Abstract 155: Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model

Despite recent extraordinary clinical success of immune checkpoint inhibitors (ICIs) in treating people affected by melanoma, a relevant number of patients still develops an adaptive resistance resulting in poor prognosis. To accelerate access to new therapies, there is a strong need for an in vitro human melanoma model mimicking the complexity of the tumor's cellular and physical microenvironment. Such model should recapitulate 3 critical features: (1) a reconstructed human melanoma-in-skin (Mel-RhS) to model melanoma cell invasion, (2) an endothelium to mimic the vascular system, and (3) circulation of, and ultimately, infiltration by, immune cells. Here, we present the development of these 3 individual biological features separately in dynamic conditions. These were constructed in standard multi-well plates (MW) by means of our microenviromentally controlled microphysiological system (MPS) (CubiX). This is a critical step towards the creation of a fully differentiated vascularized immunocompetent metastatic melanoma-on-chip model to recapitulate human pathophysiology, suitable for testing immunotherapies and studying the onset of possible resistance mechanisms. (1) Mel-RhS were constructed on transwells (8 μm pore size) and consisted of a human fibroblasts-populated collagen-fibrin dermal compartment, on top of which A375 melanoma cells and human keratinocytes were seeded. Mel-RhS were air-exposed for > 14 days to form of a fully differentiated and stratified epidermis. Melanoma nests developed and expanded into the dermal compartment in the 3D model, mimicking the initial stages of invasive melanoma. Moreover, Mel-RhS were viable under flow conditions for up to 3 days and their histology was comparable to that of the static controls. (2) The endothelial layer consisted of a monolayer of CD31+ endothelial cells on the bottom side of the transwell membrane. The shear stress provided by medium perfusion was able to induce alignment (75-80%) of cells within 24 hours (3 times faster than published protocols) as well as Von Willebrand Factor production. Real-time pH, lactate, glucose, and oxygen consumption were measured by using in-line sensors during the culture time. (3) Circulation of immune cells (MUTZ-3 progenitor cells) was achieved up to 24 hours in the presence of a healthy reconstructed human skin (RhS) model in a proof-of-concept study. The specific design of the developed MPS allowed for both immune cell flow and collection for downstream phenoty