Signals provided by the primary tumor microenvironment are important modulators of the capacity of tumor cells to invade, access the vasculature, and metastasize (Joyce and Pollard, 2009
; Nguyen et al., 2009
). However, the metastatic potential of tumor cells may be further defined in response to signals provided during their intravascular transit. Here, we have tested this hypothesis and show that platelets present in the bloodstream actively signal to tumor cells to promote their metastatic potential outside of the primary microenvironment. This effect is independent of any direct contribution of platelets to immunosurveillance, adhesion, or physical shielding functions, as tumor cells can be primed for metastasis by a transient exposure to purified platelets in vitro
. Mechanistically, a transient contact between platelets and tumor cells is sufficient to induce a prometastatic gene expression signature, induce an EMT-like transformation and invasive behavior in vitro,
and promote extravasation and metastatic seeding in the lungs in vivo
(). Considering that tumor cells would normally interact with platelets once in the bloodstream, these results suggest that tumor cells could gain a more mesenchymal phenotype and increased metastatic capacities after
leaving the primary tumor microenvironment. This implies that cells that have intravasated without losing their epithelial properties either via leaky blood vessels (Carmeliet and Jain, 2000
; Mazzone et al., 2009
) or via collective invasion mechanisms (Friedl and Gilmour, 2009
) could acquire a mesenchymal phenotype during their transit in the vasculature. In support of this idea, circulating tumor cells have been found to express epithelial markers (EpCAM, cytokeratins), suggesting that EMT is not absolutely required to access the blood flow (Nagrath et al., 2007
). Thus, interactions with platelets may be particularly important in mediating extravasation of circulating epithelial tumor cells, and to maintain or further enhance the extravasation potential of circulating mesenchymal tumor cells. In this respect, it would be interesting to define the impact of platelets on gene expression and metastatic potential of circulating tumor cells from cancer patients.
Several signaling molecules, including TGFβ, PDGF, VEGF and angiopoietin are abundant in platelets (Erpenbeck and Schon, 2010
; Sierko and Wojtukiewicz, 2007
) and may therefore impact tumor cell behavior and induce EMT. Our results show that the prometastatic effects of platelets are in large part mediated via activation of the TGFβ signaling pathway, and that abrogating either TGFβ signaling in tumor cells or TGFβ expression by platelets is sufficient to inhibit metastasis and EMT. While TGFβ has been implicated in the induction of a prometastatic phenotype in many contexts (Padua et al., 2008
; Siegel et al., 2003
), the relevant cellular source of TGFβ bioavailable to circulating tumor cells, particularly at the site of metastatic extravasation, was previously unclear.
Our results strongly indicate that platelets are an important source of bioavailable TGFβ for tumor cells in the circulation and at the site of extravasation. This finding is supported by the observation that platelets contain concentrations of TGFβ1 many-fold higher than most cell types (Assoian et al., 1983
). Furthermore, the amount of TGFβ1 produced by other cells and taken up by platelets seems minimal, as purified platelets from Pf4-cre+; TGFβ1fl/fl
mice contained less than 1% of the amount of TGFβ1 present in platelets from WT mice.
Most importantly, we show that abrogation of TGFβ1 expression solely in platelets and megakaryocytes is sufficient to inhibit metastasis and prevent the seeding of tumor cells in the lungs. Furthermore, the presence of platelet-derived TGFβ1 “in situ
” in the host bloodstream is crucial for metastasis, since pretreating tumor cells with platelets from WT mice fails to enhance metastasis formation in mice lacking TGFβ1 in their platelets. Because platelet-tumor cell interactions are transient and occur only within the first 24h ((Laubli et al., 2006
), and data not shown), we propose that platelets could provide a pulse of TGFβ1 to circulating tumor cells, which would allow them to gain a more invasive, mesenchymal-like phenotype and extravasate. Along these lines, previous studies have shown that tumor cells transiently exposed to TGFβ1 have an enhanced capacity to seed the lungs, whereas cells that are continuously exposed to TGFβ1 have decreased metastatic capacity due to the cytostatic effect of TGFβ1 (Giampieri et al., 2009
; Padua et al., 2008
). In this respect, specific therapeutic inhibition of platelet-derived TGFβ1 might result in the impairment of tumor cell extravasation at the metastatic site. Importantly, Pf4-cre+; TGFβ1fl/fl
mice maintain normal platelet counts and hemostatic functions, suggesting that pharmacological inhibition of platelet-derived TGFβ could inhibit metastasis without adverse effects on physiological hemostasis.
We also find that, although required for metastasis, activation of TGFβ signaling alone is unable to generate effects of the magnitude of those observed with platelets. Indeed, while tumor cells treated for 24h with platelets or with the pellet fraction from activated platelets undergo EMT, cells treated with the releasate of activated platelets (which contained a similar concentration of TGFβ1) do not. In line with this result, a prolonged treatment with TGFβ1 – typically 1 week or longer – is needed to induce EMT in several epithelial cancer cell lines including Ep5 cells (Labelle et al., 2008
; Mani et al., 2008
; Maschler et al., 2005
). Our data further support the existence of additional platelet-bound factors synergizing with TGFβ1. First, platelets induce the TGFβ-responsive PAI-1
reporter to levels higher than achievable with TGFβ1 alone. Second, combining exogenous TGFβ1 with platelets from either WT or Pf4-cre+; TGFβ1fl/fl
mice results in a synergistic activation of the PAI-1
reporter. Lastly, the synergistic effects on PAI-1
reporter activity as well as the induction of prometastatic genes are blocked if platelets are separated from tumor cells by a semipermeable membrane. Thus, our results clearly demonstrate that additional platelet-bound factors synergize with TGFβ1 to promote metastasis, and to induce a prometastatic EMT program in tumor cells. In particular, we show that this synergy is dependent on the activation of the NF-κB pathway, which is specifically triggered upon direct contact between tumor cells and platelets independently from TGFβ activity.
NF-κB regulates the expression of proinflammatory genes and has been associated with increased metastasis and EMT induction (Huber et al., 2004
; Lin and Karin, 2007
). For example, NF-κB promotes osteolytic bone metastasis by inducing the proinflammatory cytokine GM-CSF (Park et al., 2007
). Notably, the activation of NF-κB has also been proposed to be part of the mechanism allowing TGFβ signaling to switch from a cytostatic to a prometastatic signal (Neil and Schiemann, 2008
). In support of this idea, we found that NF-κB activation potentiates TGFβ-induced prometastatic gene expression, and that NF-κB signaling is necessary for the induction of EMT and efficient metastatic seeding upon platelet-cancer cell interactions. Thus, platelet-tumor cell contacts during metastasis potentiate tumor cell transcriptional responses to TGFβ via NF-κB activation.
In conclusion, we establish platelets as a crucial source of TGFβ bioavailable to tumor cells in the vasculature and necessary for tumor cell extravasation and metastasis formation. Importantly, our study reveals that platelets are more than physical shields and actively signal to tumor cells via the TGFβ and NF-κB pathways to potently induce a prometastatic phenotype. We thus propose a model whereby the metastatic potential of tumor cells continues to evolve outside of the primary tumor site, in response to tumor-host interactions in the bloodstream and at the site of metastasis. In particular, we identify platelet-tumor cell interactions and the signaling pathways that they trigger as fundamental determinants of cancer metastasis that may provide the basis for developing effective anti-metastatic therapies.