Commercial heparins and synthetic derivatives as potential immunomodulators in an in vitro metastatic context

Thromboses are considered a frequently underestimated risk and reason for mortality in metastatic tumor diseases, which at least partially are based on activation of platelets by cancer cells. Low molecular weight heparin (LMWH) is a guideline-based anticoagulant in thromboprophylaxis and treatment of cancer patients. The ongoing debate whether heparin can act anti-tumorigenic beyond anticoagulation is further complicated by the heterogeneous structure of heparin as a natural product. To tackle this issue and allow S/A/R, synthetic non-glycosidic heparin mimetics have been introduced in the form of copolymers of sodium 4-styrenesulfonate and itaconic acid (Poly(SS-co-IA)) at molecular masses between 10 and 20 kDa. Comparing these mimetics with commercial heparins, this project is focused on platelet-tumor cell interaction and platelet-induced immune deregulation, which has not been addressed yet. The research question is how tumor-activated platelets induce immune-suppressive activities and whether heparin derivatives can reconstitute the immune balance investigating CD4⁺ and CD8⁺ cells under in vitro conditions. The project addresses cytokines and proteins that have been shown to have immunosuppressive activities when released in higher quantities from activated platelets, whereupon TGF-β, PF-4 and MMP2 were selected. In the MMP2 activity assay, synthetic heparin mimetics (250 µg/mL) displayed superiority in the inhibition of this enzyme, while commercial heparins at adopted therapeutic concentrations did not affect the activity of MMP2. When investigating the impact of TGF-β on the differentiation of CD4⁺ cells into immunosuppressive regulatory T-cells via flow cytometry, copolymers with a lower molecular mass attenuated the differentiation rate, suggesting an immunoactivating effect. Fondaparinux, the smallest commercial heparin, confirmed this result. Using a SAW biosensor, affinity data for heparin or mimetics binding to TGF-β largely confirmed these findings. In addition, the synthetic mimetics showed a significantly lower binding affinity to PF-4 compared to UFH, interesting with regard to a lower HIT II susceptability. In summary, LMWH and heparin mimetics can reverse the immunosuppressive activities of platelets and thus provide a novel immuno-oncological perspective for their application in oncology.