Hyaluronic acid (HA) is a naturally occurring negatively charged glycosaminoglycan composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine. Its major cell surface receptor is the transmembrane glycoprotein CD44.¹ The interaction between HA and cancer cell membrane receptors is known to induce several signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread.² The influence of HA molecular weight (Mw) on the HA-CD44 interaction has been reported for solubilized HA ³. However, this Mw influence has not been addressed for surface immobilized HA. In this study, the influence of the HA Mw on the adhesion of AGS and MKN-45 cells (which overexpresses CD44)⁴, and on the CD44 expression were evaluated on Layer-by-Layer (LbL) constructs obtained through the sequential deposition of Poly-L-Lysine (PLL) and HA onto tissue culture polystyrene (TCPS).

The Zeta potential of the HA-presenting surfaces (and their PLL-presenting intermediates) produced with HAs of different Mws, i.e. 1.5MDa, 740kDa and 6.4kDa, were evaluated by Electrokinetic Analysis. The results indicate that the layers were successfully constructed, resulting in a positive Zeta potential when PLL was the last layer and a negative one when the end layer was HA (independently of the Mw). The stiffness of the surfaces (determined by AFM - Quantitative Nanomechanical Mapping) indicated significantly higher stiffness for the surfaces prepared with HA of lower Mw.

The adhesion of MKN-45 and AGS cells to the HA-presenting surfaces was evaluated. In the case of AGS cells (that present a lower CD44 expression than MKN-45) the variation of the HA Mw did not affect significantly the number of adherent cells. However, when MKN-45 cells were used it was possible to correlate the higher HA Mw with a lower cell adhesion; in addition, their pre-incubation with anti-CD44 blocking antibody, significantly reduces the number of adherent cells when HA of lower Mws were used (i.e. 6.4kDa and 752kDa). These results indicate that the HA Mw influences its binding affinity to CD44 mediating MKN-45 adhesion.

REFERENCES

1.  Wolny, P. M. et al. Analysis of CD44-hyaluronan interactions in an artificial membrane system: insights into the distinct binding properties of high and low molecular weight hyaluronan. J. Biol. Chem. 285, 30170–80 (2010).

2.  Dickinson, L. E. & Gerecht, S. Micropatterned surfaces to study hyaluronic acid interactions with cancer cells. J. Vis. Exp. (2010). doi:10.3791/2413

3.  Fuchs, K. et al. Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44. Cell Death Dis. 4, e819 (2013).

4.  Takaishi, S. et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells 27, 1006–20 (2009).