Tackling muscle fibrosis: From molecular mechanisms to next generation engineered models to predict drug delivery.

Muscle fibrosis represents the end stage consequence of different diseases, among which muscular dystrophies, leading to severe impairment of muscle functions. Muscle fibrosis involves the production of several growth factors, cytokines and proteolytic enzymes and is strictly associated to inflammatory processes. Moreover, fibrosis causes profound changes in tissue properties, including increased stiffness and density, lower pH and oxygenation. Up to now, there is no therapeutic approach able to counteract the fibrotic process and treatments directed against muscle pathologies are severely impaired by the harsh conditions of the fibrotic environment. The design of new therapeutics thus need innovative tools mimicking the obstacles posed by the fibrotic environment to their delivery. This review will critically discuss the role of in vivo and 3D in vitro models in this context and the characteristics that an ideal model should possess to help the translation from bench to bedside of new candidate anti-fibrotic agents.

PO-12 - The key role of talin-1 in cancer cell extravasation dissected through human vascularized 3D microfluidic model.

INTRODUCTION: Metastases are responsible for more than 90% of cancer related mortality. The hematogenous metastatic invasion is a complex process in which the endothelium plays a key role. Extravasation is a dynamic process involving remodeling and change in cell shape and in cytoskeleton whereby a series of strongly dependent interactions between CTCs and endothelium occurs [1]. Talins are proteins regulating focal adhesions and cytoskeleton remodeling. Talin-1 seems to be involved in the aggressiveness, motility, survival and invadopodia formation of cancer cells throughout the entire metastatic cascade [2], being up-regulated in breast cancer cells and mutated in sarcomas. Understand the implication of talin-1 in extravasation could facilitate the design of new therapies and finally fight cancer. AIM: We hypothesized that Talin-1 could be specifically involved in extravasation driving each of its steps. MATERIALS AND METHODS: We developed a human 3D microfluidic model that enables the study of human cancer cell extravasation within a perfusable human microvascularized organ specific environment[3]. For the study of extravasation we applied microfluidic approach through the development of a microfluidic device in which endothelial cells and fibroblasts generated a 3D human functional vascular networks. Microvessel characterization was performed with immunofluorescence and permeability assays. We knocked-down talin-1 in triple negative breast cancer cell line MDA-MB231 and metastatic fibro-sarcoma cell line HT1080 with SiRNA and verified by Western-blot. Cancer cells were then perfused in the vessels and extravasation monitored through confocal imaging. RESULTS: We developed a human vascularized 3D microfluidic device with human perfusable capillary-like structures embedded in fibrin matrix, characterized by mature endothelium markers and physiological permeability (1.5±0.76)×10(-6) cm/s. We focused on the role of Talin-1 in adhesion to endothelium, trans-endothelial migration (TEM) and early invasion. Adhesion to the endothelium, TEM and migration within the ECM were monitored through confocal analyses. We demonstrated that Talin-1 KD significantly reduced the adhesion efficiency and TEM in both cell lines. Early invasion was also strongly and statistically reduced by the SiRNA treatment in both cell lines. CONCLUSIONS: We proved Talin-1 function in driving the extravasation mechanism in a human 3D vascularized environment. We demonstrated that Talin-1 is involved in each part of extravasation significantly affecting adhesion, TEM and the invasion stages. Targeting this protein could thus be an effective strategy to block metastasis.

In vitro models of the metastatic cascade: from local invasion to extravasation.

A crucial event in the metastatic cascade is the extravasation of circulating cancer cells from blood capillaries to the surrounding tissues. The past 5 years have been characterized by a significant evolution in the development of in vitro extravasation models, which moved from traditional transmigration chambers to more sophisticated microfluidic devices, enabling the study of complex cell-cell and cell-matrix interactions in multicellular, controlled environments. These advanced assays could be applied to screen easily and rapidly a broad spectrum of molecules inhibiting cancer cell endothelial adhesion and extravasation, thus contributing to the design of more focused in vivo tests.