Hyaluronic acid (HA) has a key role in cancer progression. The HA’s molecular weight (Mw) is altered
in this pathological state: increased concentration of shorter fragments due to the overexpressed
hyaluronidases and ROS. Aiming to mimic this microenvironment, we developed a Layer-by-Layer
(LbL) platform presenting HA of different Mws, namely 6.4, 752 and 1500kDa, to study the influence of
HA Mw on the formation of focal adhesion sites (FAs), and the involvement of paxillin and CD44 in this
process. High paxillin expression and formation of FAs, via CD44, is observed for MKN45 cells seeded
on LbLs presenting HA 6.4kDa, with the activation of the ERK1/2 pathway, responsible for cell motility
and tumour progression. In contrast, activation of p38 pathway, usually related with cancer latency, is
observed for cells seeded on LbLs with high Mw HA, i.e. 1500kDa. Overall, we demonstrate the
suitability of the developed platform to study cancer invasiveness.