(M-496) Tumorigenic properties of uveal melanoma cells cultured in spheroids or on a reconstructed extracellular matrix

Introduction:: Uveal melanoma (UM) is the most common primary intraocular tumor in adults. It arises from the transformation of melanocytes, and despite the effective treatment of the ocular tumor, hepatic metastases lead to a poor prognosis. Invasive potential and therapeutic resistance of UM cells (UMCs) can depend on interactions with stromal cells and properties of the extracellular matrix. Our project uses the self-assembly method and UM spheroids to engineer a tumor-like environment and validate its relevance as a cancer model.


Materials and Methods:: Two types of tumor spheroids were generated: i) primary UM (UMCs from eye tumor + choroidal fibroblasts + endothelial cells) or ii) liver metastasis (UMCs from liver metastasis + hepatic stellate cells + sinusoidal endothelial cells (or HUVECs)). The organization of these mixed spheroids were studied by confocal microscopy using specific markers alpha-SMA (activated fibroblasts/stellate cells), von Willebrand factor (endothelial cells) and Ki67 (proliferation), or by transducing cells with vectors expressing fluorescent proteins eBFP, mCherry2 or eGFP. The viability and invasive capacity of mixed spheroids were assessed using a luminescent ATP detection assay, and invasion assay in Matrigel. In addition, two types of extracellular matrix sheets were produced by the self-assembly method of tissue engineering using choroidal fibroblasts or stellate cells. The resulting tissues were characterized by microscopy for collagen and fibronectin content.


Results, Conclusions, and Discussions:: We first assessed how cell types were organized in mixed UM spheroids. Higher expression of alpha-SMA (myofibroblast-like cells) and more proliferative Ki67+ cells were observed in the outer layers of the spheroids by immunofluorescence analyses for both the primary tumor and liver metastasis conditions (Fig. 1A). Before proceeding with the invasion assay, we confirmed that tumor spheroids were viable over 3 days using the ATP detection assay. Next, we embedded UM spheroids in Matrigel to compare the invasion area when stromal cells were included. We found that UMCs were more invasive in presence of stellate cells and endothelial cells (Fig. 1B). Choroidal and hepatic extracellular matrix sheets were produced in 21 days and contained collagen I and fibronectin (Fig. 1C). We succeeded in generating fluorescent UMC lines by transduction, which will be seeded into fibroblast- or stellate cell-matrix sheets to study their proliferation and invasion.

Together, our results showed that UM cells grown as spheroids with stromal cells of their microenvironment are more invasive, while they activated choroidal fibroblasts or stellate cells. We successfully generated tissue-engineered choroidal and hepatic stroma. Future experiments will combine tumor spheroids and engineered matrix sheets to investigate tumor cell invasion and matrix stiffening.

Overall, we will improve or tissue-engineered matrices by using cancer-activated fibroblasts or stellate cells to generate pathological models that better reproduce the tumor-like mechanical properties, such as stiffer and more fibrotic stroma.


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