@article {19223,
	title = {The effect of immobilized Hyaluronic Acid on CD44-overexpressing MKN45 gastric cancer cell line },
	booktitle = {12{\textordmasculine} Reuni{\~a}o do Grupo de Glucidos},
	year = {2017},
	month = {2017-09-12 00:00:00},
	address = {Aveiro},
	abstract = {<p>Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan, composed of repeating disaccharides of N-acetyl-glucosamine and glucuronic acid, connected exclusively by b-linkages.<sup>1</sup>\&nbsp;HA is one of the main ligands of the transmembrane glycoprotein surface receptor CD44, usually overexpressed by gastric cancer cells, such as MKN45.<sup>2</sup> CD44 in cancer cells interacts with HA-rich microenvironments, inducing cell signaling pathways that trigger the ability of malignant cells to migrate and to invade basement membranes {\textfractionsolidus} ECM leading to the formation of metastasis.<sup>3</sup> CD44 activates Src-kinase that regulates the phosphorilation of paxillin and consequently, the formation of invadopodia.<sup>4</sup> Herein, we studied the effect of the HA molecular weight (6.4, 752 and 1500 kDa) on the adhesion of MKN45 cells and its relationship with tumor invasiveness. We immobilized HA with different M<sub>w</sub> in a Layer-by-Layer (LbL) fashion and studied the interactions of the substrates with the gastric cancer cell line. In these developed substrates, HA is presented in an ECM-relevant manner with restricted mobility but yet able to reorganize and bind specifically other biomolecules, capable to reorganize and bind to CD44. The construction and stability of the LbL structure, i.e. lack of layers{\textquoteright} migration and the presence of the HA on the substrate{\textquoteright}s surface, was confirmed by electrokinetic analysis. The presence of HA, as end layer of the constructs, leads to negative ζ-potential values, independently of the HA Mw. To study the adhesion of MKN45 cells to the LBL surfaces, we used QCM-D technique. 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. From the DD/DF plots we can highlight the high DF and DD for the (PLL-HA<sub>752</sub>)<sub>5 </sub>and (PLL-HA<sub>1500</sub>)<sub>5</sub>. 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), and the formation of contact sites between cells and LBL surfaces. The different signal obtained for the MKN45 on (PLL-HA<sub>6.4</sub>)<sub>5 </sub>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. SEM images and Paxillin staining confirmed the first hypothesis and showed more contact sites between MKN45 and the surface (PLL-HA<sub>1500</sub>)<sub>5</sub> as compared to (PLL-HA<sub>6.4</sub>)<sub>5 </sub>and (PLL-HA<sub>752</sub>)<sub>5</sub>. 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-HA<sub>1500</sub>)<sub>5</sub> is also the one that induces a higher expression of paxillin, one of the major components of FAs.</p>},
	keywords = {CD44, gastric cancer cells, Hyaluronic acid},
	author = {Amorim, S. and Soares da Costa, D. and Freitas, D. and Reis, C. A. and Reis, R. L. and Pashkuleva, I. and Pires, R. A.}
}
