Molecular weight of surface immobilized hyaluronic acid influences CD44-mediated adhesion of gastric cancer cells

INTRODUCTION

CD44, a transmembrane glycoprotein, is the major cell surface receptor for hyaluronic acid (HA).1 HA is a negatively charged polysaccharide composed of repeating disaccharides of D-glucuronic acid and N- acetyl-D-glucosamine and is a naturally occurring glycosaminoglycan. It has been studied as a targeting motif in drug delivery systems for cancer therapy because several tumor cells are known to overexpress CD44.2 CD44 cell surface marker is reported to have increased expression in AGS and MKN-45 gastric cell lines.3 The influence of HA molecular weight (Mw) on the HA-CD44 interaction has been reported for solubilized HA.4 However, this Mw influence has not been reported for surface immobilized HA, e.g. by Layer-by-Layer (LbL). In this study, the adhesion of AGS and MKN-45 cells and the influence of the HA Mw on the CD44 expression were evaluated on LbL constructs obtained through the sequential adsorption of HA and Poly-L-Lysine (PLL).

EXPERIMENTAL METHODS
LbL construction and CD44 adsorption: the sequential adsorption (10 layers) of HA (6.4 kDa, 752 kDa or 1.5 MDa) and PLL (30-70 kDa), as well as the subsequent adsorption of recombinant CD44 (onto the constructs presenting HA as top layer) was monitored using Quartz Crystal Microbalance with Dissipation (QCM-D). Surpass Electrokinetic Analyzer were used to measure the Zeta potential of the complete LbL systems. Immunostaining: AGS and MKN-45 cells were cultured onto the LbL constructs presenting different HA Mws in the surface and the cellular CD44 expression was evaluated. Nuclei were counterstained with DAPI. Statistical Analysis: Shapiro-Wilk normality tests followed by Kruskal-Wallis with Dunn’s Post hoc test. *** Significant differences (<0.001).

RESULTS AND DISCUSSION

We evaluated the LbL deposition, the stability of the systems, and their capacity to adsorb CD44 as a function of HA Mw. The QCM-D data shows that the CD44 adsorption is directly dependent on the HA Mw. The adhesion of MKN-45 and AGS cells to the LbL constructs (produced with the different HA Mws) presents significant differences in the number of adherent cells after 24h. Surprisingly, and in contrast with the QCM-D adsorption study, the cellular CD44 expression and the number of adherent cells was higher for the surfaces presenting HA with lower Mw, i.e. 6.4kDa, for both AGS and MKN-45 cell lines.

CONCLUSION

Adhesion of AGS and MKN-45 cells is affected by HA Mw. AGS cells adhere faster than MKN-45, but AGS spread more in contact with 6.4 kDa HA. In the surfaces that present higher Mws the adhesion of AGS and MKN-45 depend on previously adhered cells, resulting in the formation of clusters and round-shaped cells, as well as, in a delayed CD44-independent cell adhesion.

REFERENCES

1. Wolny et al., J. Biol. Chem. 285:30170–80, 2010

2. Choi et al., J. Mater. Chem. 19:4102, 2009

3. Takaishi et al., Stem Cells 27: 1006-20, 2009

4. Fuchs et al., Cell Death Dis. 4:e819, 2013

ACKNOWLEDGMENTS

The authors would like to thank to EU's FP7 (FP7/2007-2013) under grant agreement no REGPOT- CT2012-316331-POLARIS, for financial support.