Abstract 5814: Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration
Authors: Mara Gilardi, Simone Bersini, Rosa Maria Moresco, Marco Vanoni, Roger D. Kamm, Matteo Moretti
Journal: Cancer Research (2017)

Abstract In extravasation cancer cells and vascular niche are involved in a tight cross-talk which has been defined as the rate-regulating event for metastases establishment [1]. Recent, animal studies support the hypothesis that metastatic deficiency lies in focal adhesion complex alterations, however, it still needs to be elucidated which are the specific regulators of each event composing extravasation. Focal adhesion proteins Talin-1 (TLN-1) and Focal Adhesion Kinase (FAK) are up-regulated in breast cancer. Both targets due to their structural and functional role, dramatically influence cancer mechanotransduction leading to endothelial junction disruption, critical in extravasation process [2]. Here, we generated ad hoc engineered models for each extravasation step allowing single cell behavior analyses through high resolution real time imaging in a reliable and quantitative way in a physiological environment. Through this novel approach we analyzed the effect of TLN-1 and FAK and their genetic and chemical inhibition in breast cancer extravasation. The 3D-microfluidic vasculature displayed maturation markers and physiological permeability (1.5±0.76*10-6cm/s) and allowed cancer cell injection through the hollow vessels. Western blot confirmed TLN-1 and FAK knock down (KD) in MDA-MB231. Both targets significantly affected morphology and proliferation (p&amp;lt;0.001). We demonstrated the involvement of both targets in adhesion to the endothelium (p&amp;lt;0.001). Trans endothelial migration (TEM) was decreased in TLN-1 KD (p&amp;lt;0.05) and in FAK KD (P&amp;lt;0.01). Both TLN-1 and FAK KD cells were trapped into the vessels and were not able to extend pseudopodia. Cancer migration tracking in 3D matrix was statistically lower in both KD (p&amp;lt;0.001). The calculated ratios of adhesion/TEM and invasion/TEM were lower in TLN-1 compared to FAK KD, demonstrating that TLN-1 plays a major role in adhesion to endothelium and early invasion while FAK was identified as the main driver in TEM. Additive (TLN-1 and FAK) KD did not show significant difference respect to the target that mostly affected adhesion and invasion demonstrating TLN-1 role in these events. Additive KD was statistically lower compared to TLN-1 KD (P&amp;lt;0.05), supporting FAK crucial role in TEM. Inhibition of TLN-1 and FAK phosphorylation revealed that actin polymerization and pseudopodia formation (required for extravasation) were dependent by their structural role and were independent by their phosphorylation. However, the inhibition of FAK activity showed FAK phosphorylation as the key driver of TEM mechanism (p&amp;lt;0.01). Concluding, our results supported by in vivo data (p&amp;lt;0.05) showed that TLN-1 and FAK inhibition may represent novel strategies challenging mechanisms leading to metastatic establishment. 1. Cell,2011.147(2):p.275-92; 2. Proc Natl Acad Sci USA,2015.112(1):p.214-9 Citation Format: Mara Gilardi, Simone Bersini, Rosa Maria Moresco, Marco Vanoni, Roger D. Kamm, Matteo Moretti. Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5814. doi:10.1158/1538-7445.AM2017-5814