrated that Platelet depletion strongly reduced the expression and tyrosine phosphorylation of the Met receptor in tumors. Met expression has been shown to result from increased tumor hypoxia. Our data demonstrated that platelet depletion decreased metastasis and was associated with decreased HIF-1a. It is well documented that tumor hypoxia is associated with vessel structure abnormalities, such as leakiness and destabilization by poor coverage of pericytes, and with excessive proliferation of tumor cells. A recent study demonstrated that platelet depletion causes a decrease in tumor proliferation and delays vessel maturation . Either a change in excessive tumor cell proliferation or impaired vessel maturation accelerates tumor hypoxia. The impaired vessel maturation may lead to an increase in the interstitial pressure due to leakage and thus alter the blood flow because of the compression of tumor vessel, thus likely reduce tumor perfusion. Although we found that platelet depletion significantly reduced blood vessel perfusion of tumors, in this study, the impaired tumor angiogenesis and vessel maturation induced by platelet depletion are not sufficient to cause significant tumor hypoxia. It seems that tumor cell proliferation could play a major role in causing hypoxia in the tumor microenvironment.
Platelet-tumor cell contact promotes the hematogenous dissemination of tumor cells by activating the NF-κB pathway . Abundant platelets were detected in the tumor microenvironment outside of the vasculature . Indeed, a previous study has shown that NF-κB is a key orchestrator of innate immunity/inflammation in many cancers . Labelle et al. identified the involvement of inflammatory cytokines in the platelet-related NF-κB pathway . HIF-1α is an inflammatory response gene. Furthermore, the presence of messengers of inflammation is strong associated with the occurrence of vascular remodeling and angiogenesis. Therefore reduced vessel density and/or function underlie, cannot rule out completely the contribution of immune/inflammatory cells for platelet-induced phenotype.