The chemokine CXCL12 (SDF-1α) has been implicated in directing the characteristic metastatic spread of breast cancer. It is well known that chemokines also promote specific leukocyte extravasation at sites of inflammation. A series of studies suggest that this latter process is only driven by chemokines which have been immobilized onto the apical surface of endothelium following binding to the heparan sulphate (HSGAG) component of proteoglycans. As such chemokines can be competitively displaced by soluble heparin, a series of experiments was performed to determine the potential of a range of heparin molecules to inhibit CXCL12-dependant migration of breast cancer cells. Firstly, HSGAG-expressing K1-Chinese Hamster Ovary cells were transfected with the CXCR4 receptor (K1-CXCR4) for CXCL12. Flow cytometric analysis demonstrated high CXCR4 expression and intracellular calcium mobilization in response to physiological levels of CXCL12. Intracellular calcium flux due to binding of CXCL12 to its receptor was inhibited with the addition of heparin to the cells (p<0.05). Chemotaxis experiments were then performed in 8μm transwell systems towards CXCL12. K1-CXCR4 showed significant levels of migration towards CXCL12 (p<0.05), demonstrating that they respond functionally to chemokine. The addition of heparin to the wells prevented chemotaxis (p<0.01), as did the low molecular weight heparins tinzaparin and dalteparin, an effect that was abrogated with the addition of protamine. Secondly, we performed radioligand binding assays using K1-CXCR4 and I125CXCL12. We demonstrated that CXCL12 binds to its receptor and that heparins (including tinzaparin, dalteparin and a non-anticoagulant low molecular weight heparin) competitively inhibited the binding of chemokine to its receptor in a dose dependant manner (p<0.01 for all heparinoids). Competitive inhibition was observed at physiological doses of heparinoid. Finally, we have investigated the effects of heparins upon an in vitro model of breast cancer. Results using a CXCR4 expressing MDA-MB-231 breast cancer cell line show that these cells migrate in response to CXCL12. Importantly, our assays provide evidence that heparin completely inhibits chemotaxis of these cancer cells (p<0.01). We have optimized a mouse model of breast cancer metastasis in CB-17 SCID mice and are currently investigating the in vivo effects of low molecular weight heparins upon breast cancer metastasis. In summary, we have shown that heparins can be used to competitively inhibit the binding of CXCL12 to its receptor and its subsequent activation. We have also demonstrated that heparin can functionally inhibit the migration of CXCR4 expressing transfectant and breast cancer cells. Heparin represents a novel method of preventing cancer metastasis and may be useful around the time of surgery when circulating cancer cell numbers are known to increase.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]