Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan, and one of the main ligands of the transmembrane surface receptor CD44, usually overexpressed by gastric cancer cells, such as MKN-45.2 CD44 is related with tumor invasiveness and progression as it activates Src-kinase that regulates the phosphorilation of paxillin and consequently, the formation of invadopodia.3 Herein, we studied the effect of the HA’s molecular weight (6.4, 752 and 1500 kDa) on CD44 binding, paxillin expression and tumor invasiveness. We applied the Layer-by-Layer (LbL) approach to deposit HA of different Mw on gold surfaces. This approach allows the surface immobilization of biomolecules with minimal or no changes of their chemistry, i.e. preserving their bioactivity. Moreover, the immobilized molecules are presented in an ECM fashion: they are spatially restricted but enough flexible to reorganize and bind to CD44. We used QCM-D technique to study the adhesion of MKN-45 cells onto these surfaces. To this purpose, a suspension of MKN45 was introduced into the QCM-D chambers and the cell adhesion onto the surfaces was monitored in-situ: changes in frequency and dissipation were plotted over time. The results demonstrated that MKN45 adhered to all studied substrates. However, a significantly less adherent cells were observed for the surfaces presenting HA of lower Mw, i.e. the substrate (PLL-HA6.4)5 DD/DF plots show similar changes in the DF for all studied Mw but very high DD for the (PLL-HA752)5 and (PLL-HA1500)5. These results can be explained with the activity of the adherent MKN45 that bind and remodel the HA of high Mw (752 kDa and 1500 kDa). The different signal obtained for the MKN45 on (PLL-HA6.4)5 is consistent with the inability of the MKN45 to reorganize the low Mw HA due either to a lower adhesion strength between the cells and the substrate or to the very tight binding between the PLL and HA. Paxillin staining confirmed the first hypothesis and showed more contact sites between MKN45 and the surface (PLL-HA1500)5 as compared to (PLL-HA6.4)5 and (PLL-HA752)5. We demonstrate that the LbL deposition is a feasible approach for presenting HA in a ECM relevant way. Adhesion of MKN45 is affected by the Mw of the immobilized HA: more cells adhered to substrates with HA of high Mw (752 and 1500 kDa). Moreover, the adhesion on these surfaces is also stronger as demonstrated by the ability of MKN45 to reorganize the LbL built up with these HAs. Finally, the substrate (PLL-HA1500)5 is also the one that induces a higher expression of paxillin, one of the major components of FAs.

REFERENCES
1. Deakin N. et al, Genes Cancer, 3, 362-370, 2012
2. Takaishi, S. et al., Stem cells, 27, 1006-1020, 2009.
3. McFarlane S. et al., Oncotarget, 6, 36762-36773, 2015.