Abstract 155: Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model
Authors: Michielon Elisabetta , de GruIijl Tanja , Waaijman Taco , Boninsegna Matteo , Prabhakaran Divyasree , Mejri Hadhemi , Corbera Antoni Homs , Gaudriault Pierre , Gibbs Susan , Fassini Dario 
Published: June 2023

Abstract
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 &gt; 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 phenotypic analysis.
In conclusion, we have individually recapitulated these major biological features of melanoma in vitro, in standard MW under dynamic culture conditions. In the future, the integration of these features into a single model would provide a unique in vitro vascularized immunocompetent human melanoma-on-chip model as a novel tool to investigate and test new therapies targeting adaptive resistance to ICIs.
Citation Format: Elisabetta Michielon, Tanja de GruIijl, Taco Waaijman, Matteo Boninsegna, Divyasree Prabhakaran, Hadhemi Mejri, Antoni Homs Corbera, Pierre Gaudriault, Susan Gibbs, Dario Fassini. Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 155.