Impiego di colture 3-D per lo studio della vascolarizzazione tumorale e la migrazione di cellule tumorali: ruolo del canali TRP

Blood vessels form extensive networks that provide oxygen, nutrients and immune control to all the cells and tissues in the body. Nevertheless, they can be involved in pathological process such as cancer progression characterized by structurally abnormal vessels defined as more permeable and leakier. These features facilitate the intravasation of cancer cells in the blood circulation. Moreover, in order to metastasise, circulating cancer cells need to extravasate from blood stream and form colonies in alternative site to the primary tumour. Metastasis formation is the leading death cause in patient suffering of many cancer, so it is of primary importance to study the mechanism involved in this process. During my research internship I focused on the study of the extravasation, particularly the one prompted by prostate cancer cell that can easily form metastasis. We performed this kind of study by reproducing blood vessel with an organ on chip approach, that enable to form 3-D vessel composed by endothelial cells. Organ on chip is an emerging technology that can fuse the easiness of in vitro culture and some aspect of the complexity of in vivo system. In fact, it is possible to reproduce the 3-D structure of an organ and introducing some physical characteristics present in vivo such as the shear stress for endothelial cells (that is achieved by microfluidic perfusion) or gradient present in a tissue. The first phase of the project was dedicated to the set-up of the technique, in which we verified the formation of good quality 3-D endothelial tubule (¿blood vessel-on-chip¿) by confocal microscopy. The presence of tight junction was verified by means of VE-cadherin staining, indicating the integrity of the vessel monolayer in the 3D structure. In a second step, I applied this technique to study cancer cell extravasation. In particular, we tested the role of TRPM8 channel in prostate cancer cell adhesion and extravasation. TRPM8, member of TRP family of channels, has been involved in prostate cancer progression indicating that its loss of expression during the carcinogenesis process lead to more invasive prostate cancer cells. By comparing cell adhesion of prostate cancer cell line PC3 to PC3 overexpressing TRPM8, I firstly demonstrated a role of this channels in cell adhesion. Finally, GFP-labelled PC3 or PC3 overexpressing TRPM8 cells were inserted into the ¿vessel-on-chip¿ and the number of cancer cells adherent was evaluated after 8h of perfusion. In accordance with adhesion experiments, I observed a significant reduction of cell adhesion in TRPM8-overexpressing PC3 cells. In conclusion, the 3 D ¿vessel on chip¿ culture approach is a useful tool to perform more complex experiment than conventional 2-D endothelial cells culture therefore reducing the gap with in vivo systems.