Advances and potentials in platelet-circulating tumor cell crosstalk

Abstract

Tumor metastasis leads to circulating tumor cells (CTCs) that separate from primary malignant tumors and enter blood circulation. CTCs survive and engage with other cells to cope with obstacles, including shear stress, disease, immune attacks, and drugs. Platelets are the best partners for CTCs. Platelets provide a good protective layer for CTCs to ensure that are not monitored and cleared by the native immune system, and protected from shear stress and survive better. Here, we review current reports on platelet-CTC interaction and the clinical relevance of their combination and summarize new techniques for CTC capture and treatment based on platelet-CTC interaction. We discuss current data, identify its shortcomings, and suggest future developments.

Introduction

Circulating tumor cells (CTCs) shed or migrate into the circulation from the primary tumor site owing to the metastatic characteristics of the tumor. CTCs undergo epithelial-mesenchymal transformation (EMT) and interact with other cell types in the circulation [1]. They adhere to the vascular endothelium and exude into the distal organs, eventually developing into macroscopic metastases [1]. CTCs circulate as individual cells or cell clusters that appear to have higher metastatic potential and shorter half-lives [2]. As a key component of the metastasis cascade, CTCs have unique phenotypic and genotypic characteristics and have been used as a non-invasive source of cancer cells for tumor phenotypic and genotypic analysis [3]. Additionally, CTCs serve as clinical indices for identifying tumor surface markers and mutations, evaluating and monitoring treatment responses, and assessing patient prognosis [1,3]. Therefore, exploring the characteristics of CTCs is critical for understanding the biology and progression of blood-borne metastatic tumors. However, the accuracy and sensitivity of CTC liquid biopsies using clinical blood collection cannot be guaranteed because of low quantification (1-10 cells per 10 ml of blood) [4]. One of the main reasons for this is that CTCs enter blood vessels through active invasion or passive shedding, and face mechanical stress, foreign microenvironments, and immune surveillance, which results in a short half-life and a high apoptosis rate.

During blood transmission, CTCs interact with all types of blood cells including platelets, neutrophils, monocytes/macrophages, endothelial cells, and cancer-associated fibroblasts [1]. These interactions are broadly classified into two categories: direct cell-cell interactions, especially within heterogeneous CTC clusters, and indirect regulation of phenotypic molecules of interacting cells [1]. CTCs survive in harsh environments and take root in distant organs, benefiting from the manipulation of the cellular function of surrounding normal cells. Among circulating cells, platelets are the most closely associated with and have the greatest influence on CTCs [5]. Under platelet shelters, CTCs are protected from the destruction of shear stress and evade surveillance by the immune system. Hence, exploring the mechanism of interaction between platelets and CTCs and using it as a theoretical basis to achieve efficient capture and targeted therapy of CTCs will greatly improve the efficacy and prognosis of patients with tumors.

Here, we review current reports on platelet-CTC interaction and the clinical relevance of their combination and summarize new techniques for CTC capture and treatment based on platelet-CTC interaction. We discuss the current literature, identify gaps in the knowledge, and suggest ideas for future studies.

Platelets in cancer

Platelets play crucial roles in human health and disease. Platelets act as “scanners” in the immune system, sense the presence of bacteria, and communicate with lymphocytes to regulate immune cell exosmosis in the circulation [6]. Therefore, platelets are important for the recovery from inflammation [7]. Platelets also play a critical role in clotting and wound healing [8]. Platelet-rich plasma-activated tendon-derived stem cells promote regeneration of ruptured Achilles tendon in rats [9]. Ristocetin-induced platelet aggregation has been used to monitor bleeding tendency in chronic lymphocytic leukemia treated with irutinib [10]. The role of platelets in tumors has been extensively. The Indian physician Sushruta discovered a relationship between tumors and blood changes as early as 1000 BC [11]. In 1878, Billroth et al. [11] discovered tumor cells in blood clots. Since platelets have not yet been discovered, only alterative characteristics caused by tumors were noticed in the blood; however, the blood components associated with tumors were unclear. Gasic et al. [12] first reported the relationship between platelets and tumor metastasis in 1968 and found that platelets caused tumor metastasis. As technology continues to evolve, the complex interactions between platelets or megakaryocytes and tumors as well as the role of platelets in promoting tumor metastasis, are being increasingly understood [13].

Tumor-platelet interaction is a vital component of cancer metastasis, and platelets have an impact on the primary site as well as on tumor cells entering the bloodstream [11]. Platelets create an environment in which multiple pro-angiogenic factors are provided to tumors, thereby stimulating the expression of these factors [14]. Furthermore, circulating vascular endothelial growth factor levels have been reported to be a prognostic factor for diagnosis and treatment evaluation in patients with tumors [15]. When CTCs enter the circulation, platelets, the first circulating cells, encounter CTCs during metastasis [16]. The ability of platelets to protect tumor cells from normal immune responses in the circulation significantly facilitates metastasis [17]. During intravascular transport, CTCs interact with clotting cascades and the innate immune system. Platelets help CTCs spread throughout the body by preventing the immune system from being discovered and destroyed in the circulation [18]. The discovery that CTCs are in a vulnerable phase of metastasis makes them an attractive therapeutic target [18]. Subsequently, platelets form cell-fibrin-platelet aggregates around CTCs surrounded by platelet-rich clots during their initial entry into the circulation, which provides physical protection from shear stress [19]. CTCs form clusters by interacting with platelets via transforming growth factor-β/transforming growth factor-β receptor type 1 (TGFβ/TGFβ1) [20]. Mani et al. compared the platelet content in patients with non-metastatic and metastatic tumors and found that the platelet content in non-metastatic patients was significantly lower than that in metastatic patients, and that the platelet content was inversely proportional to the survival rate [21]. Hovens et al. demonstrated that platelet levels, rather than circulating endothelial cells, are key predictors of early treatment failure in patients with prostate cancer after prostatectomy [22]. Furthermore, platelet-derived EVs have been shown to play an important role in tumorigenesis and metastasis based on the characteristics of platelet interactions with tumor cells in a variety of solid tumors (Figure 1), which are summarized in Table 1.

Figure 1.

Effects of bioactive molecules carried by platelet-derived extracellular vesicles on tumors.

Table 1.

Effects of bioactive molecules carried by platelet-derived extracellular vesicles (pEVs) on tumors

Cancer	Bioactive molecule	Main effects	Ref.
Breast cancer	miR-126 and miR-223	pEVs increased the sensitivity of BT549 cells to cisplatin chemotherapy.	[108]
	Ca2+	pEVs enhance the migration through the partial remodeling of calcium handling machinery to modulate motility.	[109]
Prostate cancer	MMP-2	pEVs have been found to enhance invasion of prostate cancer cells via the upregulation of MMP-2 expression.	[110]
Lung cancer	MMP-9, IL-8, VEGF, miR-223, and scatter factor	pEVs are found to act as facilitators for the formation of new blood vessels.	[111]
Ovarian cancer	miR-939	pEVs can transport microRNAs (miRNAs) like miR-939, promoting the aggressiveness of ovarian cancer cells.	[112]
Colorectal cancer	EMT markers, cyclooxygenase (COX)-2 (PTGS2) expression, and thromboxane (TX) B2 production	pEVs could drive prometastatic and prothrombotic behaviors in cancer cells, indicating potential treatment targets.	[113]

Tumor cells and secretions affect platelets. Tumor-induced platelets are a potential source of tumor-derived biomarkers available from blood biopsies. Noerholm et al. confirmed that tumor-derived substances are transferred from tumor cells to platelets, resulting from tumor-derived transcripts detected in platelets [23]. Previous studies have demonstrated the diagnostic value of platelet mRNA signals as noninvasive biomarkers for predicting tumorigenesis and monitoring tumor progression in a variety of solid tumors [24-28]. In a pan-cancer study, there were significant differences in platelet mRNA expression profiles between tumor patients and healthy volunteers, and platelet profiles were not only suitable for cancer diagnosis but also correctly identified the primary origin of pan-cancer [29]. Best et al. prospectively sequenced tumor-induced platelet mRNA profiles of platelets from healthy donors and cancer patients and were able to distinguish patients with local and metastatic tumors from healthy individuals with 96% accuracy [29]. Using clinical blood samples, the levels of platelets and distant metastases were found to be significantly higher in CTC-positive patients than in CTC-negative patients [30]. Therefore, CTCs predict that the incidence of distant metastases is high in patients with hypercoagulable lung cancer [30]. Panabieres et al. studied crosstalk between CTCs and platelets in vitro [31]. Both morphological and transcriptional alterations were observed in platelets after co-culture with CTC-conditioned medium, leading to platelet aggregation and activation [31]. Moreover, EMT-related gene expression decreased, but the expression of coding mesenchymal marker genes did not, and tumor invasion-related gene expression increased in CTCs co-cultured with platelets [31]. These findings support the hypothesis that CTC-platelet interactions help maintain CTC integrity in the bloodstream. Hence, platelets, as fundamental components of the tumor microenvironment, are considered significant in cancer biology because they contribute to tumor initiation, progression, and therapeutic responses.

Platelets: shelters for CTCs

Platelet-induced EMT of CTCs

Epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells are transformed into stromal cells via specific mechanisms. The main features of EMT include reduced cell adhesion molecule expression, cytoskeletal transformation from keratin to vimentin, and morphological characteristics of mesenchymal cells. Epithelial cells lose epithelial phenotypes (e.g., cell polarity and connection to the basement membrane), but gain interstitial phenotypes (e.g., higher migration and invasion, anti-apoptosis, and the ability to degrade the extracellular matrix) through EMT. However, some tumor progression models have suggested that tumor metastatic potential occurs entirely at the primary site, with few or no signaling events occurring during intravascular metastasis [32].

Given that multiple growth factors and cytokines are released into circulation, platelets are a source of cancer cells that may perceive additional signals beyond the original microenvironment [32]. The bidirectional transfer of lipids, proteins, and RNAs between platelets and tumor cells was analyzed to confirm their impact on tumor cell behavior and processes [32]. Increased expression of platelet and EMT markers acquired from CTCs was observed in blood samples [32]. This indicates that tumor cells and CTCs acquire highly dynamic and aggressive phenotypes, including EMT, stem-like phenotypes, and high proliferation rates, owing to platelet interactions. Moreover, TGFβ promotes metastasis by enhancing EMT and invasiveness in primary cancer, whereas platelets contain excessive growth factors and cytokines, including high concentration of TGFβ [32]. Therefore, platelet-derived factors may be involved in promoting transfer phenotypes. Hynes et al. [32] reported that platelet-derived TGFβ and direct platelet-tumor interaction synergistically activated the TGFβ/Smad and NF-kB pathways in cancer cells, which led to the transition to an aggressive mesenchymal-like phenotype and enhanced metastasis. Targeted inhibition of NF-kB signaling in cancer cells or ablation of TGFβ1 expression in platelets prevents lung metastasis [32]. Therefore, cancer cells rely on platelet-derived signals outside of the primary tumor for effective metastasis.

Matrix metalloproteinases (MMPs), a group of zinc-containing enzymes that are closely involved in angiogenesis and tumor metastasis. Specifically, MMP-2 and MMP-9 are considered the most effective for metastasis. Both enzymes cleave collagen, which is the main component of the subcutaneous matrix, thereby facilitating the transfer of tumor cells from the blood to the tissue [33]. CTCs activate by CTCs to produce microparticles and small platelet fragments that express membrane and cytoplasmic components. Co-culturing cancer cells with platelet-derived microparticles led to an increased secretion of MMP-2 in CTCs [33]. These results indicate show that platelet-related molecular channels activate EMT in CTCs.

Single-cell transcriptome analysis of CTCs provides important insights into metastatic biology. Single-CTC transcriptome analysis of gastric cancer revealed that most gastric CTCs undergo EMT, and platelet adhesion contributes to EMT progression and the acquisition of chemotherapy resistance [34]. Additionally, CTCs have prognostic value in patients with EMT-associated primary breast cancer [35]. In conclusion, EMT is an important biological process through which epithelial cell-derived malignant tumor cells acquire the ability to migrate and invade. Elucidating the molecular mechanisms of the platelet-regulated CTC EMT process, thereby exploring diagnostic and therapeutic methods based on key EMT molecules, is a key scientific issue in the study of EMT mechanisms in tumor metastasis.

Platelet-mediated CTC immune evasion

The CTCs which have been shed by primary malignancies serve as “seeds” for distant metastases. However, the mechanisms by which CTCs escape immune surveillance remain largely unclear [36]. Activated platelets gather on the surface of CTCs to form blood clots, which in turn help CTCs survive [37]. Platelets protecting CTCs from normal immune responses in the circulation may significantly facilitate metastasis [19]. The nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3) inflammasome is a key inflammatory mechanism that was recently discovered to control platelet activation and aggregation [38]. NLRP3 inflammasome expression is upregulated in circulating platelets in a mouse model of in situ pancreatic ductal adenocarcinoma [38]. Drug inhibition or gene ablation of NLRP3 in platelets results in decreased platelet activation, platelet aggregation, and tumor progression, and interference with platelet NLRP3 signaling significantly improves the survival of tumor-bearing mice [38]. Therefore, the platelet NLRP3 inflammasome plays a key role in pancreatic ductal adenocarcinoma and may serve as a novel therapeutic target.

Most CTCs in blood circulation are eliminated by shear stress and natural killer (NK) cells [39]. NK cells are cytotoxic immune cells capable of killing target cells with low or no expression of major histocompatibility complex (MHC) class I molecules [40]. As platelets transfer their own MHC class I molecules to the surface of CTCs, NK cells ignore them when CTCs are recognized, leading to immune escape [41]. CTCs and NK cells interact with HLA-E:CD94-NKG2A through immune checkpoint molecules [36]. Shi et al. [36] characterized CTC and primary and metastatic lesions in human pancreatic ductal adenocarcinoma using single-cell transcriptomes and showed that platelet-derived RGS18 promoted HLA-E expression through the AKT-GSK3b-CREB signaling pathway, and RGS18 overexpression promoted hepatic metastasis in pancreatic tumors. Thus, platelet-derived RGS18 protects CTCs from NK-mediated immune surveillance when a HLA-E immune checkpoint is involved [36].

Surgical resection is the primary treatment for most solid tumors. However, surgical injury increases the risk of tumor recurrence and metastasis [42]. Tissue trauma activates local and systemic innate immune systems, causing inflammatory responses [42]. Platelets and neutrophils play crucial roles in the early innate immune response; however, they may also contribute to the spread and distant metastasis of cancer cells [42]. Tsung et al. [42] reported that platelets activated by surgical stress enhanced platelet-tumor aggregate formation, facilitating their capture by neutrophil extracellular traps and subsequent distant metastasis. Local surgery-induced hepatic ischemia/reperfusion injury was confirmed to promote neutrophil extracellular traps to capture aggregated CTCs that eventually metastasize to the lungs, which were eliminated when platelets were depleted [42]. In summary, these results revealed that platelets help CTCs escape the immune system, and targeting the key molecular mechanisms that destroy platelets is expected to prevent tumors and postoperative distant metastasis (Figure 2). We could also make full use of the protective properties of platelets on CTCs and improve their capture and extraction efficiencies.

Figure 2.

Platelet-mediated circulating tumor cell (CTC) immune evasion.

Platelet-conferred mechanical protection on CTCs

Brain metastases commonly occur in cancer patients; however, there are limited options for effective treatment. To settle in the brain, CTCs must first become permanently lodged in brain microvessels; however, the mechanisms underlying this process are not well understood. Thrombosis often occurs in cerebral microvessels where blocked CTCs successfully extravasate and form large metastases [43]. Mechanistically, CTCs produce tissue factor-mediated thrombin that activates local plasma clotting [43]. In contrast, CTCs cannot activate platelets directly, and antiplatelet therapy reduces platelet configuration in intravascular CTC clusters but does not reduce metastatic encephaloma formation [43]. These results suggest that plasma coagulation is activated early by intravascular tumor cells in the brain and subsequently forms blood clots, leading to the discovery of a new specific mechanism that is critical for brain colonization. The pro-metastasis effect of platelets has been attributed to their ability to promote adhesion or prevent cell death in the circulation by forming a physical barrier around CTCs [32]. Platelets form cell-fibrin-platelet aggregates around CTCs to provide mechanical protection [19]. This barrier protects CTCs from NK-mediated lysis, limits their exposure to shear stress, and promotes their adhesion to endothelial cells [32]. Maftoon et al. [44] elucidated the role of platelets in CTC deformation, adhesion, and survival using highly detailed computational models. Their results illustrated that activated platelets adhered to CTCs, exacerbating metastatic spread [44]. Platelets play a vital role in thrombosis and are key factors in hemostasis and coagulation. Konstantopoulos et al. [45] described colorectal adenocarcinoma cell adhesion to tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs) in the presence or absence of platelets and red blood cells. The total number of tumor cells attached to HUVECs and the percentage of secondary adhesion to total cell adhesion depended on the platelet concentration and cell wall shear stress [45]. With enhanced platelet induction, the total number of cell tethers was almost twice that observed in the absence of platelet perfusion [45]. Together, these results revealed a novel role for platelets in promoting the binding of tumor cells to endothelial cells through secondary tether mechanisms.

Other platelet-derived biomacromolecules

Dynamic crosstalk between tumors and their microenvironment is increasingly recognized as a key regulator of malignant progression. Tumor cells secrete various cytokines that activate stromal fibroblasts and induce immune cell recruitment. Signals derived from the local microenvironment promote the invasion and metastasis of tumor cells. Mucin-4 (Muc4) is a large cell surface glycoprotein involved in the protection and lubrication of epithelial structures [46]. The abnormal expression of Muc4 affected the adhesion, proliferation, and invasiveness of tumor cells, as well as the growth rate and metastasis efficiency of xenograft tumors [46]. Reduced association of tumor cells with platelets and leukocytes using histological analysis of lung lesions suggests that Muc4 may promote metastasis by promoting the association of CTCs with blood cells, thereby increasing CTC survival in circulation [46].

Platelets contain excess growth factors and cytokines; therefore, platelet-derived factors may be involved in promoting the metastatic phenotype. Platelet-derived growth factor B has been shown to promotes and maintains vascular integrity in the tumor microenvironment by promoting pericellular recruitment [47]. Nasopharyngeal carcinoma (NPC) is a highly metastatic and aggressive malignant tumor. Distant metastases are the primary cause of treatment failure and mortality. Distant metastasis in NPC patients was positively correlated with the expression level of integrin β3 (ITGβ3) in platelet-derived extracellular vesicles in NPC patients [48]. EVs transferred from platelets to NPC cells mediate intercellular communication and induce NPC metastasis by upregulating ITGβ3 expression [48]. Mechanistically, up-regulated ITGβ3 activates the MAPK/ERK/ATF4/Nrf2 axis, inhibits ferroptosis, and promotes NPC metastasis [48]. Therefore, these findings elucidate the novel role of platelet-derived EVs in metastasis, which not only improves our understanding of platelet-mediated distant tumor metastasis but also has important implications for the diagnosis and treatment of nasopharyngeal carcinoma.

In summary, platelets are involved in the complete process of tumor metastasis (Figure 3). Metastasis is a characteristic of tumors that limits their therapeutic effects and prognosis. Platelets are key targets for inhibiting tumor metastasis, improving tumor-targeted therapy efficacy, and improving the quality of life of patients with tumors. Platelets can be potential targets for diagnosis and treatment and achieve clinical transformation by exploring the mechanism of platelets in the occurrence and progression of tumors.

Figure 3.

Platelets involve in tumor metastasis as shelters for CTCs.

Clinical relevance of CTCs combined with platelets

CTC detection will help understand tumor development as CTCs are tumor cells present in blood circulation [1]. Numerous studies on multiple solid tumors have shown that the number of detectable CTCs is inversely associated with early- and late-stage survival [1]. Platelets are a major contributor to miRNA release into the circulation [49] and may have an impact on various components of the blood. Therefore, understanding the clinical correlation between circulating CTCs and platelets is crucial for evaluating tumor development and patient prognosis.

Clinical immunology laboratory diagnostics

Platelets are shed from mature megakaryocytes in bone marrow as small pieces of cytoplasm, and megakaryocytes combined with CTCs are associated with disease progression in tumor patients [50]. In the current study on patients with metastatic breast cancer, an association was observed between a higher number of CTCs and a higher probability of megakaryocytes [51]. Platelets act as a shelter for CTCs to evade the immune system. Therefore, the mechanism of platelet-CTC interactions in tumor immune regulation must be explored to understand their clinical relevance. Preoperative platelet assessment, alone and in combination with CTCs, has prognostic potential for nonmetastatic breast cancer [52]. Żaczek et al. [52] compared preoperative platelet counts, CTCs, 65 serum cytokines, and 770 immune-associated transcripts using the NanoString technology. A high-normal platelet count was associated with lymph node metastasis and an increasing number of mesenchymal CTCs in 70 patients with operable breast cancer [52]. Patients with high platelet and CTCs counts had the shortest overall survival [52]. Similar results have been obtained in patients with renal cell cancer [53]. Mechanistically, a high platelet count was associated with high intratumoral stromal content, specifically the phenotypes associated with CD8+ T cells [52]. Increased cytokine concentrations in the bone marrow are associated with platelet activation and production [52]. Antiplatelet therapy may have good therapeutic potential in patients who have been identified as having the highest risk of disease progression.

Kimmig et al. [54] confirmed that proinflammatory markers in the blood are strongly associated with CTCs, which are precursors to metastasis, by retrospectively analyzing the clinical data, CTC, and blood count results from 171 patients with early stage breast cancer. Inflammatory indicators included the neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR), platelet-lymphocyte ratio (PLR), monocyte-neutrophil ratio (MNR), and systemic immunoinflammatory index (SII). Their combination with CTCs has important research value for tumor prognosis and classification. The potential prognostic roles of CTCs, NLR, and PLR in patients with colorectal cancer were evaluated to identify more accurate predictors in colorectal cancer. NLR and CTC counts serve as reliable prognostic factors in patients with colorectal cancer, and CTCs and PLR analyses may be clinically useful for colon cancer management and risk stratification [55,56]. Similarly, Yang et al. [57] combined CTCs and NLR to identify patients at high risk of metastatic castration-resistant prostate cancer. NLR can further classify patients into different risk groups with detectable CTCs, suggesting that NLR play a complementary role in CTC-based prognostic stratification in patients with castration-resistant prostate cancer patients [57]. Additionally, Reuben et al. [58] conducted a multivariate analysis of triple-negative tumors and found that CTCs with inflammatory indicators could predict overall survival in metastatic breast cancer; however, only CTCs and MLR remained independent prognostic factors, and the two were combined to form a prognostic score. The risk of postoperative metastasis in patients with renal cell carcinoma can be predicted using mesenchymal CTCs, MNR, or staging [53]. As a combined prognostic factor, staging and MNR may provide convenient and accurate status monitoring [53]. In summary, CTCs are of great value in tumor-related liquid biopsies. Different inflammatory cells in the blood affect CTC growth and physiological functions. Therefore, extensive and in-depth exploration of the role of blood cells, including platelets, in CTCs and the detection of the clinical correlation between the two have important translational value.

Platelets combined with CTCs to evaluate therapeutic effect and prognosis

In addition to immune regulation, platelets influence the clinical relevance of CTCs from other physiological aspects. CTCs are masked by platelets, and a positive association has been found between increased CTC aggregation and rapid disease progression during follow-up [59]. CTC clusters, along with their interstitial features and platelet marker expression, are highly associated with poor prognosis in patients with unresectable pancreatic cancer [59]. Abnormalities in blood coagulation and the “leukemia” stage with high CTC counts are common in patients with advanced and progressive breast cancer. Absolute CTC counts were significantly correlated with D-dimer levels and negatively correlated with platelet counts [60]. Therefore, there was a significant correlation among increasing CTC number, increasing D-dimer number, and decreasing platelet count, indicating that CTCs may be a direct contributor to intravascular coagulation activation. Tan et al. [30] reached the same conclusion and confirmed that CTCs predicted distant metastasis in lung cancer and that the incidence of distant metastasis was high in patients in a hypercoagulable state. Additionally, Finn et al. [61] examined the effectiveness of exercise in modulating CTC and occult platelet levels in patients with metastatic prostate cancer through the ExPeCT trial, confirming that CTCs have great potential to effectively reflect treatment effects. Preoperative chemotherapy improved short-term progression-free survival when the CTC test results were positive in patients with stage II or III esophageal squamous cell carcinoma [62]. Therefore, CTC detection can be used as an indicator to guide individualized decision making regarding preoperative chemotherapy. Velasco et al. [63] used CTCs as predictive biomarkers and further stratified an aggressive variant prostate cancer subpopulation with the worst prognosis to inform treatment decisions in metastatic castration-resistant prostate cancer patients treated with carbacasel-carboplatin combined with carbacasel alone.

Monoclonal antibodies are widely used in cancer drug therapies. Aceto et al. [64] found that high CA 15-3 tumor markers, high mean red blood cell volume, high white blood cell count, and high mean platelet volume were specifically associated with CTC clusters in patients with breast cancer treated with denosumab. Therefore, prospective studies need to validate the role of mAbs in preventing CTC production. The expression of CTCs, platelet-derived growth factor receptor (PDGFR), and PDGF ligands in soft tissue sarcoma was detected before and after olaratumab monotherapy [65]. An increase in CTC was observed on day 8 of cycle 1, and a significant decrease was observed on days 1 and 30 of cycle 3 [65]. CTCs can be used as predictors of non-small-cell lung cancer using monoclonal antibodies combined with chemotherapy as a common treatment strategy. Subjects with increased platelet endothelial cell adhesion molecule 1-negative CTCs showed significant reductions in median progression-free survival and overall survival when treated with bevacizumab combined with chemotherapy [66]. Another report demonstrated the feasibility of using CTCs and peripheral blood cells, particularly platelets, as predictive biomarkers for unresectable stage III cell lung cancer in patients treated with chemoradiotherapy and duvacizumab [67].

In summary, the detection and counting of CTCs are clinically useful non-invasive diagnostic biomarkers that can be helpful in clinical management and strategies. Thus, CTCs are potential markers for assessing disease profiles, in which CTCs can be detected in multiple cancer types (Table 2). However, CTCs represent a more diverse cell population, which limits their detection. This phenotypic diversity presents significant challenges for clinicians and researchers in enumerating, characterizing, and developing a fundamental understanding of the underlying oncology.

Table 2.

Circulating tumor cells (CTCs) combined with platelets in clinical relevance

Cancer types	Consequences	Ref.
Breast cancer	Assess prognostic potential and identify patients at highest risk for disease progression	[52]
	Predicting metastasis	[54]
	Predicting intravascular coagulation activation	[60]
Colorectal cancer	Prognosis	[55]
	Patient management and risk stratification	[56]
Lung cancer	Predictive biomarkers of chemoradiotherapy combined with durvalumab	[67]
	Predicting metastasis	[30]
Pancreatic cancer	Highly associated with poor prognosis of patients	[59]
	Evaluating treatment outcomes	[61]
Prostatic cancer	Stratifying subgroups and evaluating chemotherapy effects	[63]
	Shorter overall survival	[57]
Renal cell carcinoma	Evaluating disease progression and metastasis prognosis	[53]
Soft-Tissue Sarcoma	Evaluating olaratumab monotherapy outcome	[65]

Platelet-associated CTC capture techniques

CTCs are known as tumor “seeds” in the blood and play a critical role in tumor progression and metastasis. In 2002, Braylan et al. [68] detected hairy cell leukemia in patients with low levels of circulating malignant cells in the peripheral blood using flow cytometry. With continuous progress in medical science and technology, liquid biopsy of CTCs has become an important tool to explore disease progression in patients with tumors and evaluate the treatment effect and prognosis. However, the number of CTCs, which is approximately 1-10 cells per 10 ml blood, is the biggest obstacle to their widespread clinical use. Currently, mainstream CTC capture methods are divided into two categories: membrane surface antigens and cell size [69]. Therefore, clarifying CTC characteristics and developing efficient and specific CTC capture technologies will become a developmental direction for tumor liquid biopsies in the future.

Membrane surface antigen

Epithelial cell adhesion molecule (EpCAM) is the most commonly used membrane surface antigen, and many studies have focused on EpCAM. Liu et al. first established a method to graft carboxybetaine methacrylate with 3-aminopropyl triethoxysilane as a coupling agent, immobilized anti-EpCAM antibodies on nylon, and successfully demonstrated CTC-trapping ability in nude mouse tumor models [70]. Subsequently, the device effectively reduced protein adsorption and platelet adhesion and prolonged the plasma recalcification time, demonstrating the extraordinary biocompatibility and blood compatibility of the modified surface [71]. However, a single approach targeting EpCAM is not sufficient to achieve specific CTC capture from different tumor sources. Alpaugh et al. [72] compared the standard EpCAM CellSearch kit with EpCAM plus HER2, EGFR, and MUC-1 specific combined ferrofluid capture. The four-trapping ferrofluid reagent did not significantly improve the trapping effect [72]. Although commercial CTC detection kits mainly use the EpCAM antibody, the detection effect is very different for different tumors. Therefore, approaches using tumor-specific markers have significant limitations, such as the detection of free circulating tumor DNA in the plasma of patients with gastrointestinal stromal tumors harboring CKIT or PDGFRA activation mutations [73].

Size and morphology

CTCs have larger volumes, smaller mass density fluctuations, and shorter spatial density correlations than normal blood cell subpopulations [74]. These biophysical parameters provided a theoretical basis for specific CTC capture. Lackner et al. [75] investigated a novel hemofiltration technique for cell morphological classification, immunocytochemistry, and molecular characterization of filtered circulating non-hematological cells in patients with renal cell cancer. Furthermore, the unbiased, fast, and automated separation of CTCs using a single CTC chip enables detailed measurement of the physicochemical and biological properties of CTCs and their role in metastasis [76]. Therefore, CTCs may have different sizes and shapes than other blood components, making it possible to capture them specifically.

With the rapid development of microfluidic technology, the clinical transformation of liquid biopsies has become possible and has great potential for CTC capture. High-throughput concentration and isolation of CTC clusters from large blood volumes using microfluidic technology enables tumor cell population-specific diagnosis [77]. Zhai et al. [78] considered the influences of aspect ratio, dielectrophoretic force, channel size, flow rate, separation efficiency, and shape on cell separation, and proposed a novel two-stage, label-free, fast-continuous CTC capture device based on hydrodynamic inertial focusing and dielectrophoretic separation. In contrast, the integrated system of microfluidic combined with CTC analysis has been reported. Xiong et al. [79] developed a new wedge microfluidic chip that enriched CTCs of different sizes and identified CTCs using a three-color immunocytochemical method. The device demonstrated high performance in detecting CTCs in small blood samples from tumor patients and, combined with the advantages of low cost and mass production, has great potential for clinical applications in cancer treatment guidance and prognostic monitoring [79]. Side-based microfluidic techniques have been used to isolate CTCs from patients with metastatic breast cancer and to correlate their presence with clinicopathological data and overall survival [51]. These results make this a reality for the clinical application of microfluidic technology in CTC liquid biopsy.

Platelet-related techniques

CTCs interact with platelets to promote metastasis in circulation. Therefore, this characteristic of platelet-CTC interactions provides a theoretical basis for improving CTC capture efficiency. Two-dimensional fluid-solid interaction models have been used to study the critical conditions for CTCs to pass through narrow capillaries when platelets are attached to the capillary walls [80]. The computational framework combined with the accompanying results is a powerful tool for studying the biomechanical conditions in CTC-platelet interactions, providing a prognosis for disease progression [80]. The principles of platelet-induced CTC capture are divided into: inhibition of platelet adhesion [81], and CTC complexes that directly capture platelet interactions [82]. However, platelet-covered CTCs are extremely difficult to isolate because of masking or downregulation of surface epitopes. Toner et al. [83] developed a platelet-targeted microfluidic platform for the isolation of epithelial and mesenchymal phenotypes of lung cancer, breast cancer, and melanoma. This method first depletes unbound free platelets by sorting based on hydrodynamic size and then captures platelet-covered CTCs based on immune affinity using a chevron micromixer device [83]. Furthermore, CTC liquid biopsies require the whole blood samples to be immediately processed. Reliable blood specimen stabilization for CTC preservation provides wider geographic sharing for precise rare-cell techniques but remains challenging due to the fragility and rarity of CTCs. Toner et al. [84] also described a method that combined cryopreservation with a targeted strategy against cooling-induced platelet activation to achieve long-term preservation of CTC blood samples, and the same effect was achieved by establishing a zwitterionic magnetic microgel platform [85]. These findings are of great significance for the clinical development of CTC humoral biopsies.

In summary, despite the increasing number of studies reporting on CTC capture, there is still a long way to go to solve practical clinical problems. As blood cells are usually plastic, multi-step screening may be required to ensure accurate results, depending on the cell size or morphology for CTC capture. For membrane surface antigens, finding proteins that are expressed in all tumors and are specific to non-tumor cells is nearly impossible. However, there may be an opportunity to achieve a breakthrough by exploring CTC origin and identifying CTC-associated specific gene expression, such as the EMT-associated protein [86,87].

Platelet-associated CTC treatment

Targeting CTC is believed to be effective in inhibiting tumor recurrence and metastasis. For example, immunotherapeutic fibrin gels can “wake up” the innate immune system, thereby inhibiting the potential for local recurrence and metastasis after melanoma surgery [88]. The interaction of CTCs with platelets and other immune cells in the blood is thought to contribute to the immune escape and spread of CTCs, which greatly increases the difficulty in clearing CTCs. A metastatic complex composed of CTC clusters, platelets, and neutrophils, known as circulating tumor microemboli (CTM), is highly upregulated by hypoxia-inducing factor-1 α, and hypoxia is also thought to be an important factor promoting the colonization of CTM in the lung [89]. Therefore, the types of CTC present in the blood and the effects of different blood components on CTCs need to be explored.

Antiplatelet to inhibit CTCs

Platelet-CTC interactions promote malignant tumor progression by protecting CTCs from shear stress and immunity, helping to retain CTCs in capillary beds, allowing CTCs to successfully exit the blood and enter tissues, inducing EMT, and assisting in the establishment of metastatic sites. Platelet-mimicking modified submicron human serum albumin particles have been used to track metastatic cancers [90]. Weilbaecher et al. [91] conducted a randomized Phase II study to determine whether clopidogrel and aspirin disrupted platelet function and reduced the number of CTCs in patients with metastatic breast cancer. Baseline CTCs were lower than expected, reducing the ability to detect the effect of platelet inhibition on CTCs [91]. Therefore, antiplatelet therapy, which blocks platelet activation and aggregation, inhibits metastasis and is associated with cancer prevention. Hydroxyethyl starch (HES) inhibits platelet function, and HES 200/0.5 significantly reduces CTCs in patients undergoing radical colorectal cancer surgery and to reduce the metastatic potential of platelet-activated colon cell lines by inhibiting platelet activation [92]. The modulation of platelet activity may be a new strategy for reducing the risk of surgical metastasis. Jia et al. [93] found that multifunctional S-nitrosocaptopril (CapNO) acts on both CTCs and platelets, blocking platelet-CTC interactions and endothelial adhesion, thereby inhibiting CTC pulmonary metastasis in vivo. Furthermore, CapNO affects vasodilation, anticoagulation, the inhibition of MMP2 expression in tumor cells, and the inhibition of vascular endothelial cell adhesion molecule expression [93]. These new findings provide a basis for the use of CapNO in cancer metastatic chemoprophylaxis and suggest that modulating the CTC microenvironment is a new way to prevent cancer metastasis. Biological nanomaterials designed for platelets have the same effects as anti-CTC drugs. Lys-leu-val-ph-phe peptide motifs target tumors via hyaluronic acid-functionalized liposomes and spontaneously self-assemble to form nanofibers with a mesh structure that wraps around the tumor cells [94]. The tumor cell nanofibril coating significantly blocked tumor cell-induced platelet aggregation in vitro and prevented platelet adhesion around CTCs in vivo, thereby limiting platelet prometastasis and preventing early metastasis [94]. Blocking the physiological functions of platelets effectively inhibited CTC production and survival. Platelets play a critical role in circulation, in addition to promoting CTCs. Therefore, therapeutic approaches targeting platelet-CTC interactions must be explored.

Platelet derivatives treat CTCs

The production and targeted delivery of specific drugs based on platelet characteristics can neutralize CTCs to reduce tumor metastasis, which can be likened to a “Trojan horse” strategy. Platelets genetically modified to express surface-bound tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), a cytokine that specifically induces apoptosis in tumor cells, kills cancer cells and significantly reduces metastasis in mouse models of prostate cancer [95]. King et al. [96] developed a novel platelet-mediated TRAIL delivery system that targets CTCs and blocks metastasis via “in situ” platelet modification. The system killed over 60% of circulating CTCs from a variety of primary metastatic cancer samples [96]. Platelets protect CTCs from immune clearance and support CTC extravasation to secondary sites in circulation. When deprived, platelet baiting prevents the formation of metastatic tumors. Therefore, platelet membranes have been widely used to develop bionanomaterials that inhibit CTCs. For example, nanoplates have been developed by fusing platelets, lipid membranes, and platelet membrane-based bionic drug delivery systems [97,98]. Platelet membrane-related nanosystems can effectively bind to CTCs, improve precise drug delivery to tumors, reduce CTC survival, and inhibit tumor metastasis. Therefore, platelet-derivated CTC-targeted therapeutic systems represent a new approach to inhibit tumor growth and metastasis.

In summary, according to the mechanism of the platelet-CTC interaction, there have been many relevant studies to develop relevant therapeutic means to kill CTCs in circulation and inhibit tumor metastasis (Table 3). However, a long distance exists between antiplatelet drugs and platelet-derived biological nanomaterials. Therefore, there is a long way to go to explore the mechanism of platelet-CTC interactions and to develop effective diagnostic and therapeutic technologies.

Table 3.

Platelet-associated circulating tumor cell (CTC) capture and treatment techniques

Rationales	Techniques	Ref.
Platelet adhesion inhibition	Crosslinked polymer films prepared using bifunctional monomers	[81]
Heterotypic CTC capture	Fabrication of Channeled and Three-Dimensional Electrodes	[82]
	Microfluidic platform isolation of platelet-covered circulating tumor cells	[83]
Platelet-associated CTC stabilization	Combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation	[84]
	Zwitterionic microgel preservation platform	[85]
Antiplatelet to inhibit CTCs	Platelet-based and platelet-mimicking modified human serum albumin submicron particles	[90]
	Multifunctional S-nitrosocaptopril	[93]
	Lys-Leu-Val-Phe-Phe peptide motifs	[94]
Platelet derivatives treat CTCs	Platelets genetically modified to express surface-bound tumor necrosis factor-related apoptosis-inducing ligand	[95]
	Platelet mediated TNF-related apoptosis inducing ligand delivery system	[96]
	Nano-platesomes by fusing platelet membranes with lipid membranes	[97]

Discussion and perspective

Tumor metastasis is an extremely complex process. CTCs are the “seeds” of tumor metastasis that separate from the tumor in situ and enter blood circulation, but they need to survive and find the right “soil” to take root, sprout and grow. Therefore, CTCs survive and cooperate with other cells in the surrounding environment to cope with obstacles, including increased resistance to shear stress, diseases, immune attacks, and drugs. As the blood circulates, CTCs interact with other cells to affect their physiological functions [99,100], and platelets are the best partners for CTCs.

Platelets provide a good protective layer for CTCs, so that CTCs will not be monitored and cleared by the native immune system, in the same way that CTCs with armor will be protected from shear stress and survive better. Platelet adhesion can aggregate individual CTCs into clusters that generally have longer survival times and greater drug resistance. Currently, the interaction mechanism between CTCs and platelets has been extensively studies and the TGF-β1 and PDGFR signaling pathways are more widely recognized [101,102]. Although mechanistic exploration is always needed, achieving clinical transformation using the principle of mechanism is the direction we should look for next. Platelet- and CTC-related indicators are widely used in clinical studies for tumor grading and identification, therapeutic effect evaluation, and patient prognosis [103]. Although CTCs have absolute advantages and potential for use in liquid biopsy, CTC capture remains the biggest obstacle to their widespread clinical application [104]. There are only approximately 1-10 CTCs per 10 ml blood, which is insufficient to support additional testing and diagnostic techniques. Improving the capture efficiency of CTCs remains a popular research topic, and the results of flow cytometry [68], immunohistochemistry [105], and various materials science [106] studies are constantly enhancing the capture effect. Platelet-CTC interactions form a theoretical basis for the development of CTC capture technology. CTC capture technology for platelets has achieved promising results in terms of detection specificity and accuracy; however, there is still a long way to go before clinical conversion. Platelets are potential therapeutic targets and delivery platforms for CTCs. Antiplatelet drugs clear CTCs with good results; however, their side effects are unavoidable. Platelets not only act on CTCs, but also play an important role in the body’s blood system. Platelet-modified derivatives and bionanomaterials have shown good efficacy in the treatment of CTCs and are highly biocompatibility and safe [107]. Similarly, related products need to have more sophisticated designs and rigorous safety studies to achieve clinical translation.

In summary, we reviewed the principles and mechanisms of platelet-CTC interactions and listed current technologies for platelet-related CTC capture, detection, diagnosis, and treatment. Although the mechanism of platelet-CTC interaction still needs to be further explored, and the development of related technologies to achieve clinical translation is still a long way away, using platelets to understand and treat tumors provides a novel perspective. The role of platelets in tumor diagnosis and treatment will become increasingly important, and clinical applications will be more extensive in the future.

Disclosure of conflict of interest

None.