The platelet contribution to cancer progression pot

Complex interactions between tumor cells and circu-lating platelets play an important role in cancer growth and dissemination, and a growing body of evidence supports a role for physiol

REVIEW ARTICLE

The platelet contribution to cancer progression

N M B A M B A C E and C E H O L M E S

Division of Hematology and Oncology, Department of Medicine, University of Vermont, Burlington, VT, USA

To cite this article: Bambace NM, Holmes CE The platelet contribution to cancer progression J Thromb Haemost 2011; 9: 237–49

Summary Traditionally viewed as major cellular components

in hemostasis and thrombosis, the contribution of platelets to

the progression of cancer is an emerging area of research

interest Complex interactions between tumor cells and

circu-lating platelets play an important role in cancer growth and

dissemination, and a growing body of evidence supports a role

for physiologic platelet receptors and platelet agonists in cancer

metastases and angiogenesis Platelets provide a procoagulant

surface facilitating amplification of cancer-related coagulation,

and can be recruited to shroud tumor cells, thereby shielding

them from immune responses, and facilitate cancer growth and

dissemination Experimental blockade of key platelet receptors,

such as GP1b/IX/V, GPIIbIIIa and GPVI, has been shown to

attenuate metastases Platelets are also recognized as dynamic

reservoirs of proangiogenic and anti-angiogenic proteins that

can be manipulated pharmacologically A bidirectional

rela-tionship between platelets and tumors is also seen, with evidence

of tumor conditioning of platelets The platelet as a reporter of

malignancy and a targeted delivery system for anticancer

therapy has also been proposed The development of platelet

inhibitors that influence malignancy progression and clinical

testing of currently available antiplatelet drugs represents a

promising area of targeted cancer therapy.

Keywords: angiogenesis, cancer, metastases, platelets, TCIPA.

Introduction

Tumor cells interact with all major components of the

hemostatic system, including platelets Platelets and platelet

activation have been linked to key steps in cancer progression

(summarized in Fig 1) The contribution of platelets to

malignancy progression has been suggested to be an organized

process that underlies the pathobiology of cancer growth and

dissemination rather than a simple epiphenomenon of

neopla-sia (reviewed in [1]) Here, we highlight current insights into

how platelets contribute to cancer growth, maintenance and

propagation and identify potential targets and directions for platelet-directed anticancer therapy in the future.

Platelet structure and function Often numbering over 3–4 trillion in an individual patient with cancer, platelets represent the smallest circulating hematopoi-etic cells and are anucleate fragments formed from the cytoplasm of megakaryocytes The platelet membrane consists

of phospholipids and is covered with glycoproteins and integrins, which are essential for adhesion, aggregation and activation, the critical steps in platelet-mediated hemostasis Important platelet membrane receptors include Glycoprotein Ib-IX-V (GPIb-IX-V), Glycoprotein VI (GPVI) and Glyco-protein IIb-IIIa (GPIIb-IIIa, also as integrin aIIbb3), receptors that are essential for complete adhesion and aggregation [2,3] Additional important receptors found on platelet membranes include the protease-activated receptors (PAR), PAR-1 and PAR-4, and the P2 receptors, P2Y1 and P2Y12, which principally mediate activation and aggregation [4] Platelets also contain three types of granules: (i) dense granules containing platelet agonists such as serotonin and ADP that serve to amplify platelet activation, (ii) a granules containing proteins that enhance the activation process and participate in coagulation; and (iii) lysosomal granules containing glycosid-ases and proteglycosid-ases [5].

Many of the major structural components of platelets and platelet receptors that contribute to hemostasis have also been found to relate to malignancy progression (reviewed in Table 1) For example, in addition to coagulation-related proteins, platelets also store proteins within the alpha granule that can regulate angiogenesis and metastases [2,6] Further, platelet receptors such as GPIIb/IIIa can mediate platelet angiogenic protein release in addition to their more traditional role in fibrinogen binding At least one study has found ultrastructural changes in platelets from patients with lung cancer, including an increase in the number of platelet alpha granules [7] Interestingly, these researchers also found that the number of alpha granules was associated with survival Functionally, platelets are complex cells capable of shape change, translational protein production, protein and metab-olite release, cell-cell interactions and paracrine regulation Most of these functions relate to the processes of platelet activation and aggregation that occur following exposure to

Correspondence: Chris E Holmes, Department of Medicine,

Hematology and Oncology, University of Vermont, Burlington, VT

05401, USA

Tel.: +1 802 656 0302; fax: +1 802 656 0390

E-mail: ceholmes@uvm.edu

an in vivo stimulus In thrombosis formation, thrombin and

collagen contribute substantially, but not exclusively, to

platelet activation in vivo [2] In malignancy, tumor cells can

activate platelets by direct contact, or via release of mediators

such as ADP, thrombin, thromboxane A2 or

tumor-associ-ated proteinases [8–11] The relative importance of each

platelet activator in malignancy is unknown and some data

suggest the mechanism of platelet activation by tumor cells

may be tumor cell specific and, in some cases, mutually

exclusive [12].

Several studies have suggested an increase in platelet

activation in the blood of patients with cancer [13–19] Both

tumor secretion of activators and direct contact with tumors

have been related to this platelet activation [20–22] To date, no

differences in platelet receptor function or composition have

been described in patients with active malignancy as compared

with healthy subjects to explain this increase in platelet

activation.

Early observations on platelets and cancer

Gasic et al., in 1968 [23], first described the association between

platelet number and metastatic cancer potential This group

found neuraminidase-induced thrombocytopenia was

associ-ated with decreased metastasis of TA3 ascites tumor cells This

antimetastatic effect was neutralized by infusion of platelet-rich plasma (PRP) Thrombocytopenia experimentally induced

by a variety of mechanisms has also been associated with a reduction in the number of metastases in tumor transplant models [23,24].

Thrombocytosis is observed in 10–57% of patients with cancer, with the number varying based on cancer type [1] The relationship between elevated platelet count and malignant tumors was initially reported by Reiss et al in 1872 [25] Subsequent studies have established this relationship for common cancers, including colorectal, lung and breast cancer,

as well as gastric, renal and most urogenital malignancies [26– 32] Further, for the majority of malignancies, the extent of platelet count elevation is inversely correlated with survival, making thrombocytosis a marker of poor prognosis [26–32] Insights into the mechanisms underlying the initial observa-tions of thrombocytosis in malignancy have been forthcoming

in more recent decades Sierko & Wojtukiewicz [1] have recently summarized mechanisms underlying the humoral interaction between tumor cells, bone marrow endothelial cells (BMEC) and megakaryocytes An important driver for thrombocytosis in malignancy is the secretion of tumor-derived cytokines such as IL-1, GM-CSF, G-CSF and IL-6, which stimulate thrombopoiesis through a thrombopoietin-depen-dent mechanism, influencing largely megakaryopoietic growth and differentiation [33–38] Megakaryocytes have a similar ability to secrete inflammatory cytokines, which can in turn influence bone marrow endothelial cells to support mega-karyocytopoiesis [39,40] VEGF and b-FGF are also released

by megakaryocytes, and influence megakaryocytic maturation and transendothelial migration via an autocrine loop [41–43] Although incompletely elucidated, the interactions between tumor cells, megakaryocytes and bone marrow endothelial cells appear to promote thrombopoiesis, and may influence tumor angiogenesis.

Tumor cell-induced platelet aggregation, activation and metastases

Platelets contribute to critical steps in cancer metastasis, including facilitating tumor cell migration, invasion [44–46] and arrest within the vasculature [47–49] In cellular models of both breast cancer and ovarian cancer, invasiveness has increased following exposure to platelets [46,50] In the latter, both activated platelet membranes and platelet releasate increased invasion Platelet contents may be released into the peritumoral space following platelet activation and enhance tumor cell extravasation and metastases [51–55] An important step in metastatic dissemination is the breakdown of vessel basement membrane By releasing proteolytic enzymes such as gelatinase, heparanase and various matrix metalloproteinases (MMPs), activated platelets can directly degrade structural components, or alternatively, support this process by activating other proteinases and/or enabling tumor cells and endothelial cells to do the same [46,56–58] Moreover, modulation of proteolytic activity is accomplished by growth factors released

Platelet

1

Angio-genesis

Tumor microenvironment

Tumor cell Tumor

2

3

Fig 1 Platelets are involved in key steps of malignancy progression In in

vitroand in vivo murine models, a role for platelets has been demonstrated

in tumor metastasis, tumor growth and angiogenesis Our working

understanding of the role of platelets in malignancy involves: (i) tumor

cell-induced platelet aggregation can occur following tumor cell intravasation

into the vasculature, thereby protecting or cloaking circulating tumor

cells from physical clearance and immune surveillance, (ii) platelets

facil-itate tumor cell arrest within the vasculature, endothelial cell retraction

and subsequent tissue invasion, (iii) platelets induce endothelial cell

pro-liferation and new blood vessel formation, which are requisite for

tumor-associated angiogenesis and growth and (iv) tumor and

platelet-stromal interactions in the tumor microenvironment depend, in part, on

platelet activation and platelet protein release, which contribute to the

inflammatory response Additional platelet-related proteins and

metabo-lites that facilitate proteolysis and tissue remodelling also enhance tumor

growth and metastasis (including bony metastases)

by platelets, a topic recently reviewed by Sierko &

Wojtukiewicz [1].

Tumor cells have the ability to aggregate platelets, a finding

first reported in 1968, and referred to as tumor cell-induced

platelet aggregation (TCIPA) [23] It is now recognized that this

aggregation correlates with the metastatic potential of cancer

cells in both in vitro and in vivo models of experimental

metastasis [59,60] The mechanisms by which tumor cells

induce platelet aggregation may differ by cancer type, but have

in common the theme of conferring survival advantage In

turn, platelets can protect tumor cells in at least two ways: by

coating them and thereby directly shielding them from physical

stressors within the vasculature and by permitting evasion from

the immune systems effector cells For example, platelets have

been shown to protect tumors from NK cells and TNF-a cytotoxicity [61,62] Timar et al [63] have raised the hypothesis that some malignant cells can acquire a platelet-like phenotype, with expression of similar adhesion molecules and receptors This concept of platelet-mimicry has been suggested to relate

to the perceived lack of tumor-directed immune surveillance Recently, platelet-derived transforming growth factor-b has been shown to down-regulate the activating immunoreceptor NKG2D on NK cells and impair NK cell antitumor activity [64].

Tumor-platelet aggregates have the ability to disseminate and embolize within the pulmonary microvasculature and have been directly observed to do so in murine models [65] A brief discussion of several major mechanisms of TCIPA and tumor

Table 1 Key platelet components and their contribution to hemostasis and malignancy

Platelet

component

Principal role in thrombus formation

Role in malignancy

Reference

GPIIb/IIIa

(aIIbb3)

Activation allows fibrinogen binding and platelet plug

reinforcement

Tumor cell and platelet interaction (via fibronectin, fibrinogen and VWF) demonstrated in numerous cell lines; inhibition decreases TCIPA and platelet-mediated angiogenic growth factor release

Decreased pulmonary metastasis following inhibition of receptor by antibody and receptor antagonists

[3,60,71,86–88,90,160, 165,166]

GP Ib-IX-V Binding of von

Willebrand factor;

anchors platelet to subendothelium

Limited data to suggest role in TCIPA; conflicting data on tumor cell-platelet interactions

Pulmonary metastasis decreased in mice lacking GPIb but increased when GPIb functionally inhibited by monovalent, monoclonal antibodies

[71,91,92]

collagen

Not studied to date 50% reduction in

pulmonary metastases in GPVI-deficient mice

[93]

P-selectin Mediates

platelet-leukocyte tethering;

triggers leukocyte activation

Facilitated interaction between tumor cells and endothelial cells via sialylated fucosylated carbohydrates

Deficiency or blockade of P- selectin inhibits the formation of melanoma metastases

[94–100]

P2Y

receptors

ADP-mediated platelet aggregation

ADP-mediated VEGF release from platelets; ADP induced TCIPA

ADP depletion associated with reduced metastases

[67–71,124,133,163,167]

PAR

receptors

Thrombin mediated platelet activation

Selective release of angiogenesis influencing proteins; induces TCIPA

Promote metastases [11,122,123,168]

Alpha

granules

Storage of proteins that enhance adhesive process: fibrinogen, VWF, MMP-II, P-selectin, factor V, PF-4, platelet activating factor

Uptake and storage of angiogenic proteins that are selectively packaged and released: VEGF, b-FGF endostatin, angiostatin, TSP-1; storage and release of proteolytic enzymes and metastasis influencing proteins

Maintenance of intra-tumor vascular integrity

[6,117,120–122,126,141]

Platelet

microparticles

Enhances thrombosis and secondary hemostasis

Increased tumor cell invasiveness, metastasis, MMP-2 up-regulation and angiogenesis; increased leukemia, prostate and breast cancer invasion/

migration

Increased chemo-invasiveness and metastases formation in lung cancer models

[101–108]

cell-induced platelet activation follows as their understanding is

pivotal to the development of selective agents targeting the

pharmacologic inhibition of these central pathways (see also

[66] for extensive review).

Adenosine diphosphate (ADP) is contained in platelet dense

granules and is considered a secondary mediator of

aggrega-tion The major ADP receptors, P2Y1 and P2Y12, are both

involved in platelet aggregation Stimulation through these

receptors also leads to shape change and thromboxane A2

generation by platelets [67] ADP contributes to TCIPA

induced by various tumor cell lines, including neuroblastoma,

melanoma and breast carcinoma [68,69] The P2Y12receptor

plays a central role in platelet activation and in TCIPA [70,71].

For example, by generating ADP, MCF-7 breast carcinoma

cells activate and aggregate platelets via the P2Y12receptor [71].

Thrombin has a multifaceted role in hemostasis and

represents a key link between primary and secondary

coagu-lation responses Thrombin has also been linked to

tumori-genesis and angiotumori-genesis, with thrombin signaling being a

major contributor to metastatic tumor dissemination [72].

Thrombin has been detected in situ in numerous tumor types,

including small cell lung cancer, renal cell, melanoma and

ovarian cancer [73–75] Tumor-enhancing effects of thrombin

include induction of TCIPA, increased tumor-cell adhesiveness,

promigratory and chemotactic effects, and up-regulation of

VEGF expression by tumor cells [76–79] Importantly,

throm-bin is also the most potent platelet activator, and exerts its

function via the platelet PAR receptors, PAR-1 and PAR-4.

Secretion of ADP and thrombin by human tumor cells

activates platelets and recruits them to participate in TCIPA

[11] Following thrombin-mediated platelet activation, up to

300 biologically active molecules can be released and deposited

ad lib at sites of vascular injury, at the site of a wound or within

the tumor and tumor vasculature [6].

Cathepsin B, cancer procoagulant factor and the matrix

metalloproteinases (MMPs) are contributors to TCIPA.

Cathepsin B and cancer procoagulant factor can induce

platelet aggregation when released by tumor cells [80,81].

MMPs have demonstrated a similar ability to induce TCIPA in

vitro [82] MMPs can be released by both platelets and cancer

cells in vivo (reviewed in [83]) Jurasz and colleagues have

identified enhanced generation of MMP-2 as the potential

cause of human platelet aggregability in the setting of

metastatic prostate cancer [66].

Thromboxane A2 (TXA2) and its receptor (TX) also play

integral roles in platelet-tumor aggregation It has been shown

that TX mediates platelet aggregation induced by murine and

tumor cell lines [84] TXA2 can be generated by platelets as a

result of activation induced by other platelet agonists, an

observation that highlights the complex and interrelated nature

of platelet functional responses in the tumor.

Platelet adhesion receptors also play a critical role in

tumor-platelet cross-talk and the process of hematogeneous metastasis

(recently reviewed in [85]) The role of the GPIIb-IIIa receptor

in TCIPA has been established for decades, and numerous

metastatic models have highlighted the importance of this

receptor in the tumor-platelet interaction model [86–89] A recent role for the GPIIb/IIIa receptor in the release of proangiogenic proteins and fibrinogen has also been elucidated [87,90] The involvement of the integrin receptor GPIba in tumor metastasis, on the other hand, has been more difficult to define [59,86,91,92] Recently, the GPVI surface receptor, a member of the immunoglobulin superfamily, which principally binds collagen, has become a subject of active investigation Importantly, a 50% reduction in experimental pulmonary metastases in GPVI-deficient mice was reported by Jain et al [93] Clinically, patients with GPVI deficiency exhibit a mild bleeding tendency, suggesting that this receptor could poten-tially be inhibited without major hemostatic consequence Finally, P-selectin is expressed on activated platelets and endothelial cells and has been identified as an important mediator of the interaction between these cells and the vessel wall [94] This facilitated interaction also applies to tumor cells

as P-selectin can bind to different tumor cell lines through binding of sialylated fucosylated carbohydrates [95,96] In a similar manner, P-selectin appears to facilitate interactions between tumor cells and the surrounding endothelium, at least

in the case of melanoma [97] Deficiency or blockade of P-selectin has inhibited the formation of metastasis in various other experimental models [97,98] This effect is most pro-nounced in mucin-producing cancers [99,100].

Platelet microparticles and malignancy When platelets are activated or exposed to high shear stress, they release particles expressing membrane receptors and cytoplasmic constituents termed platelet microparticles (PMPs) A growing body of literature supports the direct involvement of PMPs in malignant cell proliferation and growth PMPs have the ability to induce chemotaxis of many hematopoietic cells and increase their adhesive affinity to fibrinogen [101] PMPs express multiple proteins and chemo-kine receptors, which can be transferred to surrounding cell membranes, including malignant cells, which then benefit from enhanced invasiveness [102–105].

In vitro, PMPs have been shown to induce proliferation and tube formation of human umbilical vein endothelial cells,

to increase trans-matrigel chemoinvasion of lung cancer cell lines, and to increase invasiveness of breast cancer cells [105].

In vivo, angiogenesis can be observed in the heart of ischemic rats when PMPs are injected into myocardium [106] Injection

of murine Lewis lung cancer cells coated with platelet PMPs was associated with significantly more metastatic lung disease [107] Janowska-Wierczorek et al [105] have recently demon-strated that PMPs promote adhesion of tumor cells to endothelium, induce chemotaxis and chemoinvasion, and up-regulate MMP production MMP-2 up-regulation and increased malignant cell invasiveness have also recently been reported in prostate cancer [108] PMPs appear to represent

an important aspect of the functional interaction between tumors and platelets and may represent a novel treatment approach in the future.

The role of platelets in angiogenesis

Evidence supporting the link between platelets and

angiogen-esis has accumulated since Pinedo and Folkman first raised this

hypothesis [109] The growth of solid tumors and formation of

metastases depend on the generation of neovessels, and it is

recognized that tumor cells cannot grow beyond 2–3 mm in

size without a new vascular network [110] These vessels are

needed not only to sustain and nourish the developing tumor

cells, but also to allow delivery of proteases and cytokines that

permit further invasion, extravasation and dissemination This

elaborate delivery and transportation system exists secondary

to an altered balance between angiogenesis stimulators and

inhibitors These proteins are released by many components of

the tumor microenvironment, including the tumor itself This

tumor microenvironment is comprised of stromal fibroblasts,

resident macrophages and mast cells, mononuclear cells and

platelets [111–115].

Platelets contain over 30 important angiogenesis regulating

proteins Platelets are now recognized as the major source of

VEGF (a pro-angiogenic protein) in serum as the platelet pool

comprises > 80% of total circulating VEGF in patients with

cancer as well as healthy individuals [116,117] Of interest is the

observation that in some cancers, platelet-derived VEGF better

predicts tumor progression than serum levels of VEGF [118].

Platelets also contain proteins that inhibit angiogenesis,

including platelet factor-4 (PF-4), TSP-1 and endostatin

[119,120].

Under normal physiologic conditions, platelets have been

suggested to release angiogenic proteins to promote wound

healing These pro-angiogenic proteins are later

counterbal-anced by the release of angiogenic inhibitors from stromal cells

and platelets, to stop uncontrolled growth in later stages of

healing in non-malignant wounds [121] These angiogenic

mediators are packaged into distinct alpha granule

popula-tions, and selective release based on selective engagement of

platelet receptors has been proposed [122] Ma and colleagues

first introduced the concept of differential release of platelet

angiogenic proteins, by demonstrating that PAR-1 activation

was associated with VEGF release and suppression of

endost-atin, while PAR-4 activation, conversely, stimulated endostatin

release and suppressed release of VEGF [123] These

investi-gators subsequently treated rats with established gastric ulcers

with an oral PAR-1 antagonist or vehicle In this model,

significant healing of ulcers did not occur in the rats treated

with the PAR-1 antagonist [123].

Subsequently, the ADP receptors, P2Y1 and P2Y12, have

been demonstrated to participate in the regulation of

angio-genic protein release, though this pathway of platelet activation

appears to release less VEGF than thrombin-mediated

activa-tion [124] ADP-mediated platelet activaactiva-tion is associated with

a net increase in the release of VEGF in healthy individuals,

with no effect on endostatin release This VEGF release can be

abolished by selectively inhibiting the P2Y12receptor [124].

The source and mechanism of platelet-derived angiogenesis

proteins remain under active investigation in both healthy

individuals and patients with cancer Recent studies have offered insight For example, in the circulation, platelets have been shown to uptake and store proteins that regulate angiogenesis [1,125,126] In addition to protein uptake, Zaslavsky et al [120] have recently demonstrated that the platelet source of TSP-1 is megakaryocyte derived, suggesting that enhanced production or endocytosis by marrow precursor cells may contribute to the platelet angiogenic protein content Based on the findings that VEGF-A was regulated by Il-6 in a megakaryoblastic cell line, Salgado et al [127] bring forward the hypothesis that higher VEGF levels in cancer patients may partly result from an IL-6 mediated up-regulation of the expression of VEGF-A in platelet precursors.

In vitro, proangiogenic effects of platelets were observed by Pipili-Synetos et al [128], who noted that platelets stimulated endothelial cell proliferation and growth of capillary-like structures in Matrigel assays An additional in vivo model of angiogenesis showed a reduction of retinal neovascularization

in mice with induction of thrombocytopenia as well as inhibition of platelet aggregation by a highly specific alpha-IIbbeta3 receptor antagonist or aspirin [129] This resulted in a 35–50% reduction of retinal neovascularization, further supporting the platelet contribution to angiogenesis [129] Kisucka et al also examined the role of platelets in four

in vivo animal models of angiogenesis using both a cornea and Matrigel assay They report that platelet-depleted mice experienced a significant reduction in corneal neovasculariza-tion and developed hemorrhage, and postulate that platelets support angiogenesis through release of growth factors and platelet-vessel wall interactions [130] Brill has also demon-strated the role of platelet microparticles in models of angiogenesis [106].

Importantly, a clear understanding of the contribution of platelets specifically to tumor-associated angiogenesis remains under investigation For example, while platelets enhance angiogenesis as in the examples above, platelet-endothelial interactions in tumor microvessels have been found to be reduced in murine models of tumor angiogenesis [131] The platelet as a scavenger of VEGF and therefore a potent anti-angiogenic cellular component of the tumor microvasculature could also be considered.

A complex and bidirectional relationship between tumor cells and platelets

There is growing evidence to suggest that the interplay between platelets and tumors is neither passive nor unidirectional (Fig 2) Complex relationships between host, tumor and platelet within the cancer patient will need to be carefully delineated and significant research efforts are required if antiplatelet therapy is to be used successfully in the clinical setting The platelet role in coagulation-mediated cancer progression, the platelet contribution to the tumor-stromal interaction and the contribution of platelets to inflammation and its subsequent role in malignancy progression are just several examples of these relationships [132]) Shared tumor cell

and platelet agonists and receptors offer both opportunity and

potential obstacles for drug targeting For example, drugs that

inhibit the P2Y receptors on platelets may also interact with

endothelial and cancer cell P2Y receptors and contribute to the

overall impact of the drug [133–135] The well-delineated role

of thrombin signaling and activation of PARs found on

malignant cells is another example of shared targets between

tumor cells and platelets (reviewed in [136,137].

Some evidence suggests that platelets can be conditioned in

vivo by tumor cells to deliver anti-angiogenic proteins [121,138].

In a murine model, Kerr et al [138] have recently demonstrated

that platelets preferentially store tumor-derived GM-CSF,

TPO, TNF-a, TGF-bı and especially MCP-1 over host-derived

proteins An emerging concept in the literature focuses on the

platelet as a reporter of malignancy For example, both platelet

associated PF-4 and TSP-1 have been associated with early

cancer growth and been proposed as biomarkers of early tumor progression [120,139].

Platelet granule proteins not only promote growth of tumor vessels, but prevent tumor hemorrhage, presumably by main-taining the integrity of the existing tumor vascular supply [140,141] Though the precise mechanism underlying this phenomenon has not been fully elucidated, this appears to occur independently from thrombus formation The prevention

of tumor hemorrhage by platelets has more recently been found

to relate, in part, to their ability to modulate vascular damage

by tumor-infiltrating leukocytes [142]; an observation that further illustrates the complex tumor-stromal interaction, including the ability of platelet to influence inflammatory responses [140,143–145] These observations suggest that the mechanism underlying the maintenance of neoplastic vessels by platelets may be distinct from that used for maintenance of host vessels, rendering pharmacologic inhibition of the former plausible Selective platelet storage and release of stimulatory, inhibitory and regulatory proteins represents a novel concep-tual framework to be explored in the understanding of tumor angiogenesis.

Antiplatelet therapy in the treatment of cancer

In 1989 and 1993, Dr Leo Zacharski and colleagues, writing for the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis Sub-committee on Hemostasis and Malignancy, published an update of clinical trials using antiplatelet therapy and antico-agulants in cancer [146,147] At the time, over 20 studies, most

of them pilot studies with 50 patients or fewer, were reported

in the literature using an antiplatelet drug in the treatment (not prevention) of cancer The majority of these studies focused on the drug dipyridamole in non-randomized studies, which reported variable response rates An analogue of dipyridamole (RA-233, mopidamol) has also been studied in prospective randomized studies, with no survival benefit demonstrated in small cell and ovarian cancer but an approximate 100-day improvement in survival in non-small-cell lung cancer patients [148–150].

The remaining prospective studies using antiplatelet therapy focused on the use of aspirin in renal cell and small cell lung cancer and showed no effect [151] Aspirin use has been most extensively studied in colorectal and breast cancer, with demonstrated efficacy in the colorectal cancer prevention setting [152] Aspirin-mediated inhibition of platelet aggrega-tion is well documented, and recently aspirin has also been shown to attenuate platelet protein release [153] In vivo data suggesting a possible inhibitory role in the formation of metastasis were initially reported by Gasic et al [154], who observed metastatic inhibition of MCA6 ascites sarcoma cells

in mice, in the presence of aspirin In a more recent publication, aspirin but not indomethacin suppressed the formation of lung metastasis in a metastatic hepato-cellular murine model [155] Antimetastatic effects of aspirin, however, have not been seen consistently in all laboratory models.

Fig 2 A diagramatic representation of the multiple bidirectional

inter-actions between platelets and tumor cells Platelets and tumor cells express

many of the same receptors, illustrating the concept of platelet mimicry

These receptors, such as GPIb and GPIIbIIIa, may participate in TCIPA

by promoting arrest of tumor cells in the vasculature and by promoting

interactions with bridging proteins such as VWF, fibronectin and

fibrin-ogen Tissue factor expression is up-regulated in tumor cells, leading to

thrombin generation Tumor cells also have the ability to directly secrete

platelet agonists such as ADP and thrombin, which activate platelets in the

tumor microenvironment In turn, activated platelets secrete growth

factors and proteinases that can regulate tumor growth and invasion

Activated platelets shed microparticles, which facilitate cell invasion and

angiogenesis ADP, adenosine diphosphate; PAR, proteinase-activated

receptors; P2Y, P2Y receptors; GPIb, Glycoprotein Ib; GPIIbIIIa,

Glycoprotein IIbIIIa; VWF, Von Willebrand factor; Lpa,

lysophos-phatidic acid; Txa2, thromboxane; PMP, platelet microparticle

Clinical data evaluating the impact of aspirin therapy on

cancer survival have begun to emerge Fontaine et al [156]

have recently reported preliminary data suggesting that aspirin

used in combination with the surgical treatment of

non-small-cell lung cancer is associated with increased survival Similarly,

in a prospective observational study of women diagnosed with

breast cancer, as reported in the Nurses Health Study, aspirin

use was associated with decreased risk of breast cancer

recurrence and death [157] Additionally, aspirin use was

found to decrease the proangiogenic effects of tamoxifen in

patients with breast cancer [116] Importantly, the clinical

benefits of the drug are likely to also relate to its

anti-inflammatory effects.

A review of selected published human clinical studies using

antiplatelet therapy in the treatment of cancer is found in

Table 2 This table does not contain data related to the role of

antiplatelets (such as aspirin) in cancer prevention or the

potential antiplatelet (p-selectin inhibition) effect of heparin

(the latter recently reviewed in [85]) Additional pilot studies of

antiplatelet drugs alone or in combination with additional

chemotherapy have been reviewed by Hejna et al [158] The table highlights the paucity of clinical trial data using currently available antiplatelet agents Importantly, while we have recently reported on the use of aspirin therapy in women with breast cancer receiving tamoxifen therapy [116,159], there is a paucity of data to support the combination of antiplatelet therapy with existing tumor-targeted therapy.

Despite the limited number of prospective randomized trials, the laboratory data using antiplatelet therapy continue

to accumulate Early laboratory studies focused on prosta-cyclin and prostaprosta-cyclin analogues, which have been previ-ously reviewed [158] In addition, blockade of the GPIIb/IIIa receptor using the monoclonal anitbody 10E5, an inhibitor

of human platelet GPIIb/IIIa, decreased lung colonization of cancer cells [160] A challenging aspect of the administration

of GPIIb/IIIa antagonists in the clinical setting has been the need for intravenous administration of these agents, which are now widely used in high-risk acute coronary syndromes Recently, however, an oral inhibitor of GPIIb/IIIa, XV454, has halted experimental metastasis formation in a murine

Table 2 Clinical outcomes associated with the use of platelet inhibitors in patients with cancer Limited clinical data are available on the impact of platelet inhibitors on clinical outcomes in patients diagnosed with cancer Murine model data are reviewed in the text and in Table 1

Platelet inhibitor

or modulator

Mechanism of platelet inhibition

Type of cancer(s) studied Protocol designs Observations in clinical studies Reference Aspirin Inhibits platelet thromboxane

production and platelet aggregation (anti-neoplastic effects of this drug are also anticipated to rely on COX-2 tissue and tumor inhibition)

Colon cancer Double blind No difference in overall survival [169]

randomized

No effect on survival [170] Renal cell

carcinoma

Prospective randomized

No significant response or effect on survival

[151] Breast cancer Prospective

observational study

Decreased recurrence and mortality from breast cancer

[157]

NSCLC (early stage)

Retrospective analysis

Increased survival post-resection

[171] Prostate

cancer

Retrospective analysis

Improved PSA control in patients undergoing radiation

[172] Benoral

(aspirin-acetaminophen

conjugate)

Breast cancer Double blind No significant response or

improved survival

[173]

Clopidogrel P2Y12 receptor antagonist;

inhibits platelet aggregation induced by ADP

Prostate cancer Retrospective

analysis

Improved PSA control in patients undergoing radiation

[172]

RA-233

(Mopidamole)

Dipyridamole derivative;

increase in platelet cyclic AMP; decreased platelet aggregation

Colon cancer Double blind No significant response [149] NSCLC

(early stage)

Double blind Improvement in survival in

limited stage/resected disease;

no effect in disseminated disease

[149,150]

randomized

No significant response [150] Ovarian cancer Prospective

randomized trial

No effect on survival or recurrence

[148]

Dipyridamole Increase in platelet cyclic

AMP; decreased platelet aggregation

Colon cancer (advanced)

Prospective randomized trial

No impact on survival or response

[174]

NSCLC (advanced stage)

Prospective non-randomized

No significant response compared with historical controls

[175]

model of lung cancer [87] Integrilin, a commercially

available platelet-specific aIIbb3 integrin antagonist, was

administered to mice after establishment of bony metastases

in a study by Boucharaba and colleagues, evaluating the role

of platelet-derived lysophosphatidic acid This resulted in

thrombocytopenia, decreased circulating Lpa plasma levels

and a significant reduction in the number of osteolytic bony

metastases [51].

Wenzel et al have recently reported successful in vivo

reduction of pulmonary metastases in a murine model of

breast cancer using the platelet aggregation inhibitor cilostazol.

By administrating liposomal cilostazol intravenously, they

observed decreased ex vivo platelet aggregability and decreased

platelet-tumor complex formation [161] Similar results were

obtained using liposomal dipyramidole [162] Few studies

evaluating the common ADP receptor inhibitors, clopidogrel

and ticlopidine, have been reported and they demonstrated

limited success [163].

Conclusion

Platelets play a multifaceted and important role in cancer

biology (Table 3) The existing research suggests a compelling

biological rationale for attempting to disrupt tumor-platelet

cross-talk, with the goal of down-regulating tumor invasion,

angiogenesis and spread In the laboratory, platelet receptors,

both constitutive and activation dependent, such as GP1b/IX/

V, P-selectin and alphaIIb-beta3 integrin, can promote the

progression and metastases of various tumor types and are

obvious targets for further clinical study [164] Additionally,

control of the platelet reservoir of angiogenic proteins, which

are both secreted and sequestered in a selective manner,

represents an approach to angiogenic control within the tumor

microenvironment.

The study of platelet inhibitors in the clinical setting will

require a careful consideration of not only cancer type but stage

of disease targeted Importantly, appropriate trial endpoints must be chosen that are not by design predicated on direct and toxic tumor effects and secondary rapid cell kill and tumor shrinkage A potential barrier that surrounds chronic admin-istration of antiplatelet agents in the setting of active malig-nancy is directly related to the paramount role that platelets play in maintaining hemostasis Currently available oral antiplatelet agents irreversibly inhibit their target, making the risk of bleeding more difficult to mitigate Future work in the development of novel agents would ideally yield a molecule able to inhibit platelet-tumor interaction while maintaining sufficient platelet function to prevent bleeding Potential new classes of agents include antibodies against P-selectin, platelet-specific oral integrin inhibitors, PAR-1 antagonists and block-ade of platelet-derived LPA.

How should we combine antiplatelet therapy with con-ventional cancer cell-directed therapy? Will other host factors that influence platelet activation, such as diabetes, be important in patient selection [135]? Existing antiplatelet drugs, such as aspirin and clopidogrel, remain understudied

as adjuvants to conventional chemotherapeutic and hor-monal therapies, particularly in animal models and the clinical setting Increasing our translational database on the anticancer biology of antiplatelet strategies to include com-bination therapy and studies directed at prevention vs low burden vs high burden disease are imperative for the successful clinical translation of results Importantly, we have learned much from the use of antiplatelet therapy in the treatment of cardiovascular disease, such as the concept of drug resistance These considerations might be applied prospectively in oncologic studies Future clinical trials formally addressing the role of antiplatelet therapy will need rigorous attention to patient selection, combination therapy with existing agents and trial endpoints but offer the hematologic community a significant opportunity to poten-tially improve cancer outcomes.

Table 3 Overview of important platelet-cancer cell interactions and their potential influence on cancer progression A full discussion of these interactions is found in the text These observations reflect in vitro and murine model data

Platelet activation

Increased in patients with cancer

Facilitated by contact with tumor cells

and tumor release/production of platelet

agonists such as ADP and thrombin

Platelet activation enhances tumor cell-induced platelet aggregation, releases chemotactic cytokines, proteolytic enzymes and platelet microparticles that can support cancer growth and extravasation as well as angiogenesis

Platelet activation provides a procoagulant surface to facilitate cancer-related coagulation

Inhibition of key platelet activation and aggregation receptors decreases metastases Tumor-cell-induced platelet aggregation (TCIPA) Platelet aggregation correlates with metastatic potential in in vivo and in vitro models Protection of tumor cells from environment Platelets provide mechanical shielding from physical stressors

Platelet-derived proteins down-regulate immune cells, thereby impairing their antitumor activity

Production of platelet microparticles (PMPs) Transfer of receptors to tumor cell membranes, which may increase invasiveness May

regulate MMP production and influence invasion Release of angiogenic proteins Platelets contain pro- and anti-angiogenic proteins packaged into distinct alpha

granules, which can be differentially released to support angiogenesis Prevention of tumor hemorrhage Platelets maintain tumor vascular integrity and reduce tumor hemorrhage

Platelet-enhanced metastases Platelets facilitate tumor cell migration and extravasation

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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