While being the rarest skin cancer, melanoma is also the deadliest. To further drug discovery and improve clinical translation, new human cell-based in vitro models are needed. Our work strives to mimic the melanoma microenvironment in vitro as an alternative to animal testing. We used the self-assembly method to produce a 3D human melanoma model exempt of exogenous biomaterial. This model is based on primary human skin cells and melanoma cell lines while including a key feature for tumor progression: blood and lymphatic capillaries. Major components of the tumor microenvironment such as capillaries, human extracellular matrix, a stratified epidermis (involucrin, filaggrin) and basement membrane (laminin 332) are recapitulated in vitro. We demonstrate the persistence of CD31+ blood and podoplanin+/LYVE-1+ lymphatic capillaries in the engineered tissue. Chronic treatment with vemurafenib was applied to the model and elicited a dose-dependent response on proliferation and apoptosis, making it a promising tool to test new compounds in a human-like environment.

This protocol describes a unique in vitro method for the generation of a 3D human lymphatic network within native connective tissue devoid of any exogenous material such as scaffolds or growth factors. In this five-stage protocol, human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts spontaneously organize into a stable 3D lymphatic capillary network. Stage 1 involves the isolation of primary fibroblasts and LECs from human skin. Fibroblasts are then cultured to produce connective tissue rich in extracellular matrix (stage 2), onto which LECs are seeded to form a network (stage 3). After stacking of tissue layers and tissue maturation at the air-liquid interface (stage 4), the 3D construct containing the lymphatic microvascular network can be analyzed by microscopy (stage 5). Lymphatic vasculature generated by this approach exhibits the major cellular and ultrastructural features of native in vivo human dermal lymphatic microvasculature and is stable over many weeks. The protocol for generating a 3D construct takes 6 weeks to complete, and it requires experience in cell culture techniques. The system described here offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function in a human cell context.

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Purpose: Radiation skin injuries are difficult to quantitatively assess. Various scoring scales exist based on visual images and can be used in quantitative form for histological scoring. As an alternative to human scoring systems, an automated, quantitative system is proposed to provide unbiased scoring of radiation skin injury biopsy samples, with comparisons to human-based scoring systems. Materials and methods: A unique algorithm was developed and tested on a sample pool obtained from in-vivo beta radiation experiments with a porcine model. The grading results achieved by the developed algorithm and those provided by an expert histopathologist are compared. Results: The extent of the epidermal necrosis is quantified in terms of the number of dead cells and their respective distribution across the length of the samples. The accuracy of the grading performed by the automated algorithm is comparable to that of a trained histopathologist, as demonstrated by statistically significant difference between the grades. Conclusions: This study demonstrates the feasibility of the proposed method as a potential tool designed to aid in the histopathological analysis of the tissues affected by beta radiation exposure. An expanded study with a larger sample pool is recommended to further improve the accuracy of the proposed algorithm