Cell-surface and cell-cell receptors have been studied extensively to investigate the switch from benign noninvasive to metastatic tumors. However, one of the major limitations has been the lack of studies that explore the effect of the cellular microenvironment on this transition. Here, we propose to use microfluidic platforms to control the spatial distribution of surface bound extracellular matrix (ECM) and cadherin proteins, thereby manipulating the microenvironment that the cell experiences. Our proposal seeks to elucidate the synergies that are shared between integrins and cadherins. These receptors will be engaged at varying degrees while cell migration and GTPase expression levels are monitored.
Specific Aim 1. To test the hypothesis that cadherins and integrins differentially modulate cell migration behavior. To do this, we will quantify the migration rate of EC expressing cells on substrates patterned with EC, ECM (fibronectin or laminin) or a combination of these proteins at various surface densities. We will therefore quantify the various influence of these extracellular components on cell migration.
Specific Aim 2. To test the hypothesis that integrins and cadherins differentially modulate cell adhesion and migration by differentially influencing the competing signaling pathways. To achieve this, we will generate countergradients or gradients of EC and ECM to determine if increased engagement alters signaling molecule activation and cell migration. Specifically, expression of RhoA, Rac1 and Cdc42, which are involved in E-Cadherin adherens junctions and integrin-ECM focal adhesion sites, will be monitored using immunofluorescent staining.
Specific Aim 3. To test the hypothesis that greater cadherin engagement through the substratum will lead to increase expression of EC, NC and corresponding increase in vimentin. To achieve this, we will quantify the expression levels of EC, NC and vimentin using immunofluorescent staining. In conjunction, in situ monitoring of the expression levels of green fluorescent protein (GFP) tagged EC, NC and vimentin will be assessed. This research seeks to understand the effects of tumor microenvironment on cellular behavior. In particular, we will explore how cell invasiveness and migration is affected when exposed to controlled environments. ? ? ?
| Silvestre, Jonathan; Kenis, Paul J A; Leckband, Deborah E (2009) Cadherin and integrin regulation of epithelial cell migration. Langmuir 25:10092-9 |
