Development of a vascularized in vitro skin model mimicking metastatic melanoma

Introduction: Metastatic melanoma represents the last step of melanoma progression with an extremely poor prognosis. Effective therapies for this disease are still lacking and their development requires new models able to replicate the complexity of this tumour and the interactions between the tumour cells and their surrounding environment. To this aim, this work focusses on the development of a vascularized model able to replicate melanoma progression and invasion to be used as a novel tool for the screening and validation of new therapies. Experimental methods: The model is composed by a dermal compartment, made of fibroblasts (HFF1) embedded in a collagen (COL)-hyaluronic acid (HA) hydrogel, and an epidermal compartment, made of keratinocytes (HACAT). The gel is realized with bovine COL type I, methacrylated bovine COL type I, and methacrylated HA. HFF1 were cultured in the gel up to 28 days and their viability monitored by cell titer blue assay. Cell morphology and distribution were assessed by confocal microscopy. Melanoma spheroids (SK-MEL28) were prepared under low adhesive conditions and embedded in the gel to mimic the initial phase of the pathology and their behaviour was monitored by confocal microscopy. COL deposition by the embedded cells was analysed by immunofluorescence. To mimic the presence of the vascular network, a microfluidic chip was developed to induce perfusion, a crucial stimulus to obtain endothelial cells (HUVEC) maturation. The chip is composed of three compartments connected in series through a microfluidic channel. HUVEC were cultured within the microfluidic device for up to 48 hours and cell morphology was observed by confocal microscopy. Results and discussion: The COL/HA hydrogel was successfully obtained. Human fibroblasts were able to colonize the entire volume of the hydrogel, recreating the in-vivo like architecture. Keratinocytes were also successfully cultured on the gel and their maturation was achieved by maintaining the gel at the air liquid interface for 14 days. Tumour spheroids, in the cellularized hydrogel, were able to invade the surrounding space, interacting with fibroblasts. Immunofluorescence images showed that beginning from day 7, fibroblasts were able to produce their own collagen, confirming their ability to produce new ECM. HUVECs showed a preferential cytoskeleton orientation when cultured under dynamic conditions after 24 hours of culture. Conclusion: A 3D COL/HA-based matrix was realized allowing the cells to grow in a physiological-like environment. Fibroblasts were viable and able to recreate an in vivo-like structure and synthetize native ECM proteins when cultured within the hydrogel. The microfluidic chip allowed HUVEC maturation. In the future, the presence of different compartments in the chip will be used to host different tissue models to mimic the process of melanoma metastasis. Acknowledgments: Carlotta Mattoda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorato su tematiche green" for co-financing her Ph.D scholarship.