Tumor cells rely on platelet-derived TGFβ and direct contact with platelets for induction of transition into an invasive phenotype and associated gene upregulation.

Direct signaling between blood platelets and tumor cells that have migrated to the vasculature plays a key role in epithelial-mesenchymal-like transition and enhances the development of metastases in vivo, scientists claim. In vitro and murine studies by a team at the Howard Hughes Medical Institute found that platelet-derived TGFβ and direct platelet-tumor cell contact work synergistically to activate the TGFβ/Smad and NF-κB pathways in cancer cells, which prompts the cells to develop an invasive mesenchymal-like phenotype, and leading to enhanced metastasis in vivo.

Reporting their findings in Cancer Cell, Richard O. Hynes, M.D., Myriam Labelle, M.D., and Shahinoor Begum, M.D., say either blocking TGFβ1 expression selectively in platelets, or inhibiting NF-κB signaling in cancer cells, reduces the development of lung metastases in experimental mice. The results are published in a paper titled “Direct Signaling between Platelets and Cancer Cells Induces an Epithelial-Mesenchymal-Like Transition and Promotes Metastasis.”

Research has shown that signals provided by a tumor’s local microenvironment promote the invasive abilities of cancer cells, the Howard Hughes team explains. In primary cancers, growth factors and cytokines secreted by stromal cells have been found play key roles in inducing epithelial-mesenchymal transition (EMT), a reversible process that promotes cell motility, invasion, and dissemination of cancer cells out of the tumor microenvironment.

Current thinking assumes that the tumors acquire their metastatic potential at the primary tumor site, and then travel through the vasculature to new sites. However, the authors postulate, given that multiple growth factors and cytokines are released into the blood stream, it’s possible—although as yet unknown—that cells may pick up additional signaling cues outside the primary tumor site.

Transforming growth factor-β (TGFβ) is already known to promote metastasis by enhancing EMT and invasiveness in primary carcinomas: once cancer cells reach the site of metastasis, TGFβ enhances cell malignancy. While in the case of bone metastases the source of TGFβ is the bone matrix itself, in other cases, the source of TGFβ available to tumor cells at the site of metastatic seeding remains unknown.

Platelets, meanwhile, contain high concentrations of TGFβ, and could potentially be involved in promoting a metastatic phenotype, the team continues. Indeed, defective platelet function or reduced platelet counts have been associated with decreased metastasis formation in various transgenic mouse models.

To see whether platelets can directly prime tumor cells for metastasis, the researchers co-incubated MC38GFP mouse colon carcinoma cells and Ep5 breast cancer cells with purified platelets in vitro. After 40 hours the platelets were washed away, and the tumor cells injected into the tail veins of mice. The results showed that animals given pretreated cancer cells developed a significantly increased number of metastatic foci in the lungs.

When they looked more closely at both MC38GFP and Ep5 cells co-cultured with platelets, the team found that both cell types underwent morphological changes similar to an EMT, and consistently upregulated the mRNA expression of mesenchymal markers and transcription factors involved in EMT, including Snail, vimentin (Vim), fibronectin (Fn1), and plasminogen activator inhibitor-1 (PAI-1; Serpine1). In contrast, the epithelial marker claudin 1 (Cldn1) was downreglated in platelet-treated cells. Interestingly, the human breast epithelial cell lines MCF10A and HMLER also displayed a more mesenchymal morphology when exposed to platelets, and increased the expression of EMT-associated genes.

Comparing the gene expression profiles of platelet-treated and untreated Ep5 cells showed that the treated cells had significantly upregulated several genes known to play prominent roles in EMT, ECM remodeling, and the promotion of metastasis. Platelet-specific mRNAs (assessed by separate microarray analysis), were excluded from the results. “Bioinformatic analysis using GeneGo canonical pathway maps and gene set enrichment analysis (GSEA) confirmed that platelets strongly activate EMT-related genes and revealed TGFβ-dependent pathways as being the most significantly upregulated following platelet treatment,” the authors write.

Particularly interesting was the finding that previously defined gene signatures associated with cancer stem cells, poor prognosis, and metastasis were also enriched in platelet-treated cells, “suggesting that platelets induce an overall more aggressive phenotype in tumor cells.”

They moved on to investigate whether platelet-derived TGFβ alone could activate TGFβ/Smad signaling in tumor cells. Initial tests found increased levels of active and latent TGFβ1 in the medium derived from tumor cell-platelet co-cultures, after the platelets were removed, while Ep5 and MC38GFP cells treated with platelets also showed increased phosphorylation of the TGFβ signaling effector Smad2 and Smad-binding element (SBE)-dependent transcription.

Significantly, adding a TGFβRI inhibitor (SB431542) or a TGFβ1-blocking antibody abolished platelet-induced cell invasion, and treatment using SB431542 also inhibited the upregulation of EMT markers induced by platelets in Ep5 and MC38GFP cells.

Studies in mice engineered to lack TGFβ1 specifically in megakaryocytes and platelets (Pf4-cre+; TGFβ1fl/fl mice) indicated both that platelets are a major source of TGFβ1 in the circulation, but also that platelet-derived TGFβ1 is key for the platelet-induced activation of Smad signaling in cancer cells. And when MC38GFP cells were injected into the tail veins of either wild-type or Pf4-cre+; TGFβ1fl/fl mice, the transgenic animals developed far fewer lung metastases than their littermate controls. Conversely, when tumor cells that had been treated using either TGFβ1-deficient platelets or with “normal” platelets were injected into wild-type mice, the animals receiving tumor cells treated using TGFβ1-deficient platelets developed far fewer metastases.

“Thus, whereas a platelet pretreatment primes tumor cells for metastasis in wild-type mice in a TGFβ1-dependent manner, the presence of platelet-derived TGFβ1 in the host bloodstream is also required for efficient metastasis,” the researchers remark.

Subsequent experiments designed to investigate the role of platelet-derived TGFβ1 in the early steps of metastatic seeding showed that Pf4-cre+; TGFβ1fl/fl mice given tumor cell injections retained far fewer cancer cells in the lungs by two days after initial injection than wild-type animals. Evaluation of lung tissue by confocal microscopy and 3-D rendering in addition showed that at the 21 hour point, although wild-type and Pf4-cre+; TGFβ1fl/fl mice both demonstrated tumor cells in the intravascular and extravascular compartments, smaller numbers of cells were located extravascularly in the lungs of Pf4-cre+; TGFβ1fl/fl animals, suggesting that tumor cell extravasation was impaired.

Another question to answer was whether exposure to the releasate from activated platelets would prime tumor cells for metastasis. Firstly, the team activated platelets and then the releasate separated from the platelets by centrifugation. Studies using both the releasate and pellet fractions showed that higher concentrations of active TGFβ1 were present in the conditioned medium of tumor cells treated with the releasate than with the pellet from activated platelets, but similar amounts of total TGFβ 1 were found in the conditioned medium of tumor cells treated with either fraction.

However, the researchers continue, while conditioned medium of tumor cells treated with releasate contained higher concentrations of TGFβ1 in vivo, higher numbers of lung metastases were only observed when tumor cells were preincubated with the pellet fraction, and now with releasate from activated platelets. Tumor cell retention in the lung was also only increased in mice administered with cells treated using platelets or pellet fraction, and not with cells treated using releasate. Importantly, microarray analysis of pellet- or releasate-treated Ep5 cells revealed that treatment with the pellet fraction induced gene expression changes very similar to those recorded after treatment with whole platelets. Treatment using releasate only resulted in partial gene expression changes.

The evidence thus far pointed to a mechanism by which platelet-induced effects on cancer cell EMT, invasion, and metastasis were mediate by secreted TGFβ, but also required additional platelet-bound factors, which acted in synergy with the secreted TGFβ to promote metastasis. This synergistic activity was confirmed using tests with an engineered cell line, which demonstrated that platelet-derived TGFβ1 is required to induce the expression of prometastatic genes and metastasis, but that the platelets themselves provide other signals that synergize with TGFβ signaling on direct platelet-tumor cell contact.

The researchers used a set of luciferase reporter assays for different pathways involved in cancer, to screen for their activation in Ep5 cancer cells incubated with platelets. This indicated that the JNK and NF-κB pathways are involved, and that while the JNK pathway appeared to be activated by the platelet releasate, activation of the NF-κB pathway only increased when cells were incubated with either platelets or the pellet fraction from activated platelets. Supporting this finding, the team demonstrated that secretion of the NF-κB pathway target MCP-1 was increased in the supernatant of Ep5 and MC38GFP tumor cells treated with platelets or the pellet fraction. And a number of the genes that were highly upregulated in the platelet-induced gene signature have previously been identified as NF-κB target genes. “Furthermore, GSEA analysis revealed that NF-κB-related gene signatures are enriched in platelet- or platelet pellet-treated Ep5 cells, but not in platelet releasate-treated cells.”

Notably, mice injected with with platelet-treated Ep5 cells that had been engineered using an IκBα that blocked NF-κB function (Ep5-lkBSR cells) developed far fewer metastases than animals injected with control, platelet-treated Ep5 cells. Mice receiving the Ep5-lkBSR cells also showed less retention of cancer cells in the lungs. In vitro, the Ep5-IkBSR cells didn’t take on a mesenchymal morphology or demonstrate increased invasion in response to platelet treatment.

The authors claim their results demonstrate that platelets represent a crucial source of TGFβ to tumor cells in the vasculature, which is necessary for tumor cell extravasation and metastasis formation. “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 metastatic seeding in the lungs in vivo,” they conclude. “Because platelet-tumor cell interactions are transient and occur only within the first 24 hours, 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…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.”

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