Hyaluronan (HA) is a linear non-sulfated glycosaminoglycan present in the extracellular matrix and known to modulate cellular interactions and fate. Its main receptor, CD44, is usually overexpressed in cancer cells and has been correlated with cancer aggressiveness [1]. Herein, we report on the utility of HA colloidal gradients to better understand CD44-HA interactions and the possibility to apply these platforms for tumor cells sorting.

Our platform was build on a colloidal gradient of gold nanoparticles (20 nm) immobilized on aminated glass. HA end-on functionalized with alkanethiol (C₁₁SH) was then bound to the gold gradients by simple immersion. We have studied these platforms prior and after passivation, where the HA-free spots on the substrates were blocked with albumin. The gradients were characterized by x-ray spectroscopy, scanning electron microscopy, and histochemistry with labelled lectin. Three breast cancer cell lines with different CD44 expression levels (Sk-Br-3^-, MDA-MB-231⁺ and MDA-MB-468⁺⁺) were studied in HA gradients. Different cells’ response was observed through the gradient and the passivation impact tremendously the results. Prior passivation we observed more adherent cells at low densities of HA^-, indicating that the cells might adhere to unfunctionalized substrates. At these HA densities, cells presented a round morphology and a low expression of CD44. The increase of HA density was marked by the increase of CD44 expression and formation of filopodia associated with a spindle-like morphology. Of note, colocalization of CD44 and actin was observed at the filopodia’s tips. These changes were more pronounced in cells that overexpress CD44. In passivated platforms cell density was proportional to HA in the surface. Moreover, cell morphology was greatly affected, in HA^- region cells have a round-shape morphology wih constricted cytosqueletum, with HA increasing cells start to adhered by filopodia resulting in spindle-like morphology. Individual cell motility was analyzed. Different motilities in HA gradient was observed, not only according to CD44 expression levels, but also according to the density of HA at the surface. At low densities cells presented higher motility and longer persistent length, which decreased with the increase of HA density. The results suggest that cells recognize differently HA present in the gradient via CD44, which can be further explored as a cell sorting approach for CD44⁺ cells

[1] Afratis N. et al The FEBS J. 279:1177-1197, 2012