Abstract
Purpose :
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. Our hypothesis is that the invasive potential and therapeutic resistance of UM cells (UMCs) depend on interactions with stromal cells and properties of the extracellular matrix (ECM). Our study aims to determine the impact of UMCs in stromal cell activation, and to define the role of the ECM produced by choroidal fibroblasts or hepatic stellate cells (HSteCs) on UM cell proliferation and invasion.
Methods :
Two types of tumor spheroids were generated i) primary UM (UMCs from eye tumor + choroidal fibroblasts + endothelial cells) and ii) liver metastasis (UMCs from liver metastasis + HSteCs + sinusoidal endothelial cells). The organization of these mixed spheroids were studied by confocal microscopy using the following markers: Melan-A (UMCs), alpha-SMA (activated fibroblasts/stellate cells), CD31 (endothelial cells) and Ki67 (proliferation). Viability and invasion capacity of the mixed spheroids were analyzed using an ATP consumption assay, and the measurement of the invasion area in Matrigel. Two types of matrices were produced by tissue engineering using choroidal fibroblasts or HSteCs. These matrices were devitalized and characterized by confocal microscopy (collagens, fibronectin), before seeding UMCs to study their morphology, proliferation and ECM remodeling.
Results :
Higher expression of alpha-SMA and more proliferative cells were observed in the outer layers of the spheroids by immunofluorescence analyses. Choroidal and hepatic matrices were produced in 21 days, and contained fibronectin and collagen I. UMCs seeded onto these reconstructed stromas remodeled the ECM.
Conclusions :
We characterized the viability and cellular organization in both choroidal/hepatic spheroid models, and successfully generated tissue-engineered choroidal and hepatic matrices. With the optimization of these 3D models, we will better understand the steps involved in the progression of ocular melanoma to a metastatic stage. These complex models may replace the 2D model using tumor cells in monolayer for future drug screening, which will consider the impact of the microenvironment on UM therapeutic resistance.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.