Abstract PR07: Dissection of cancer cells extravasation through human vascularized 3D microfluidic model: The major role of talin-1

Abstract

Abstract Cancer cells spread from a primary tumor to secondary loci is responsible for more than 90% of cancer related mortality. Hematogenous metastasis is a complex process [1]. It includes a chain of events that can be summarized as follows: migration from primary tumor site and intravasation of the primary tumor cancer cells into the blood flow, dissemination through the circulation, extravasation in different organs, survival in the new microenvironment and colonization with generation of a new tumor. Recently our group presented a microfluidic 3D model reproducing the effects of the CXCL5-CXCR2 interaction between bone cells and metastatic breast cancer cells observed in vivo [2]. We further developed a human 3D microfluidic model that enables the study of human metastatic breast cancer cell extravasation within a perfusable human microvascularized bone-mimicking microenvironment [3]. Understanding the cellular and molecular events implicated in extravasation could facilitate the design of new therapeutic strategies targeting cancer cells only in order to couple these new developed therapies with already existing treatments and finally fight cancer. Talin-1 is a cytoplasmic adaptor essential for integrin-mediated adhesion to the ECM. Talin-1 links the actin cytoskeleton to integrin at the plasma membrane, regulates the focal adhesion pathway in normal cells and it is up-regulated in triple negative breast cancer cells such as MDA-MB231 and it is mutated in sarcomas. Based on the above described models, we developed a human vascularized 3D microfluidic device where human perfusable capillary like structures were embedded in fibrin matrix, characterized by mature endothelium markers (VE-cadherin, ZO-1) and physiological permeability (1.5±0.76)*10-6cm/s. Since these models can provide quantitative data on cancer cell extravasation, we focused on the role of Talin-1 in extravasation through blood vessels and in early invasion in two cell lines whose Talin-1 expression was silenced by SiRNA: triple negative metastatic breast cancer cells (MDA-MB231) and the metastatic fibro-sarcoma cell line (HT1080). Talin-1 silencing was confirmed by western blotting with specific antibodies. Adhesion to the endothelial wall, extravasation and ensuing migration out from the wall within the extracellular matrix was monitored by means of confocal high resolution real time imaging analyses. We demonstrated that silencing of Talin-1 expression dramatically and in a statistically significant way reduced both the adhesion efficiency and extravasation in both MDA-MB 231 and HT1080. Cell migration was also strongly and statistically reduced by the SiRNA treatment in both analyzed cell lines. In summary, we are the first to dissect the role of Talin-1 in each step of extravasation, demonstrating that targeting this protein could be an effective strategy to block metastasis. These data identify Talin-1 as a promising target for the development of new anti-metastatic therapies based on Talin-1 inhibition.