Contribution of Glycosaminoglycan binding in CCL21-mediated Migration of Breast Cancer cells

Abstract

Lymph node metastasis constitutes a key event in Breast Cancer progression and it is a process at least partially mediated by the chemokine CCR7. Chemokine function is dependent upon their binding to both cell-surface glycosaminoglycans (GAGs) molecules and to their specific receptors; thus, the role of GAGs in CCR7-mediated lymph node metastasis was investigated by creating a non-GAG binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ98-134) was synthesized, at 50nM it had similar potential to mobilize intracellular calcium compared to wild-type CCL21 (WT-CCL21). Next, a series of experiments was performed to determine how deletion of the GAG-binding site altered the ability of CCL21 to stimulate chemotaxis within a concentration gradient generated by free solute diffusion. Both WT-CCL21 and mut-CCL21 had a similar potential to stimulate chemotactic migration of PBMC (P>0.05). However, 4T1-Luc cells exhibit reduced migration at 30 & 50nM (p<0.001). Interestingly, this effect was greatly exacerbated in trans-endothelial migration, with the mut-CCL21 failing to increase cell migration above the background level at 30 nM in PBMC and 4T1-Luc cells (p> 0.001 vs WT). This difference could potentially be attributed to reduced GAG binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind heparan sulphate compared to the WT-CCL21. Finally, a murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20µg mut-CCL21 once the tumor was visible. Mice were imaged weekly with IVIS to assess bioluminescence and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p=0.0148), suggesting the potential to target chemokine binding to GAGs as a therapeutic option.