Tumor Cell-induced Platelet Aggregation Research Articles

Identification of Podoplanin Aptamers by SELEX for Protein Detection and Inhibition of Platelet Aggregation Stimulated by C-Type Lectin-like Receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet-tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (Kd) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Cancer-Associated Stroke: Thrombosis Mechanism, Diagnosis, Outcome, and Therapeutic Strategies.

Cancer can induce hypercoagulability, which may lead to stroke. This occurs when tumor cells activate platelets as part of their growth and metastasis. Tumor cells activate platelets by generating thrombin and expressing tissue factor, resulting in tumor cell-induced platelet aggregation. Histopathological studies of thrombi obtained during endovascular thrombectomy in patients with acute stroke and active cancer have shown a high proportion of platelets and thrombin. This underscores the crucial roles of platelets and thrombin in cancer-associated thrombosis. Cancer-associated stroke typically occurs in patients with active cancer and is characterized by distinctive features. These features include multiple infarctions across multiple vascular territories, markedly elevated blood D-dimer levels, and metastasis. The presence of cardiac vegetations on echocardiography is a robust indicator of cancer-associated stroke. Suspicion of cancer-associated stroke during endovascular thrombectomy arises when white thrombi are detected, particularly in patients with active cancer. Cancer-associated stroke is almost certain when histopathological examination of thrombi shows a very high platelet and a very low erythrocyte composition. Patients with cancer-associated stroke have high risks of mortality and recurrent stroke. However, limited data are available on the optimal treatment regimen for stroke prevention in these patients. Thrombosis mechanism in cancer is well understood, and distinct therapeutic targets involving thrombin and platelets have been identified. Therefore, direct thrombin inhibitors and/or antiplatelet agents may effectively prevent stroke recurrence. Additionally, this strategy has potential benefits in cancer treatment as accumulating evidence suggests that aspirin use reduces cancer progression, metastasis, and cancer-related mortality. However, clinical trials are necessary to assess the efficacy of this strategy involving the use of direct thrombin inhibitors and/or antiplatelet therapies.

Discovery of 5-Hydroxy-1,4-naphthoquinone (Juglone) Derivatives as Dual Effective Agents Targeting Platelet-Cancer Interplay through Protein Disulfide Isomerase Inhibition.

In this study, a series of 2- and/or 3-substituted juglone derivatives were designed and synthesized. Among them, 9, 18, 22, 30, and 31 showed stronger inhibition activity against cell surface PDI or recombinant PDI and higher inhibitory effects on U46619- and/or collagen-induced platelet aggregation than juglone. The glycosylated derivatives 18 and 22 showed improved selectivity for inhibiting the proliferation of multiple myeloma RPMI 8226 cells, and the IC50 values reached 61 and 48 nM, respectively, in a 72 h cell viability test. In addition, 18 and 22 were able to prevent tumor cell-induced platelet aggregation and platelet-enhanced tumor cell proliferation. The molecular docking showed the amino acid residues Gln243, Phe440, and Leu443 are important for the compound-protein interaction. Our results reveal the potential of juglone derivatives to serve as novel antiplatelet and anticancer dual agents, which are available to interrupt platelet-cancer interplay through covalent binding to PDI catalytic active site.

Design, Synthesis, and Antitumor Evaluation of an Opioid Growth Factor Bioconjugate Targeting Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge with high lethality and limited effective drug treatments. Its heightened metastatic potential further complicates the prognosis. Owing to the significant toxicity of current chemotherapeutics, compounds like [Met5]-enkephalin, known as opioid growth factor (OGF), have emerged in oncology clinical trials. OGF, an endogenous peptide interacting with the OGF receptor (OGFr), plays a crucial role in inhibiting cell proliferation across various cancer types. This in vitro study explores the potential anticancer efficacy of a newly synthesized OGF bioconjugate in synergy with the classic chemotherapeutic agent, gemcitabine (OGF-Gem). The study delves into assessing the impact of the OGF-Gem conjugate on cell proliferation inhibition, cell cycle regulation, the induction of cellular senescence, and apoptosis. Furthermore, the antimetastatic potential of the OGF-Gem conjugate was demonstrated through evaluations using blood platelets and AsPC-1 cells with a light aggregometer. In summary, this article demonstrates the cytotoxic impact of the innovative OGF-Gem conjugate on pancreatic cancer cells in both 2D and 3D models. We highlight the potential of both the OGF-Gem conjugate and OGF alone in effectively inhibiting the ex vivo pancreatic tumor cell-induced platelet aggregation (TCIPA) process, a phenomenon not observed with Gem alone. Furthermore, the confirmed hemocompatibility of OGF-Gem with platelets reinforces its promising potential. We anticipate that this conjugation strategy will open avenues for the development of potent anticancer agents.

A change of rhodocytin's suprastructure turns the agonist into an antagonist of tumor cell induced platelet aggregation.

Cancer cells circulating in the blood attach to platelets by direct cell-cell interactions via several receptor-counterreceptor contacts and indirectly by fibrin bridges which connect the two cell types by distinct integrin receptors. In the microenvironment of these tumor cell platelet aggregates (TCPAs), the tumor cells are shielded from the shear stress of the blood flow and from attack by the immune system. This supports hematogenous metastasis and tumor cell induced thrombosis. Platelet activation is triggered by binding of podoplanin on cancer cells to the platelet receptor Clec-2. Therefore, we hypothesize that targeting this initial step will prevent the entire cascade leading to the formation of TCPAs. Rhodocytin, a heterodimeric (αβ)2 C-type lectin from the Malayan pit viper Calloselasma rhodostoma, binds to Clec-2 and thereby induces TCPA formation. Remarkably, mutations in rhodocytin that disturbed formation of oligomers, blocked the podoplanin-Clec-2 axis and prevented platelet activation. Therefore, we used lysine reactive chemicals to modify rhodocytin isolated from the crude snake venom. Blue native gel electrophoresis and far western blotting showed a change of rhodocytin's suprastructure triggered by acetylation and PEGylation. Mass spectrometry analysis of altered lysines suggested that their modifications interfered with the formation of rhodocytin tetramers. When tested in assays for tumor cell induced platelet aggregation, we found that derivatization turned rhodocytin from an agonist into an antagonist. This observation indicates that Clec-2 is a valid target receptor molecule to curb TCPA formation and to prevent hematogenous metastasis and tumor cell induced thrombosis in cancer patients.

Tumor cell-induced platelet aggregation accelerates hematogenous metastasis of malignant melanoma by triggering macrophage recruitment

BackgroundTumor cell-induced platelet aggregation (TCIPA) is not only a recognized mechanism for paraneoplastic thrombocytosis but also a potential breakthrough alternative for a low response to immune checkpoint inhibitors (ICIs) in hematogenous metastasis of malignant melanoma (MM). However, there is no TCIPA-specific model for further investigation of the relationship among TCIPA, the tumor immune microenvironment (TIME), and metastasis.MethodsWe developed a TCIPA metastatic melanoma model with advanced hematogenous metastasis and enhanced TCIPA characteristics. We also investigated the pathway for TCIPA in the TIME.ResultsWe found that TCIPA triggers the recruitment of tumor-associated macrophages (TAMs) to lung metastases by secreting B16 cell-educated platelet-derived chemokines such as CCL2, SDF-1, and IL-1β. Larger quantities of TAMs in the TCIPA model were polarized to the M2 type by B16 cell reprocessing, and their surface programmed cell death 1 ligand 1 (PD-L1) expression was upregulated, ultimately assisting B16 cells in escaping host immunity and accelerating MM hematogenous metastasis.ConclusionsTCIPA accelerates MM lung metastasis via tumor-educated platelets (TEPs), triggering TAM recruitment, promoting TAM polarization (M2), and remodeling the suppressive TIME in lung metastases.

Effect of factor XI inhibition on tumor cell-induced coagulation activation

BackgroundCancer-associated thrombosis is a frequent complication in patients with malignancies. While factor XI (FXI)/FXIa inhibition is efficacious in preventing postoperative venous thromboembolism, its role in tumor cell-induced coagulation is less defined. ObjectivesWe thus aimed to provide mechanistic insights into FXI/FXIa inhibition in tumor cell-induced coagulation activation. MethodsProcoagulant activity (PCA) of 4 different tissue factor (TF) expressing tumor cell lines was analyzed by single-stage clotting and thrombin generation assay in the presence of a FXIa inhibitor, BMS-262084 (BMS), an inhibitory FXI antibody (anti-FXI), or peak and trough concentrations of rivaroxaban or tinzaparin. Further, tumor cell-induced platelet aggregation was recorded. Recombinant human TF served as positive control. ResultsAlthough BMS and anti-FXI potently inhibited FXIa amidolytic activity, both inhibitors efficiently mitigated recombinant human TF- and tumor cell-induced fibrin clot formation and platelet aggregation only in the presence of low TF PCA. The anticoagulant effects showed an inverse correlation with the magnitude of cellular TF PCA expression. Similarly, BMS markedly interfered with tumor cell-induced thrombin generation, with the most prominent effects on peak and total thrombin. In addition, anticoagulant effects of FXIa inhibition by 10 μM BMS were in a similar range to those obtained by 600 nM rivaroxaban and 1.6 μM tinzaparin at low TF PCA levels. However, rivaroxaban and tinzaparin also exerted marked anticoagulant activity at high TF PCA levels. ConclusionOur findings indicate that FXI/FXIa inhibition interferes with tumor cell-induced coagulation activation only at low TF PCA expression levels, a finding with potential implications for future in vivo studies.

New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

A Supramolecular Nitric Oxide Nanodelivery System for Prevention of Tumor Metastasis by Inhibiting Platelet Activation and Aggregation.

Tumor cell-induced platelet aggregation (TCIPA) is known as a critical step in hematogenous tumor metastasis. The endogenous nitric oxide (NO) plays an important role in anticoagulation, which might have great potential to inhibit TCIPA. Herein, a glutathione-sensitive supramolecular nanocarrier is prepared via host-guest interaction for effective delivery of NO and chemotherapeutic agent gemcitabine (GEM). NO could be effectively released in tumor cells and inhibits platelet activation and aggregation. The inhibition of TCIPA by NO could effectively attenuate the migration and invasion of tumor cells in vitro. Furthermore, the in vivo experiments demonstrate that the NO and GEM co-delivered supramolecular nanocarriers can suppress the growth of primary tumor. More importantly, although NO-containing nanocarriers cannot inhibit the growth of primary tumors effectively, they can significantly inhibit tumor metastasis. This NO-based nano-delivery system not only provides new inspiration for multifunctional applications of NO in cancer therapy but also shows great potential in clinical antimetastatic applications.

Inhibition of Metastatic Cancer in Mice via Atopaxar: A Research Protocol

Introduction: When cancer becomes metastatic, tumour cells intravasate out of the primary tumour and spread to other organs, causing about 90% of cancer deaths. One way circulating tumour cells (CTCs) metastasize is by interacting with platelets, resulting in tumour cell-induced platelet aggregation (TCIPA) that shields CTCs from immune attack. Previous studies suggest that tumour cells promote metastasis and induce TCIPA by activating protease-activated receptor-1 (PAR-1) on platelets. Therefore, this study aims to investigate whether administering Atopaxar, a PAR-1 antagonist that has not yet been studied in cancer as other PAR-1 antagonists have, can limit metastasis in mouse models. Methods: We will assess the effectiveness of Atopaxar and a placebo (or control) on adult C57BL mice inoculated with GFP-transfected Lewis lung carcinoma cells. Flow cytometry of blood samples taken 7, 14, and 21-days post-inoculation will be performed to quantify the number of GFP+ cells and activated CD8+ (cytotoxic) T cells in the samples. Results: We expect that the Atopaxar treated mice will have reduced numbers of CTCs and higher numbers of cytotoxic T cells, suggesting that the inhibition of TCIPA via Atopaxar will correlate with reduced shielding of CTCs and metastasis rates. Discussion: These results could provide novel insight into the use of PAR-1 antagonists in confining cancer to its primary site in patients and inhibiting CTCs’ function as a seed for metastases. Conclusion: Since CTCs will usually be present in the blood even after removal of a secondary tumour, limiting metastasis can significantly improve the prognosis and wellbeing of patients.

Proteomics profiling identifies extracellular vesicles’ cargo associated with tumour cell induced platelet aggregation

BackgroundCancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells’ EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard.MethodsEVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach.ResultsBoth TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs’ cargo identified candidate proteins (including uPAR and PDGFRβ) that may be involved during this process.ConclusionsTNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.

Tumor Cell-Induced Platelet Aggregation as an Emerging Therapeutic Target for Cancer Therapy.

Tumor cells have the ability to induce platelet activation and aggregation. This has been documented to be involved in tumor progression in several types of cancers, such as lung, colon, breast, pancreatic, ovarian, and brain. During the process, platelets protect circulating tumor cells from the deleterious effects of shear forces, shield tumor cells from the immune system, and provide growth factors, facilitating metastatic spread and tumor growth at the original site as well as at the site of metastasis. Herein, we present a wider view on the induction of platelet aggregation by specific factors primarily developed by cancer, including coagulation factors, adhesion receptors, growth factors, cysteine proteases, matrix metalloproteinases, glycoproteins, soluble mediators, and selectins. These factors may be presented on the surface of tumor cells as well as in their microenvironment, and some may trigger more than just one simple receptor–ligand mechanism. For a better understanding, we briefly discuss the physiological role of the factors in the platelet activation process, and subsequently, we provide scientific evidence and discuss their potential role in the progression of specific cancers. Targeting tumor cell-induced platelet aggregation (TCIPA) by antiplatelet drugs may open ways to develop new treatment modalities. On the one hand, it may affect patients’ prognosis by enhancing known therapies in advanced-stage tumors. On the other hand, the use of drugs that are mostly easily accessible and widely used in general practice may be an opportunity to propose an unparalleled antitumor prophylaxis. In this review, we present the recent discoveries of mechanisms by which cancer cells activate platelets, and discuss new platelet-targeted therapeutic strategies.

Effect of Anti-Podoplanin on Malignant Glioma Cell Viability, Invasion and Tumor Cell-Induced Platelet Aggregation

The transmembrane receptor podoplanin (PDPN) is a platelet aggregation-inducing factor, which is widely expressed in various malignant tumors such as squamous cell carcinomas, mesotheliomas, glioblastomas. Podoplanin regulates a pathway leading to cell invasion and migration. Glioblastoma multiforme (GBM) is the most aggressive and invasive tumor of the central nervous system. A high level of PDPN expression has been reported to be associated with reduced survival, cancer aggression and migration. Aim of study to determine the effect of anti-podoplanin antibody on cell-platelet aggregation, cell invasion and viability in Uppsala 87 malignant glioma (U87MG) cell lines. The expression of podoplanin on U87MG cells was measured using flowcytometry. The dimethyl-thiazol diphenyl-tetrazolium bromide (MTT) assay was used to measure U87MG cell proliferation after treatment by anti-podoplanin monoclonal antibody (NZ-1.3). The invasion of cancer cells was assessed by using a novel microfluidic based assay. The results show reduction of cell viability and cell migration after treatment with anti-podoplanin antibody. After co- treatment of U87MG cells and platelets with and without anti-podoplanin antibody, the cell-platelet aggregation was significantly reduced in anti-podoplanin treated cell. Podoplanin is involved in aggregation of gliobastoma cells, and their viability and invasion and its neutralizing antibody can inhibit this process. So, blocking of podoplanin may be representing a promising therapeutic approach to glioblastoma multiform cancer therapy.

Curcumin-Based Inhibitors of Thrombosis and Cancer Metastasis Promoting Factor CLEC 2 from Traditional Medicinal Species Curcuma longa.

The CLEC-2 receptor protein belongs to the C-type lectin superfamily of transmembrane receptors that have one or more C-type lectin-like domains. CLEC-2 is a physiological binding receptor of podoplanin (PDPN), which is expressed on specific tumour cell types and involved in tumour cell-induced platelet aggregation and tumour metastasis. CLEC-2 and podoplanin-expressing tumour cells interact to increase angiogenesis, tumour development, and metastasis. CLEC-2 is a hemi-immunoreceptor tyrosine-based activation motif (hemi-ITAM) receptor located on platelets and a subset of dendritic cells that are expressed constitutively. This molecule is secreted by activated platelets around tumours and has been shown to inhibit platelet aggregation and tumour metastasis in colon carcinoma by binding to the surface of tumour cells. Pharmacokinetic studies were carried using a DrugLiTo, and molecular docking was performed using AutoDock Tools 1.5.6 (ADT). Twenty-nine bioactive compounds were included in the study, and four of them, namely, piperine, dihydrocurcumin, bisdemethoxycurcumin, and demothoxycurcumin, showed potential antagonist properties against the target. The resultant best bioactive was compared with commercially available standard drugs. Further, validation of respective compounds with an intensive molecular dynamics simulation was performed using Schrödinger software. To the best of our knowledge, this is the first report on major bioactive found on clove as natural antagonists for CLEC-2 computationally. To further validate the bioactive and delimit the screening process of potential drugs against CLEC-2, in vitro and in vivo studies are needed to prove their efficacy.

Preferential interaction of platelets with prostate cancer cells with stem cell markers

BackgroundProstate cancer (PCa) may be initiated by CD133+/CD44+ expressing stem cell-like cells (PCSC), which are also thought to drive metastasis. Platelets also contribute to metastasis via tumor cell-induced platelet aggregation (TCIPA), which in part enhances cancer cell invasion. Moreover, activated platelets secrete stromal derived growth factor-1α (SDF-1α) that can mobilize CSCs via the CXCR4 receptor. However, the potential reciprocal interactions between CSCs and platelets have not been investigated. ObjectiveTo characterize the mechanisms behind PCSC-platelet interaction. MethodsFluorescence Activated Cell Sorting was utilized to separate DU145 and PC3 PCa cells into CD133+/CD44+, CD133+/CD44-, CD44+/CD133-, and CD133-/CD44- subpopulations and to measure their CXCR4 surface expression. PCa subpopulation TCIPA experiments were performed using aggregometry and immunoblot was used to measure prothrombin. Platelet SDF-1α secretion was measured by ELISA. Modified-Boyden chamber assays were used to assess the role of SDF-1α:CXCR4 pathway in platelet-PCSC interactions. ResultsDU145 and PC3 expressing both CD133 and CD44 stem cell markers accounted for only small fractions of total cells (DU145: CD133+/CD44+ 3.44 ± 1.45% vs. CD133+/CD44- 1.56 ± 0.45% vs. CD44+/CD133- 68.19 ± 6.25% vs. CD133-/CD44- 20.36 ± 4.51%). However, CD133+ subpopulations induced the greatest amount of aggregation compared to CD44+/CD133- and double-negative DU145, and this aggregation potency of CD133+ PCa cells corresponded with high levels of prothrombin expression. Additionally, CD133+ subpopulations expressed significantly higher level of CXCR4 compared to CD133-/CD44- and CD44+/CD133-. Disruption of SDF-1α:CXCR4 pathway reduced platelet-induced PCSC invasion. ConclusionsCD133+/CD44+ and CD133+/CD44- PCSCs have highest platelet aggregation potency, which could be attributed to their increased prothrombin expression. Reciprocally, platelet-derived SDF-1α stimulates PCSC invasion.

Detection of Tumor Cell-Induced Platelet Aggregation and Granule Secretion.

Hematogenous metastatic spread of cancer is strongly dependent on and triggered by an intensive interplay of tumor cells with platelets. Immediately after entering the blood vascular system, tumor cells are surrounded by a platelet cloak, which protects them physically from shear stress and from attacks by the immune surveillance. Furthermore, tumor cell binding activates platelets, which in turn release growth factors and chemokines to recruit myeloid cells into the platelet/tumor cell microemboli, eventually create a permissive microenvironment in the early metastatic niche. Although the molecular mechanisms of tumor cells to activate platelets appear versatile being a matter of further research, interference with platelet activation turns out to be an attractive target to efficiently inhibit tumor metastasis. Some experimental assays are generally recognized to follow tumor cell-induced platelet activation (TCIPA), which provide an insight into the molecular mechanisms of TCIPA and allow searching for potential inhibitors. In this chapter, we describe the two most prominent experimental assays to follow TCIPA, namely platelet aggregation and platelet granule secretion, experimentally realized by dense granules´ ATP quantification. Although light transmission aggregometry and ATP detection from dense granule secretion are two age-old techniques, they are still highly relevant to provide reliable information concerning platelet activation status since all tumor cell-derived molecular triggers are covered and monitored in the experimental outcome.

A mechanistic and empirical review of antcins, a new class of phytosterols of formosan fungi origin

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients. © 2019

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Taiwan. Many risks factors induce liver chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Mulberry fruits containing polyphenols to remove free radicals and mitigate inflammation has been reported to not only against gastric cancer, melanoma and leukemia but also prevent liver injury induced by alcohol or CCl4 in previous researches. The aim of this study is to examine whether Mulberry could inhibit hepatocarcinogenesis. In animal experiment, diethylnitrosamine (DEN) was used to induce hepatic tumorgenesis. After injecting DEN, the rats treated with mulberry water extracts (MWE) had less and smaller tumor than others without MWE. Moreover, MWE reduced the serum ALT and AST, HCC marker, cleavage caspases, Ser-15-p53 and Ser46-p53 induced by DEN. Further, we observed that mulberry polyphenol extracts (MPE) inhibited the cell growth of HepG2 cell and Hep3B cell. By using flow cytometry and western blotting methods, MPE induced HepG2 cell apoptosis by increase subG1 cells and the elevated expression of caspase-3/8/9. Instead of apoptosis, MPE caused Hep3B cells autophagy by inhibiting Akt and mTOR phosphorylation. Comprehensively, mulberry extracts has a potential to be a health supplement to prevent hepatocarcinogenesis in the future. © 2019

Extract of Caulis Spatholobi, a novel platelet inhibitor,efficiently suppresses metastasis of colorectal cancer by targeting tumor cell-induced platelet aggregation

Tumor cell-induced platelet aggregation (TCIPA) is the core mechanism potentiating high viability for circulatory tumor cells,which is the rate-limiting factor for metastasis.Additionally,as supported by the successful application of aspirin,the pro-malignant effects during tumor-platelets interaction can be largely neutralized by pharmacological deactivation of platelets.Caulis Spatholobi is widely used as an anti-coagulation herb in traditional Chinese medicine,indicating its potential against TCIPA.In our study,three fractions of Caulis Spatholobi extracts were firstly prepared.In colorectal cancer(CRC) model,the anti-metastatic potential was evaluated both in vitro and in vivo followed by the detection of their platlet regulatory effects.Results showed that all three extracts significantly suppressed the invasion and metastasis of CRC.Mechanistically,by blocking platelet-derived PDGF-B releasing,they reversed the enhanced epithelial mesenchymal transition during MC38-platelets interation.Further,ethyl acetate fraction shows the most promising efficacy for the future application in treatment.Overall,our study have for the first time proved CaulisSpatholobi extracts,especially the ethyl acetate fraction,as a potent TCIPA inhibitor during metastatic progression,which provided a novel candidate for pharmacologically blockage of metastasis in CRC.

1970P - Aspirin and ticagrelor for the prevention of tumour cell induced platelet aggregation

BackgroundTumour cell induced platelet aggregation (TCIPA) increases the metastatic potential of cancer. Mechanisms include protection of tumour cells from immune destruction, interaction of platelet receptors with tumour ligands to facilitate adhesion, enrichment of the tumour microenvironment to promote tumour cell extravasation and proliferation. Reducing these interactions using antiplatelet agents could reduce metastatic progression. This study investigated the effect of Ticagrelor and aspirin on TCIPA in metastatic breast and colorectal cancer patients, compared to healthy controls. MethodsParticipants in this randomised, crossover study received aspirin or Ticagrelor for 2 weeks, followed by 2 weeks washout and crossover to the other monotherapy, before completing 2 weeks of dual therapy. Platelet rich plasma was prepared from blood samples at each time point. Light transmission aggregometry measured spontaneous aggregation of the platelets over 30minutes without the addition of exogenous agonists. Flow cytometry measured the activation markers P-selectin and fibrinogen binding on unstimulated platelets. Results20 healthy, 10 breast and 6 colorectal cancer participants completed the study. Untreated platelets from colorectal patients had higher spontaneous aggregation (14.8±2.6%) than breast cancer (8.7±1%) or healthy platelets (8.1±0.9% p=0.007). This was reduced by Ticagrelor (7.7±3.3% p=0.01). Untreated platelets from patients with colorectal cancer had higher expression of P-selectin compared to platelets from healthy donors (14.2±1.3% vs 21.5±3.6% p=0.03). Untreated platelets from breast patients had increased fibrinogen binding (49.5±7.8%) compared to healthy platelets (31.4±4.2% p=0.03). This was reduced by Ticagrelor (26.7±5.2% p=0.04) and dual therapy (31.4±6.9% p=0.01). ConclusionsThis study demonstrated that platelets from breast and colorectal cancer patients are hyperactive compared to healthy donors. Ticagrelor reduces aggregation in platelets from patients with colorectal cancer, and as monotherapy and dual therapy can reduce platelet activation in patients with breast cancer. The pilot study indicates Ticagrelor may reduce TCIPA in cancer patients and could be used to decrease metastatic spread. Clinical trial identificationEudraCT: 2014‐004049‐29, Start date:10‐03‐2015. Legal entity responsible for the studyUniversity of Leicester. FundingAstraZeneca. DisclosureA. Thomas: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Pierre Fabre; Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen. A. Goodall: Research grant / Funding (institution): Hoffmann La Roche; Research grant / Funding (institution): AstraZeneca. D. Adlam: Research grant / Funding (institution): Abbott Vascular Inc; Research grant / Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.