The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways.

An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets' ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.

Unfractionated and Low Molecular Weight Heparin Reduce Platelet Induced Epithelial-Mesenchymal Transition in Pancreatic and Prostate Cancer Cells.

The interaction with platelets is of crucial importance for tumor cells passing through hematogenous metastasis. Platelets protect cancer cells from immune surveillance and exhibit many other prometastatic effects. Notably, platelets can change the epithelial tumor phenotype, a process termed epithelial-mesenchymal transition (EMT), which confers stem cell-like properties onto tumor cells associated with an increased motility and drug resistance. The aim of the study is to investigate the impact of heparin on the platelet induced EMT program in pancreatic and prostate tumor cells. Platelet activation and interaction with cancer cells were determined by static adhesion assays. Applying ELISAs, the platelet release of EMT inducing mediators was quantified. EMT marker protein expression by tumor cells was explored by western blot and qPCR. Our data show that different tumor cell entities have different platelet binding capacities and also that a weak interaction is sufficient to change tumor cell phenotype. Additionally, unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) reduced tumor cell platelet interaction. Subsequently, attenuated platelet-derived mediator release resulted in reduced EMT marker protein and transcription factor expression by the cancer cells and decreased cell migration. These data suggest that heparin reduces platelet induced EMT program and prevents the formation of cancer cells with stem cell-like properties. This additional mechanism argues for the use of heparin in oncological applications.

The mechanisms how heparin affects the tumor cell induced VEGF and chemokine release from platelets to attenuate the early metastatic niche formation.

Metastasis is responsible for the majority of cancer associated fatalities. Tumor cells leaving the primary tumor and entering the blood flow immediately interact with platelets. Activated platelets contribute in different ways to cancer cell survival and proliferation, e.g. in formation of the early metastatic niche by release of different growth factors and chemokines. Here we show that a direct interaction between platelets and MV3 melanoma or MCF7 breast cancer cells induces platelet activation and a VEGF release in citrated plasma that cannot be further elevated by the coagulation cascade and generated thrombin. In contrast, the release of platelet-derived chemokines CXCL5 and CXCL7 depends on both, a thrombin-mediated platelet activation and a direct interaction between tumor cells and platelets. Preincubation of platelets with therapeutic concentrations of unfractionated heparin reduces the tumor cell initiated VEGF release from platelets. In contrast, tumor cell induced CXCL5 and CXCL7 release from platelets was not impacted by heparin pretreatment in citrated plasma. In defibrinated, recalcified plasma, on the contrary, heparin is able to reduce CXCL5 and CXCL7 release from platelets by thrombin inhibition. Our data indicate that different chemokines and growth factors in diverse platelet granules are released in tightly regulated processes by various trigger mechanisms. We show for the first time that heparin is able to reduce the mediator release induced by different tumor cells both in a contact and coagulation dependent manner.