  Within the tumor microenvironment (TME), cancer cells use mechanotransduction
pathways to convert biophysical forces to biochemical signals. However, the
underlying mechanisms and functional significance of these pathways remain
largely unclear. The upregulation of mechanosensitive pathways from biophysical
forces such as interstitial flow, leads to the activation of various cytokines,
most importantly transforming growth factor-$\beta$ (TGF-$\beta$). TGF-$\beta$
is a critical inducer of epithelial-mesenchymal transition (EMT) in cancer
cells that leads to increased cell motility and invasion in a Smad dependent
manner. Current research models have limited ability to investigate the
combined effects of biophysical forces (such as interstitial flow) and
cytokines (TGF-$\beta$) in a 3D microenvironment. We used a 3D-matrix based
microfluidic platform to demonstrate the potentiating effect of interstitial
flow (IF) on exogenous TGF-$\beta$ induced upregulation of the Smad-signaling
activity and the expression of mesenchymal marker vimentin in A549 lung cancer
spheroids. To monitor this, we used stably integrated fluorescent based
reporters into the A549 cancer cell genome. Our results demonstrate that
interstitial flow enhances exogenous TGF-$\beta$ induced Smad-signaling
activity in lung cancer spheroids embedded in a matrix microenvironment. In
addition, we observed an increased cell motility for A549 spheroids when
exposed to interstitial flow and TGF-$\beta$. Our 3D-microfluidic model
integrated with real-time imaging provides a powerful tool for investigating
cancer cell signaling and motility associated with invasion characteristics in
a physiologically relevant TME.
