It is widely acknowledged that platelets can be activated by various factors released by tumor cells like TF, adenosine diphosphate (ADP), thromboxane A2 (TXA2) and high-mobility group box 1 (HMGB1) (35, 36). Tumor cells can activate platelets via direct interactions or secretion of biologically active proteins leading to tumor cell-induced platelet aggregation (TCIPA) (37, 38). During the TCIPA process, platelet αIIbβ3, α6β1, platelet P-selectin, platelet Toll-like receptor (TLR) 4 and platelet CLEC-2 bind to protein molecules on the surface of the corresponding tumor cells, enhancing platelet activation and tumor cell malignant behavior (36, 39–42). In addition, the biological characteristics of platelets during the TCIPA process are subjected to significant changes, with tumor cell-induced platelet extracellular vesicle formation, granule release and alterations in platelet RNA profiles (37, 43). Moreover, activated platelets undergo various cellular responses, including morphological changes and translocation of membrane glycoproteins, and eventually release extracellular vesicles (EVs) containing bioactive substances (44). EVs are mainly composed of exosomes and microvesicles (MVs). Exosomes are intraluminal vesicles, 30-100nm in diameter, formed by the inward budding of endosomal membranes during maturation of multivesicular endosomes (MVEs), while MVs 100-1,000nm in diameter are generated by the outward budding and fission of the plasma membrane (45, 46). EVs induce different biological signals depending on the cell of origin. In this regard, platelet-derived exosomes originate from the extracellular secretion of multivesicular bodies and alpha granules, and MVs are produced by surface shedding (44). Furthermore, the number and proteomic profile of platelet-derived microvesicles (PMVs) exhibit variations with different stimuli (including pathologies). For instance, it has been shown that integrin α6 levels in shear stress-originated PMVs were significantly elevated compared to thrombin-induced PMVs (47). Importantly, integrin α6 has been documented in vascular endothelial growth factor-A (VEGF-A) and fibroblast growth factor-2-driven angiogenesis, promoting tumor growth in vivo and in vitro (48). Nevertheless, the proteomic alterations of platelet-derived MVs in PC remain unclear, emphasizing the need for further investigation. The effect of platelets on cancer cells may be attributed to the ability of exosomes to shuttle selected molecules, since EVs containing protein, mRNA and miRNA with biological functions can be delivered to PC cells (49). Similarly, tumor cell-derived exosomes can transfer mutated RNA to platelets by shuttling, a process that may involve plasma membrane fusion, clathrin-mediated endocytosis, and phagocytosis (50). The tumor microenvironment (TME) contains various cells, cytokines and extracellular matrix components and is the main place for the interaction between the body and the tumor. Tumor cells and platelets maintain a complex, bidirectional interaction in the TME. During TCIPA, activated platelets aggregate near tumor cells to form tumor platelet clots, protecting tumor cells from T cell immune responses and NK cell surveillance and ensuring that tumor cells can persist in circulation and metastasize to distant locations (38). Moreover, platelets contain many bioactive molecules that promote the proliferation, migration and invasion of PC cells (24, 41).

It has been established that various cancer cells can activate platelets by secreting “activators” that result in venous thrombosis, the leading cause of death in cancer patients, especially in PC (30). Furthermore, studies have shown that cancer cells can secrete coagulants or fibrinolytic substances to induce platelet aggregation (51, 52). Mounting evidence suggests that membrane vesicles released by tumor cells called tumor-derived microvesicles (TMVs) incorporate large amounts of TF produced by tumor cells (52–55). TMVs are generated by outward budding and division of the plasma membrane, followed by vesicle release into the extracellular space. Using in vitro experiments, Geddings et al. (52) demonstrated that TMVs from human PC cells BxPc-3 and L3.6pl cells could interact with resting platelets to induce TF delivery and platelet aggregation in human and mouse plasma. In addition, after intravenous injection of TMV into mice, they found that femoral vein thrombosis and platelet deposition in the lungs was significantly increased. Using mouse models, researchers established that both TMV and TF could significantly activate platelets and increase the aggregation ability of platelets to form thrombosis by reducing the recalcification time (52, 56). In a study by Stark et al. (55), PC microvesicles (pcMVs) derived from human PC cell line L3.6pl were injected into mice by intravenous injection. It was found that pcMVs could selectively promote thrombus growth in regions with slow and turbulent flow and significantly shortened whole blood clotting time, leading to the formation of large thrombi upstream of the stenosis. Furthermore, activated platelets can directly activate neutrophils and induce the formation of neutrophil extracellular traps (NETs) (57). Conversely, NETs can induce thrombin production and activate platelets to release ATP and ADP, causing a cascade of reactions and promoting platelet aggregation (58, 59). It has been established that during NETs formation, DNA, TF, myeloperoxidase and histones are released, and DNA upregulates platelet aggregation through the platelet receptor for advanced glycation end products (RAGE) (60). Thrombosis is showed in Figure 2 . However, NETs were not observed in the thrombi in a study where a minimally invasive laser-induced injury model was used, although neutrophils were present at the injury site (61). Interestingly, activated platelets gather into clusters and form thrombi in the TME; however, the thrombi are different from those caused by benign diseases. Histological analyses confirmed that pcMV-thrombi had a composition distinct from nonmalignant thrombi. In contrast, a study reported that the luminal area in the pancreatic TMV-thrombi was filled with a loose fibrin (-proto) network, while neutrophils, monocytes and platelets were significantly reduced (55). Portal vein thrombosis is well-recognized as the most common type of thrombosis in patients with advanced PDAC, followed by mesenteric vein thrombosis and splenic vein thrombosis, suggesting its value as an indicator of poor prognosis (62, 63). Nevertheless, it has been reported that thrombocytosis has little to do with the increased incidence of thromboembolism (6) since the hypercoagulable state of the whole body is not related to an absolute increase in specific factors but the presence of activated coagulation factors (64). Overall, high expression levels of TMV, NET and TF in PC tissue participate in platelet activation and aggregation, which coupled with procoagulant molecules enzyme heparanase (HPSE), podoplanin (PDPN) and the fibrinolytic system, contribute to the high incidence of thrombosis, making it an excellent model for studying cancer-associated hypercoagulable states (65).

Ponert et al. (66) demonstrated that different cancer cells could easily bind to activated platelets in vitro, thereby accelerating the adhesion between platelets and tumor cells; this phenomenon was particularly prominent in PC cells. In another in vitro experiment, investigators observed that after many platelets aggregated by binding to PC PANC-1 cells, the migration, invasion and proliferation capacity of PANC-1 cells was significantly enhanced (24, 67). The cytoplasm of platelets contains many biologically active proteins, such as growth factors, chemokines, cytokines and proteases, which are secreted by activated platelets (68). The main documented molecular pathways are shown in Figure 3 .

The enzyme cytidine deaminase (CDD) has been reported to participate in the mechanism of gemcitabine resistance by intracellular metabolism of gemcitabine (109) after its upregulation by platelet releasate (110). Human ENT1 (hENT1) is well-known for enhancing the cellular uptake of gemcitabine, thereby enhancing its toxic effects in PC cells (111). Moreover, Slug is a master regulator of EMT that is highly expressed in CD133⁺ human PC cell lines (Capan-1) and enhances the migration and invasion of PC cells, resulting in gemcitabine resistance (112). In vitro experiments have shown that platelet-derived ADP and ATP induced high Slug expression via P2Y1 and P2X7 receptors on the surface of human PC cell lines AsPC-1 and BxPC-3. Importantly, a study demonstrated that Slug could effectively inhibit hENT1 expression, stimulate CDD expression, and enhance the resistance of PC cells to gemcitabine by inhibiting the uptake of gemcitabine by PC cells and accelerating the metabolism of gemcitabine (35, 113). Activated platelet-derived TGF-β1 stimulates PI3K/Akt and MEK/Erk signaling in PC cells, resulting in decreased cisplatin sensitivity (114). Nonetheless, the regulatory role of platelets on the efficacy of PC chemotherapy remains unclear, warranting further study. The above findings suggest that activated platelets can mediate drug resistance in PC cells to a certain extent.

The varying degrees of fibrosis associated with PC account for the difficulty of providing effective treatment for this patient population. Tumor cells are hidden in a thick fibrotic matrix that acts as a barrier and is responsible for the poor response to chemotherapy drugs. Over the years, research on the mechanism of platelet fibrosis in PC has been limited to PSCs. PSC has been acknowledged to play a key role during pancreatic fibrosis in chronic pancreatitis and the pro-fibrotic reaction of PC by producing the stromal reaction. Studies have shown that activated platelets could effectively activate PSC and promote the formation of connective tissue (75). In a mouse model experiment, Vonlaufen et al. (115) demonstrated that the PC group co-cultured with PSC exhibited a faster growth rate, larger volume and more fibrotic bands containing activated PSC. Moreover, PSC migration was significantly increased by PC cells in vitro. On the contrary, the secretion of PSC could induce PC cell proliferation and migration and inhibit apoptosis. PDGF and TGF-β released by activated platelets have been recognized as effective stimulators for PSC proliferation to accelerate extracellular matrix synthesis (115). Fitzner et al. (116) elucidated that the activation of rat PSCs in vitro was related to increased expression of galectin-1, and galectin-1 could mediate PSC function. PDGF stimulated the expression of the lectin galectin-1 resulting in high proliferation rates and synthesis of more collagen. Targeting platelets against PC fibrosis is a potential therapeutic approach.

When tumors and associated blood vessels are destroyed, cancer cells escape and slough off into the circulation to form circulating tumor cells (CTCs), which become the seeds for distant metastasis of tumors (117). Interestingly, platelets can couple to tumor cells, increase vascular permeability and induce extravasation of tumor cells (118). Tumor cells entering the circulation must deal with high shear rates and immune surveillance, such as NK cell attacks. Eventually, only a small proportion of tumor cells enter the blood circulation for metastasis, making this process very inefficient (119). NK cells play important roles in cancer immune surveillance by mediating direct cytotoxicity and releasing immunomodulatory cytokines to form an adaptive immune response and prevent tumor progression and metastasis. During hematogenous metastasis, cancer cells are quickly encapsulated by platelets, similar to cancer cells putting on a “protective suit”, making it impossible for NK cells to recognize tumor cells allowing distant metastasis ( Figure 2 ). Interestingly, researchers found that thrombocytopenia could effectively inhibit the ability of cancer cells to metastasize in mice models. This phenomenon was reversed by the depletion of NK cells and secretion of TGF-β by activated platelets, thereby inhibiting immunoreceptor natural killer group 2, member D (NKG2D), indispensable for antitumor activity of NK cells (120). Okazaki et al. (121) noted that platelets could preferentially adhere to mesenchymal cells rather than epithelial cells in the TME of mouse models of infectious disease. Interestingly, cancer cells could be wrapped by activated platelets to escape immune surveillance and promote metastasis.

Neutrophils are important innate immune cells in the blood circulation that play important roles in innate and adaptive immunity. Activated platelets can recruit neutrophils by releasing chemical mediators such as CXCL4 (122) and directly interact with integrin αMβ2 on neutrophils via GPIb or integrin αIIβ3 to activate and regulate the functions of neutrophils (123, 124). After activation by various stimuli (e.g., infection, surgery, activated platelets), neutrophils can release reticular ultrastructures composed of protein-studded chromatin called NETs (52, 125, 126). NETs play a double-edged role. On the one hand, they play a positive role in the invasion of pathogenic microorganisms. On the other hand, NET amplifies platelet activation, aggregation and thrombin activation, promotes intravascular coagulation, and promotes the attachment of cancer cells to the blood vessel wall, resulting in enhanced tumor migration (127). In this regard, it has been reported that after surgical stress, activation of the TLR4-ERK5-integrin GPIIb/IIIa axis leads to platelet activation and formation of microaggregates with tumor cells and tumor platelet-neutrophil complexes, enhancing immune escape and leading to distant metastasis of tumor cells (128). Importantly, thrombomodulin effectively prevents PC metastasis to the liver by degrading HMGB1 and thus inhibiting the induction of NETs (129). Furthermore, tumor-produced CXCR1 and CXCR2 chemokine receptor agonists induce neutrophils and granulocyte myeloid-derived suppressor cells (MDSCs) to generate NETs that encapsulate tumor cells and protect them from the cytotoxicity of CD8⁺ T cells and NK cells by hindering the contact between immune cells and surrounding target cells (130). NETs can suppress T-cell responses through metabolic and functional exhaustion, promoting tumor growth (131). However, the role of activated platelets in evasion of immune surveillance and immune cytotoxicity by PC cells remains to be elucidated. The studies of potential markers associated with platelets in PC are summarized in Table 1 .

At present, the application of platelets to enhance antitumor therapeutic effects is widely used in preclinical studies and clinical trials of PC, emphasizing the inhibition of platelet activation and abnormal pathways of cancer cells associated with activated platelets. The efficacies of single antiplatelet drugs and a combination of antiplatelet and chemotherapy drugs have been assessed in these studies. Aspirin is widely acknowledged as a derivative of salicylic acid with antiplatelet properties. It has a significant inhibitory effect on platelet aggregation and effectively prevents thrombosis by inhibiting the production of cyclooxygenase. Accordingly, it is widely used in clinical practice to prevent transient ischemic attacks, myocardial infarctions, and artificial heart thrombi formation after valve surgery. Low-dose aspirin can effectively reduce the incidence and mortality of colorectal cancer (132). In this regard, low-dose aspirin taken every other day has been reported to effectively reduce the risk of colorectal cancer in healthy women (133, 134). A study where activated platelets and PANC-1 cancer cells were co-cultivated demonstrated that the proliferation ability was increased by upregulating the expression of c-MYC. After treatment with aspirin, the proliferation rate of cancer cells was significantly reduced, and the expression of c-MYC was suppressed (72). Clopidogrel exhibits a similar antiplatelet effect as aspirin by inhibiting ADP receptors on the surface of platelets. Using an orthotopic PC mouse model, Mezouar et al. (135) revealed that clopidogrel could directly inhibit platelet activation, significantly reducing thrombus formation, tumor growth, and metastasis without increasing the risk of bleeding. Additionally, studies have shown that the activation of EMT progression in PC cells further promoted chemotherapy resistance of PDAC (136). Low molecular weight heparin (LMWH) can reduce direct contact and interaction between platelets and tumor cells through the action of antithrombin, thereby reducing EMT progression induced by platelets (55). However, the risk of treatment-related bleeding is greatly increased by the long-term use of LMWH. Receptor tyrosine kinase (RTK) represents the largest class of enzyme-linked receptors, acting as a receptor and an enzyme that can bind to ligands and phosphorylate tyrosine residues of the target protein. It consists of a ligand-binding site in the extracellular domain, the single-pass hydrophobic α helix region and an intracellular domain with tyrosine-protein kinase (PTK) activity (137). RTK mediates the connection between cells and controls a wide range of complex biological functions, including cell growth, movement, differentiation, and metabolism. Therefore, the dysregulation of the RTK signal leads to various human diseases, including cancer (138).

It has been established that PDGFRs and VEGFRs belong to the RTK supergene family, are widely distributed in the membrane of PC cells and vascular endothelial cells in the TME, and interact with abundant PDGF and VEGF released by platelets. Many experiments have been designed to explore the roles of PDGFR or/and VEGFR in PC tumorigenesis. Current evidence suggests that platelet-derived endothelial cell growth factor harbors angiogenic activity in vitro and in vivo and contributes to angiogenesis and remodeling in the TME (138). The platelet-derived endothelial cell growth factor is overexpressed in most human cancers and associated with increased microvessel density, tumor aggressiveness and poorer patient prognosis (139). In nude mouse orthotopic tumor model experiments, the phosphorylation of PDGFR in tumor and tumor-associated endothelial cells was found to be significantly inhibited by the administration of GN963, a tyrosine kinase inhibitor against PDGFR and Src kinases. Importantly, the activity of Src and Akt kinases in tumor cells was reduced, resulting in a decrease in microvessel density and cell proliferation and increased apoptosis of tumor and tumor-associated endothelial cells (140).

Pericytes are embedded in the basement membrane of capillary endothelial cells and regulate the proliferation and differentiation of endothelial cells associated with angiogenesis. It has been shown that the surface of pericytes is rich in PDGFR-β (141). The specific inhibition of the PDGFR-β signal eliminates PDGFR-β(+) progenitor perivascular cells and mature pericytes around tumor blood vessels, resulting in excessive expansion of blood vessels, endothelial cell apoptosis and low pericytes coverage in PC (142). Subsequently, eliminating pericytes is conducive to tumor vascular degeneration and significant tumor growth inhibition. Moreover, inhibitors of VEGF signaling can block VEGF-mediated endothelial cell survival, tube formation and downstream signaling, inhibit angiogenesis, and reduce tumor vascular distribution (143, 144), exhibiting no harm to the integrity of blood vessels in normal tissues and organs (145). However, due to the complexity of tumor metabolism in time and space, the efficacy of antitumor drugs is greatly affected. Bergers et al. (146) revealed that SU5416, an inhibitor that targets VEGFR in endothelial cells, was effective for early angiogenic lesions but not for large, well-vascularized tumors in mouse models of PC. In contrast, SU6668, a selective kinase inhibitor of PDGFR, has been shown to prevent further end-stage tumor growth, causing pericyte detachment and tumor blood vessel destruction. The combination of SU5416 and SU6668 was more effective than mono drug therapy at all stages of pancreatic islet carcinogenesis. Other antiplatelet drugs have also been used for the treatment of PC. For example, integrin α-2 is the most expressed integrin molecule on the platelet membrane and mediates platelet adhesion and aggregation. Integrin α-2 inhibitor significantly reduces the microvessel density of PC in mice and effectively inhibits tumor growth (147). In a mice model of PC with liver metastasis, the number of multiple metastatic nodules on the liver surface was significantly reduced after injection of prostaglandin before tumor formation, which might be due to inhibition of platelet aggregation by prostaglandin E1 and I2 (148).

At present, although gemcitabine is still the standard first-line treatment for patients with advanced PC, the benefits of these drugs for the survival of patients with PC are below expectations (136), which may be accounted for by activated platelets weakening the therapeutic effect of antitumor drugs. Platelets interact with PC cells and stimulate the PI3K/Akt and MEK/Erk signaling by releasing activated platelet-derived TGF-β1, causing cancer cell tolerance to cisplatin (114). The activation of platelets in the TME account for increased chemotherapy resistance of pancreatic ductal adenocarcinoma. In an orthotopic PC model in nude mice, the combination of VEGF receptor antibody and gemcitabine inhibited primary pancreatic tumor growth and the incidence of lymphatic metastasis and liver metastasis to a great extent compared to monotherapy, improving the survival rate of mice (149, 150). PKI 166, an EGFR protein tyrosine kinase inhibitor, combined with gemcitabine, could effectively reduce microvessel density, inhibit cell proliferation, increase tumor cell and endothelial cell apoptosis, and significantly inhibit lymph node and liver metastasis (151). Ticagrelor, which inhibits platelet activation through the ADP-P2Y12 axis, can significantly reduce the proliferation ability of PC cells but not normal pancreatic cells. The combination of ticagrelor and gemcitabine significantly has been found to reduce tumor growth in vivo (83). In addition, gemcitabine combined with anticoagulants (e.g., dalteparin) can significantly reduce the incidence of vascular thromboembolism in advanced PC and reduce mortality due to vascular thromboembolism (152, 153). Importantly, it has been shown that radioimmunotherapy combined with imatinib (a potent inhibitor of PDGF-β) significantly inhibits the growth of PC compared to radioimmunotherapy alone and does not produce any obvious side effects (154). Experimental studies have shown that SU6668 could increase the radiosensitivity of tumor blood vessels, which contributed to tumor growth inhibition and enhanced tumor response to radiotherapy (155). Adjuvant chemoradiotherapy has been established to play a minimal role in controlling advanced PC and does not improve patient prognosis (156). It remains unknown whether antiplatelet therapy combined with chemoradiotherapy will benefit patients. Overall, antiplatelet therapy can inhibit platelet-related cancer-promoting pathways, and combinations of chemotherapy and radioimmunotherapy can effectively enhance antitumor efficacy (157).

The barrier function of tumor vascular endothelial cells is strengthened by adhesion of the covering activated platelets, limiting penetration of chemotherapeutic drugs in the tumor cell yielding a poor antitumor effect. To overcome this problem, Cao et al. (158) constructed TM33 peptide-modified gelatin/oleic acid nanoparticles loaded with TNA that could specifically bind to P-selectin on the surface of activated platelets and release the target drug TNA into the extracellular space under the stimulation of MMP-2 secreted by activated platelets, to induce high local TNA exposure. Platelet activation was inhibited by the high concentrations of TNT on the surface of tumor blood vessels, which improved blood vessel penetration and allowed antitumor chemotherapy drugs to leak into tumor cells. Most importantly, TNT did not cause additional side effects, such as bleeding, without changing the biological functions of platelets. In addition, a small dose of nanoparticle-antitumor drugs coated with platelet membrane could selectively adhere to cells in the TME and improve the bioavailability of the antitumor drug after local delivery. Side effects of systemic high-dose administration, including rapid white blood cell consumption and temporary local immune insufficiency, were not observed (159). Geng et al. (160) constructed a platelet camouflage nanoprobe with active targeting properties, which could escape macrophage phagocytosis and specifically bind to CD44 on the surface of most cancer cells, showing great potential for accurate diagnosis and effective treatment of cancer. Accordingly, platelet-related nanotechnology treatments can reduce the damage caused by drugs to vital human organs and accurately target PC lesions. Nano-combined targeted drug technology has great potential in the treatment of PC.

Although antiplatelet drugs combined with chemotherapeutics effectively improve the antitumor effect, the adverse events caused by these drugs should not be ignored. Common adverse events encompass fatigue, anorexia, dysphonia, nausea and decreased platelet count (161), while serious adverse events include peptic ulcer disease and gastrointestinal bleeding (133, 162). More importantly, it remains controversial whether antiplatelet drugs combined with chemotherapeutics will bring survival benefits compared with chemotherapeutics alone in advanced PC patients (161), raising awareness on the need to develop precise and individualized treatments for this patient population.

Organoids are three-dimensional (3D) cell cultures that contain key properties of the organs they represent. These in vitro culture systems include self-renewing stem cells that can differentiate into multiple organ-specific cell types, exhibiting a similar spatial organization to their counterparts and reproducing some of their functions. Accordingly, they can mimic human development. and disease potential, thus providing a physiologically relevant system (163). It has been shown that PC organoids can be rapidly generated from resected human or mouse tumors and biopsies with success rates as high as 75-83%. A comprehensive transcriptional and proteomic analysis of pancreatic organoids could reveal key genes and pathways altered during disease progression (164). Given that 85% of PC patients are not indicated for surgery (165), PC organoids can be generated from limited amounts of cellular material provided by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to detect differences in gene profile expression and find diagnostic and personalized treatment approaches (164). Moroever, a comprehensive genomic, transcriptomic and therapeutic analysis of PC patient-derived organoids (PDOs) could identify molecular and functional subtypes of PC, predict treatment response, and facilitate precision medicine for this patient population (166). Interestingly, establishing a platelet co-culture model with PC organoid can simulate the crosstalk between PC, extracellular matrix and platelets in the TME and reveal the underlying mechanisms of the interaction between PC cells and platelets (167). However, exploring the mechanisms of PC development through platelets (platelet-related therapy) and organoid co-culture models is at an early stage, warranting more experimental data to substantiate current findings.