Quantification of the antimetastatic effects of heparins by Single-cell force spectroscopy
H. Schillers, A. Schmidt, S. Bobe, N. Martins-Castanheira (1Münster, Germany)
Time: 17:15 - 18:15
Objective: It is well known that platelets support cancer cells in nearly every step of metastasis. Heparin exhibits antimetastatic effects as shown in animal models and clinical studies. Unfractionated heparin consists of a broad range of polysaccharides with varying weights (3-30kDa) whereas low molecular weight heparins (LMWH) contain shorter chain lengths (up to 6kDa). Goal of this project was to test the efficiency of unfractionated heparin and LMWH to reduce platelet-tumor cell adhesion on the single cell level and to elucidate the fate of platelets upon tumor cell adhesion.
Methods: We used human non-small cell lung cancer cells (A549) and freshly isolated platelets, forming a dense layer on collagen coated dish. Single cell force spectroscopy (SCFS) was used to obtain the max. adhesion force (FA) and the detachment work (WD) between A549 cells and activated platelets. Heparin-Na 25000 (Ratiopharm) and Tinzaparin (Innohep®, Leo Pharma) was used in concentrations between 0.001 – 100 U/mL. In another approach, A549 cells were stained with LysoTracker and incubated with FITC-CD42a-labeled platelets. Confocal laser scanning microscopy was used to visualize localization of platelet CD42a protein within A549 cells.
Results: Adhesion between platelets and A549 cells show values of 1060pN for FA and 4 fJ for WD. The maximum decrease of adhesion is induced by 10 U/mL of unfractionated heparin (FA 38%, WD 29%) and 0.01 U/mL Tinzaparin (FA 38%, WD 50%) respectively. A sigmoidal dose-response fit revealed IC50 values of 8.4 U/ml for unfractionated heparin and 0.01 U/mL for Tinzaparin. Confocal microscopy exhibited clear colocalization of the platelet specific protein CD42a in acidic compartiments of A549 cells already after 30min of incubation. Frequently, the A549 cells exhibited formation of phagocytotic protrusions and invaginations around platelets. Internalized CD42a followed a gradient of size and density originating from these invaginations.
Conclusion: We established a method to quantify the effect of heparins on platelet-A549 cell adhesion on single-cell level and demined EC50 values. Furthermore, we could visualize the uptake of platelets by A549 cells. Since platelet-tumor cell adhesion is a crucial step for metastasis, our approach represents a valuable method to investigate early steps of metastasis and to test the efficiency of substances to block platelet-tumor cell interactions.