Engineered vascularized in vitro skin model mimiking melanoma development and progression

Introduction Metastatic melanoma represents the final stage of melanoma progression and is associated with an extremely poor prognosis. Effective treatments for this disease remain limited, necessitating the development of advanced models that can accurately replicate the tumour’s complexity and its interactions with the surrounding microenvironment [1]. The present study aims at developing a vascularized in vitro model mimicking melanoma progression and invasion, as a platform for the screening and validation of advanced therapeutic strategies. Materials and Methods The model comprises a dermal compartment, formed by a collagen (COL)-hyaluronic acid (HA) hydrogel embedding human dermal fibroblasts (HFF1), and an epidermal compartment, consisting of a thick layer of human keratinocytes (HACAT). The hydrogel is obtained using bovine type I COL, and methacrylated HA. HFF1 cells were cultured within the gel for up to 21 days, monitoring cell viability through the CellTiter-Blue assay. Cell morphology and distribution were also analysed through confocal microscopy. Native extracellular matrix (ECM) proteins deposition was evaluated using immunofluorescence (IF). To replicate the early stages of melanoma progression, melanoma (SK-MEL28 cells ) spheroids were generated under low-adhesion conditions and embedded within the hydrogel. Their behaviour in terms of invasion and interaction with ECM components was monitored via confocal microscopy and IF. To simulate the presence of the vascular network, a microfluidic chip was designed. The chip comprises three compartments connected in series through a microfluidic channel. Endothelial cells (HUVEC) were co-cultured with the dermal matrix under dynamic conditions for up to 8 days exploiting the microfluidic device. The development of self-assembled capillaries within the hydrogel was monitored using confocal microscopy. Results The COL/HA hydrogel supported HFF1 proliferation, as cells were found to fully colonize the matrix, recreating a 3D architecture and depositing native ECM proteins (e.g., human collagen and fibronectin). HACAT were also successfully cultured on the hydrogel, achieving maturation after 7 days at the air-liquid interface as confirmed by the expression of cytokeratin 14 . Tumour spheroids demonstrated an invasive behaviour withing the dermal hydrogel, as well as interactions with HFF1 in the surrounding matrix, as indicated by the expression of activation markers (namely α-SMA) and the enhanced deposition of ECM proteins by HFF-1. Perfusion within the microfluidic system facilitated the alignment of HUVECs along the flow direction, promoting their maturation. In comparison to the samples maintained in static culture, confocal analysis showed an upregulation of CD31, ZO-1, and VE-cadherin and a preferential cytoskeleton orientation along the flow direction in the samples cultured in dynamic conditions, confirming the importance of the flow stimulus to obtain a functional endothelial barrier. After 6 days of perfused co-culture, a self-assembled capillary network formed within the dermal model. By day 8, the network exhibited increased branching, confirming the successful establishment of a vascular network within the dermal construct. Discussion The presented results confirmed the development of a vascularized full-thickness melanoma model replicating key aspects of disease progression and invasion. The COL/HA hydrogel provided a supportive microenvironment for HFF1 viability, ECM deposition, and epidermal maturation, closely mimicking in vivo conditions. Additionally, the inclusion of tumour spheroids within the hydrogel enabled the study of tumour-stromal cells interactions, highlighting the role of HFF1 in ECM remodelling [2]. The system was also successfully integrated within a microfluidic system achieving endothelial cell alignment and capillary sprouting. The resulting capillary-like network, absent in static cultures, demonstrated the importance of dynamic conditions in achieving vascularization on in vitro models [3]. Conclusions These findings underscore the potential of this model as a valuable tool for studying melanoma progression and response to therapies. This platform offers a more physiologically relevant alternative to traditional in vitro models. Future studies will focus on incrementing the model complexity, by connecting additional tissue mimics to investigate the process of melanoma metastases. Carlotta Mattoda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorato su tematiche green" for co-financing her Ph.D scholarship. Part of this project has received funding from the the project “D34H—Digital Driven Diagnostics, prognostics and therapeutics for sustainable Health care” (project code: PNC0000001), Spoke 4 funded by the Italian Ministry of University and Research (MUR). References [1] Patton, E. E. et al., 2021, 10.1016/j.ccell.2021.01.011. [2] Romano, V. et al., 2021, 10.3390/ijms22105283. [3] Meng, F. et al., 2022,10.1016/j.medntd.2022.100143.