Platelets are involved in the development and progression of cancer through several mechanisms. Platelet activation at the site of tissue damage contributes to the initiation of a cascade of events which promote tumorigenesis. In fact, platelets release a wide array of proteins, including growth and angiogenic factors, lipids and extracellular vesicles rich in genetic material, which can mediate the induction of phenotypic changes in target cells, such as immune, stromal and tumor cells, and promote carcinogenesis and metastasis formation. Importantly, the role of platelets in tumor immune escape has been described. These lines of evidence open the way to novel strategies to fight cancer based on the use of antiplatelet agents. In addition to their ability to release factors, platelets are able of up-taking proteins and genetic material present in the bloodstream. Platelets are like ‘sentinels’ of the disease state. The evaluation of proteomics and transcriptomics signature of platelets and platelet-derived microparticles could represent a new strategy for the development of biomarkers for early cancer detection and/or therapeutic drug monitoring in cancer chemotherapy. Owing to the ability of platelets to interact with cancer cells and to deliver their cargo, platelets have been proposed as a ‘biomimetic drug delivery system’ for anti-tumor drugs to prevent the occurrence of off-target adverse events associated with the use of traditional chemotherapy.
Several lines of evidence sustain the role played by platelets in tumorigenesis, both at early stages of cancer development and also during the metastatization [1–3]. Many platelet-dependent mechanisms in tumorigenesis and cancer progression have been proposed [2–4]. Platelets are involved in the early stages of tumorigenesis via the interaction with the stromal cellular component . Also, platelets play a crucial role in promoting the colonization of distant organs by cancer cells. These effects are mediated by the direct interaction of platelets with target cells and/or the release of mediators (including lipids and proteins) [2,5]. These events cause phenotypic changes in immune, epithelial, endothelial and cancer cells which contribute to cancer promotion [5,6]. Platelets induce epithelial–mesenchymal transition (EMT) [7–9], which is the process of transdifferentiation of epithelial cells into motile mesenchymal cells and contributes pathologically to fibrosis and cancer progression . Several lines of evidence show that the release of platelet microparticles (MPs) is an important mechanism for cancer promotion induced by activated platelets. Platelet-derived MPs are rich in proteins and genetic material, including microRNAs (miRNAs), which can be delivered to other cells . Interestingly, cancer cells can acquire platelet-derived proteins from MPs, a phenomenon called platelet mimicry of cancer cells. This is an important event which can activate the coagulation cascade or the platelets and facilitate the hematogenous dissemination of cancer cells .
Platelets are also capable of taking up proteins and RNAs from the blood circulation during their lifespan [12–15]. Moreover, it has been described the uptake of extracellular vesicles (EVs) by circulating platelets . Through these mechanisms, the platelet content is influenced by the clinical conditions of the individuals, and the assessment of platelet RNA and protein profile has the potential for blood-based cancer diagnostics [14,15,17].
This review provides an overview of novel platelet functions implicated in the process of tumorigenesis, supporting the use of platelet molecular signature as a source of new biomarkers for cancer detection and monitoring the efficacy of anticancer treatments.
Platelets are the second most abundant cell type in the blood; in fact, every day thousands of platelets are produced starting from megakaryocytes, present in the bone marrow and derived from hematopoietic stem/progenitor cells . Platelets are characterized by nucleus loss . They play a fundamental role in hemostasis, repair and inflammation  and can be considered metabolically active cells, as they are equipped with functional organelles, such as the endoplasmic reticulum, the Golgi apparatus, mitochondria and the granules (dense and α-granules) . The platelets in the resting state appear to be similar to the smooth disks; as a result of the activation process, platelet structure is transformed into spinous spheres, a consequence of the changes of calcium in their cytoskeleton . Following a vascular lesion, platelets are activated through the interaction with extracellular matrix components [including collagens and von Willebrand factor (vWf)]. Initial tethering of platelets is by the glycoprotein (GP)Ib/V/IX platelet complex  through the binding of the GPIb to the vWf . Then, platelets adhere via the direct interaction with collagen, mediated by two primary surface platelet receptors, i.e. the integrin α2β1 and GPVI . GPVI is the primary collagen receptor which belongs to the immunoglobulin superfamily [25,26] and is non-covalently associated with the Fc receptor γ-chain (FcRγ chain) . Its activation leads to tyrosine phosphorylation of many downstream proteins and the release of second-wave agonists, most importantly thromboxane (TX)A2 and ADP . TXA2 is a lipid autacoid which is generated mainly by platelets from arachidonic acid (AA) via the cooperative activity of cyclooxygenase (COX)-1 and TXA2 synthase (TXAS) . Then, it is released from activated platelets and binds to its G-protein-coupled receptors, TPα and TPβ, two isoforms (generated by alternative splicing of a single gene) which differ in their C-terminal tails . Released TXA2 acts as a positive feedback loop in the amplification of platelet activation via the induction of multiple downstream pathways, including phospholipase (PL)C, and intracellular Ca2+ . ADP represents the preferential agonist of the G-protein-coupled purinergic receptors, the P2Y1 and P2Y12 . P2Y1 is a Gq-coupled receptor acting through the stimulation of PLC and the phosphatidylinositol-signaling pathway. P2Y12 is a Gi-coupled receptor which decreases intracellular cyclic adenosine monophosphate (cAMP) levels by inhibiting the adenylate cyclase .
A strong platelet agonist is thrombin which acts by cleaving and activating protease-activated receptor (PAR)-1 and PAR-4. In turn, these receptors activate G proteins (Gq, G12/13 and Gi), leading to the increase of cytosolic Ca2+ concentrations and the decrease in intracellular cAMP content .
All the mechanisms, described above, facilitate the recruitment and activation of additional platelets at the wound site and converge on the expression of GPIIb–IIIa (integrin αIIbβ3) on platelets which can bind fibrinogen and vWf . Occupation of this receptor by fibrinogen results in platelet aggregation via the cross-linking of adjacent stimulated platelets .
As a result of platelet activation, various substances are released from platelet secretory granules into the circulation. The α−granules contain a wide array of proteins, including vWf, fibrinogen, P-selectin, endothelial cell adhesion molecules PECAM-1 (also named CD31), CD40L, platelet factor 4 (CXCL4), β-thromboglobulin, thrombospondin, platelet-derived growth factor (PDGF), coagulation factor V, integrin αIIbβ3 and different pro-inflammatory cytokines [33,34]. Dense granules contain many prothrombotic mediators that act in an autocrine and paracrine manner to stimulate platelet activation . Also, platelets transport and deliver their cargo to other cells through the release of EVs, including exosomes (30–100 nm in diameter) and microvesicles (MVs), called MPs (100–1000 nm in diameter) . Platelet MPs are known to express CD42b, CD41 and CD62P . They contain proteins, mRNAs and miRNAs that can be delivered to target cells . Shai et al.  have shown that the proteome of the platelet MPs may differ depending on the platelet stimulation pathway.
Platelets not only release molecules to the neighborhood but, also, can absorb them from the blood, acting as sponges . Given the presence of the open canalicular system, platelets passively seize the material encountered in the circulation (for example, bacteria, viruses, proteins, antibodies and nucleic acids) [13,40,41]. Moreover, they use the receptor-mediated active absorption . Altogether, these events lead to the changes in platelet content, a phenomenon called ‘platelet education’. Thus, it has been shown that the assessment of the molecular content of blood platelets has the potential of being a liquid biopsy-based molecular diagnostics, in patients with several types of cancer [14,41].
In addition to the role of platelets in thrombus formation, numerous findings have shown a central action of platelets in cell–cell communication, thus leading to the activation of different cell types, including stromal cells and cancer cells . Several lines of evidence suggest that platelets can also promote the immune escape of the tumor cells by many mechanisms [5,6]. These further roles of platelets contribute to cancer development and open the way to use antiplatelet agents in the prevention and treatment of the disease [43,44].
Role of platelets in tumorigenesis
The participation of platelets in cancer development has been suggested by the results obtained in clinical studies and meta-analysis of randomized clinical trials (RCTs) with aspirin that showed the reduction of the probability of colorectal adenoma recurrence and colorectal cancer (CRC) incidence and mortality, even at the low doses used for the prevention of cardiovascular events (75–100 mg daily) [43,45–49]. The chronic administration of low-dose aspirin for at least 5 years reduces the risk of developing gastrointestinal cancers (of the esophagus, stomach and colon) by ∼20% . It also protects against other cancers, such as breast, lung and prostate, but the reduction in risk is less pronounced . The finding that aspirin decreases long-term post-trial cancer deaths  suggests that the drug interferes with mechanisms which come into play early in tumorigenesis. Since low-dose aspirin, given once daily, targets platelet COX-1 primarily, via the irreversible inactivation of the enzyme and consequently the inhibition of TXA2 biosynthesis [50,51], it has been proposed that the inhibition of platelet function by aspirin represents the mechanism which counteracts tumorigenesis [52,43,44]. Platelet activation is the first event which comes into play to ensure the restoration of tissue damage/dysfunction [2,4,43,44,53]. However, if the platelet response is not controlled, it contributes to the development of a chronic inflammatory response through the release of several mediators, such as prostanoids, growth and angiogenic factors, cytokines, chemokines and MPs, which activate the cells of the stromal compartment, including leukocytes and fibroblasts. This phenomenon contributes to enhancing the concentration of growth factors and inflammatory mediators in the stromal microenvironment [2,4]. Among them, there is prostaglandin (PG)E2 whose biosynthesis is dependent on the induction of COX-2 [4,54,55] (Figure 1A). This cascade of biological events culminates with the expression of COX-2 also in epithelial cells [4,56–58] which contributes to the expression of the anti-apoptotic protein Bcl-2 and increased resistance to apoptosis . This survival advantage may promote the accumulation of genetic mutations leading to the ultimate loss of proliferative control. Moreover, PGE2 induces the transactivation of epidermal growth factor receptor (EGFR) [60,61]. Whether this mechanism participates in the EGFR-dependent CRC progression has not been demonstrated.
The crucial roles of platelets in cancer.
Further analyses of RCTs with aspirin used for cardiovascular prevention by Rothwell and colleagues showed a short-term effect of the drug, even at low doses, on cancer incidence and mortality associated with the interference of cancer metastasis [48,65,66]. The antiplatelet effect of aspirin may play a role, in this setting, since there is a considerable amount of evidence available which indicates that platelets are involved in the development of metastasis [1–3,5]. It is noteworthy that the role of platelets to support metastasis dissemination was first suggested almost 50 years ago by Gasic et al.  who showed the reduction of metastasis formation in mice with neuraminidase-induced thrombocytopenia . The results of numerous subsequent studies have allowed identifying many mechanisms by which platelets promote metastasis [1–3,5,68]: (i) the formation of platelet aggregates surrounding tumor cells which may support tumor cell survival and protection from immune elimination; (ii) the enhancement of the adhesion of tumor cells to the endothelium thus leading to tumor cell arrest and extravasation; (iii) the synthesis of lipid products and the release of proteins from α-granules during platelet activation that may enhance tumor vascularization and facilitate tumor cell dissemination into the bloodstream and (iv) the induction of a migratory and invasive phenotype of cancer cells [7–9] (Figure 1B).
In the bloodstream, platelets activate cancer cells which on the other hand activate platelets through a direct interaction [3,5]. There are many platelet molecules involved in this cross-talk, such as the adhesion molecules integrins , the platelet collagen receptor GPVI that interacts with the galectin (Gal)-3 expressed in cancer cells  and the platelet P-selectin that interacts with mucin-type glycoprotein and other cancer cell receptors [70–72] (Figure 1B). Moreover, the cancer–platelet interaction leads to the release of many soluble molecules from platelet α-granules [such as PDGF and transforming growth factor (TGF)-β] and the synthesis of eicosanoids, including TXA2, PGE2 and 12-hydroxyeicosatetraenoic acid (HETE) [2,7–9,73]. The direct interaction and the released products promote some changes in the gene expression program of tumor cells, leading to the development of EMT and a migratory phenotype [7–9]. Platelet-induced EMT is characterized by the up-regulation of mesenchymal markers (such as Vimentin, Twist1, Snail and Zeb) and the down-regulation of epithelial markers (such as E-cadherin) in cancer cells [7–10]. Additionally, cancer cells may acquire the expression of megakaryocyte/platelet proteins (‘platelet mimicry’), promoting further platelet aggregation and the activation of the coagulation cascade. These events play a role in the hematogenous dissemination of cancer cells [9,11]. Low-dose aspirin and other antiplatelet agents (such as the antagonists of the ADP receptor P2Y12) may counteract metastasis formation by preventing EMT and by inhibiting cancer cell-induced platelet activation [7,9].
Platelet-derived microparticles in cancer
Recent evidence suggests the role of platelet MPs in cancer and metastasis . The procoagulant activity of platelet MP surface is 50–100-fold higher than that of activated platelet surface . Their prothrombotic potential depends on: (i) their surface binding sites for coagulation factors; (ii) the exposure of phosphatidylserine due to its translocation from the inner to outer layer during platelet activation  and (iii) the increased expression of surface tissue factor [76–78]. Platelet MPs induce the adhesion, survival and proliferation of tumor cells by transferring their membrane receptors, such as cytokine receptors and platelet–endothelium adhesion receptors (including CD41, CD61 and CD62) . However, at the tumor microenvironment, platelet MPs may interact with other cell types, including immune, inflammatory and endothelial cells . In addition to proteins, platelet MPs can deliver bioactive lipids, such as AA and sphingosine-1 phosphate, to monocytes and endothelial cells, inducing COX-2 overexpression and enhancing AA metabolism .
Platelet MPs can also contain short non-coding RNA sequences known as miRNAs that, once delivered, modify the biological responses of the target cell . Mir-939, transferred by platelet MPs to ovarian epithelial cancer cells, can induce the proliferation and migration of SKOV3 cells . Platelet MPs infiltrate solid tumors and deliver miR-24 to tumor cells in vivo and in vitro inducing cell apoptosis and suppressing tumor growth .
The assessment of the number, type and the molecular content of platelet MPs has been suggested to give relevant information on the clinical condition of an individual and open the avenue to the development of novel disease-associated biomarkers. Thus, the plasma levels of platelet MPs, together with vascular endothelial growth factor (VEGF), interleukin-6 and RANTES, were increased in patients with gastric cancer at stage IV, and their evaluation identified the patients with metastatic cancer . The characterization of circulating MPs in patients with CRC and pancreatic cancer in comparison with those with inflammatory bowel or pancreatic diseases and healthy subjects revealed that platelet-derived MPs have a distinct signature in cancer patients . In non-small-cell lung carcinoma (NSCLC) patients, the levels of four types of circulating MPs (mainly originated from endothelial cells and platelets) were increased in respect to control subjects, decreased after the therapy and predicted the clinical outcome of these patients .
Platelets as new players in cancer immunotherapy
Platelets may contribute to immune escape of cancer cells through various mechanisms (Figure 1B,C) [87–89]. Recent findings have shown that platelets may transfer their molecules of major histocompatibility complex class I (MHC-I) to cancer cells, which acquire the capacity to escape the immune attacks (Figure 1B) . Moreover, the platelet surface TGF-β-docking receptor glycoprotein A repetitions predominant (GARP) activates latent TGF-β, and GARP/TGF-β complex suppresses the immune response to cancer cells mediated by regulatory T cells  (Figure 1C).
It has been recently reported that T-cell therapy against hepatocellular carcinoma (HCC) is improved by the concurrent treatment with antiplatelet agents, such as aspirin (which inhibits COX-1) and clopidogrel (which blocks P2Y12 receptor) . Platelets, through the interaction with T cells, may promote their local accumulation and induce pathological events involved in HCC, such as liver injury and fibrosis . The mechanism involves the contribution of platelet CD154 (CD40 ligand) .
Platelet signature as a cancer diagnostic tool
The early diagnosis of cancer represents the most critical challenge in cancer treatment. Recently, a novel strategy for early cancer detection, known as CancerSEEK, has been reported . It may be used for the screening of multiple different cancers (ovarian, liver, stomach, pancreas, esophageal, colorectal, lung and breast cancers) within the general population. CancerSEEK is a non-invasive blood-based test for the assessment of the levels of eight known serum proteins and 16 mutated genes in circulating cell-free DNA (cDNA) . The results of this test, obtained in more than 1000 individuals, gave positive results in ∼70% in all cancer types. This test identified the molecular blood signature of the tumors with specificity >99%; however, ∼80% of detected cancers were at advanced stages (II or III stage). Thus, it remains to demonstrate whether the test is useful for the early detection of cancer .
The assessment of the content of RNAs and proteins in platelets might help to cancer diagnosis at early stages. In fact, platelets can uptake and store in their intracellular granules several molecules from the tumor microenvironment, including blood, to form the so-called tumor-educated blood platelets (TEPs). TEPs are characterized by a different pattern of proteins and RNAs (mRNAs and miRNAs) for the early detection of cancer [14,15]. The sequencing of TEP mRNAs of 283 individuals allowed to distinguish patients with localized and metastatic tumors (n = 228) from healthy subjects (n = 55) with an accuracy of 96% . Also, the RNA-seq analysis of circulating TEPs, coupled with particle-swarm optimization algorithms, differentiates NSCLC patients, healthy subjects and individuals without cancer but with inflammatory not cancer-related conditions .
The improvements in mass spectrometry techniques coupled with novel bioinformatic strategies have allowed the identification of the platelet proteome of healthy individuals, which comprises >5000 proteins . The pioneers in the study of platelet protein content related to cancer patients were Judah Folkman and co-workers who showed that in mice bearing human xenografts, the levels of different angiogenic proteins sequestered by platelets were altered due to tumor growth, even before the tumor was macroscopically evident . The analysis of several angiogenic proteins (VEGF, bFGF, PDGF, PF4, TSP-1 and endostatin) in platelets isolated from CRC patients and healthy individuals revealed that the levels of PDGF, PF4 and VEGF were independent predictors of CRC . Sabrkhany et al.  performed the platelet proteomic analysis in patients with lung or pancreatic cancer at early stages and healthy individuals. By using a mass spectrometry-based technique, they identified 4384 unique proteins, and 85 of them were significantly changed in early-stage cancer versus healthy controls; additionally, the levels of 81 proteins resulted to be normalized after surgical removal of the tumor . Also, a platelet proteomics analysis has been performed in ovarian cancer patients, and the study identified a group of platelet proteins as a set of biomarkers differently expressed between ovarian cancer cases and benign lesions .
Platelets as a delivery system for anticancer drugs
The traditional chemotherapy represents the main choice for the treatment of several cancers. However, it is associated with serious adverse effects, which in some cases limit its use. A strategy to overcome this limitation is the development of an efficient drug delivery system to specifically target cancer cells. Considering that platelets can interact with tumor cells through several mechanisms, it has been proposed that platelets might function as a ‘biomimetic drug delivery system’ of antitumor drugs . Up to date, there are several preclinical studies which use platelets as the cargo of different drugs for cancer treatment. Xu et al.  reported a novel platelet-based delivery system for the treatment of lymphoma by the loading of platelets with doxorubicin (DOX-platelet) and then conjugated with anti-CD22 monoclonal antibody to facilitate the internalization of DOX-platelets to cancer cells. They showed that DOX-platelet–CD22 treatment of tumor-bearing mice enhanced antitumor activity and attenuated cardiotoxicity of DOX, thus suggesting that DOX-platelet–CD22 could be a promising strategy for lymphoma treatment . Li et al.  functionalized synthetic silica particles with the membrane of activated platelets and performed surface conjugation of tumor-specific apoptosis-inducing ligand cytokine, TRAIL. These ‘biomimetic’ particles were incorporated into microthrombi associated with circulating tumor cells in the lung vasculature and significantly reduced lung metastasis in a mouse model of breast cancer . Dai et al.  loaded the platelets with kabiramide (KabC), an inhibitor of actin polymerization and platelet aggregation. Studies with fluorescence microscopy revealed that KabC-platelets, coupled with transferrin and cyanine 5 dye, bind specifically to RPMI8226 multiple myeloma cells and K562 leukemia cells, and accumulated within RPMI8226 cell-derived myeloma xenotransplants in immuno-compromised mice . Hu et al.  reported the synthesis of polymeric nanoparticles enclosed in the plasma membrane of human platelets which maintain the immunomodulatory properties and adhesion antigens of platelets. These nanoparticles, compared with uncoated ones, were characterized by a reduced cellular uptake by macrophage-like cells. Moreover, they did not present particle-induced complement activation in plasma and present several ‘platelet-mimicking properties’, such as the adhesion to the damaged vasculature and enhanced binding to platelet-adhering pathogens . In a rat model of coronary restenosis and a murine model of systemic bacterial infection, the use of these functionalized nanoparticles to deliver docetaxel and vancomycin, respectively, enhanced the chemotherapeutic efficacy associated with these drugs .
PDL-1 (programmed death-ligand 1) is an immune checkpoint protein, and the binding to its receptor PD-1 on activated T cells inhibits their anti-tumor activity . Recently, Wang et al.  showed that the conjugation of an anti-PDL1 antibody on the surface of platelets was associated with the reduction of post-surgical tumor recurrence and metastasis in animal models. By using mice bearing partially removed primary melanomas (B16-F10) or breast carcinomas (4T1), the authors showed that the anti-PDL1 antibody was released by platelet-derived MPs.
Despite the role of platelets in cancer, particularly in tumor metastasis, was first suggested a long time ago , the subsequent research work in this field did not allow to provide the appropriate experimental and clinical evidence to translate into the development of therapeutic strategies which target the platelet function. It is important to recognize the studies of Rothwell and his colleagues [48,49,65,66], which through the analyses of the vast amount of clinical information from placebo-controlled RCTs with aspirin for cardiovascular prevention, have provided several pieces of clinical evidence in support of the anticancer effect of low-dose aspirin. The evidence was considered appropriate to recommend aspirin use for the primary prevention of cardiovascular disease and cancer by the U.S. Preventive Services Task Force . However, many questions remain to be addressed: (i) is an anticancer benefit associated with the use of other conventional antiplatelet agents, such as clopidogrel; (ii) can its coadministration with low-dose aspirin improve the anticancer efficacy and (iii) what is the appropriate aspirin dose.
The evidence of the efficacy of the antiplatelet agent aspirin has catalyzed the work of researchers with different expertise who have uncovered the multi-faceted roles played by platelets in tumor development and metastatic dissemination [1–3,6,104]. Platelets act through the cross-talk with stromal cells and cancer cells mediated by the release of several soluble mediators and MPs (Figure 1) [2,6,104]. Platelet-derived MPs can promote changes in the gene expression profile of cancer cells by the delivery of miRNAs. Also, MPs can transfer platelet proteins to cancer cells . This phenomenon, called ‘platelet mimicry’, has a pivotal role in the promotion of cancer-associated thrombosis and the development of hematogenous dissemination of cancer cells [9,11]. Recently, preclinical and clinical evidence pointed out that targeting ‘platelet mimicry’ in different cancer types may provide a synergic therapeutic strategy for cancer management .
In addition to the release of biologically active mediators, platelets can take them up, acting as sponges . This new aspect of platelet biology opens the way to the development of novel biomarkers of disease status based on the analysis of platelet signatures. The proteomics and transcriptomics analysis of platelets and platelet-derived MPs may identify cancers also at early stages, before their detection by conventional tests [12–15]. Owing to the capacity of platelets to specifically interact with tumor cells, platelets can act as a ‘biomimetic drug delivery system’ of antitumor drugs . This approach represents a new frontier in nanomedicine which may potentially improve the risk/benefit ratio in the development of targeted therapies for several diseases.
The biomedical research should exploit the new knowledge on the role of platelets in cancer and focus on the development of safer antiplatelet drugs that interfere with the molecular mechanisms involved in the cross-talk of platelets with the cells of the microenvironment and/or tumor cells. These new pharmacological agents may potentially stop cancer at the earliest stages of development, thus preventing tumor metastasis.
cyclic adenosine monophosphate
platelets with doxorubicin
epidermal growth factor receptor
glycoprotein A repetitions predominant
major histocompatibility complex class I
non-small-cell lung carcinoma
platelet-derived growth factor
programmed death-ligand 1
randomized clinical trials
transforming growth factor
vascular endothelial growth factor
von Willebrand factor
This work was supported by the Associazione Italiana per la Ricerca sul cancro [grant no. IG-20365 (to P.P.)]; the financial support from ‘G. d'Annunzio’ University of Chieti-Pescara, Italy (ex 60% funds to P.P.).
We apologize to our colleagues for not being able to reference all primary work due to space limitations.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
These authors contributed equally to this work.