Abstract
Purpose :
Uveal melanoma (UM) is the most common intraocular malignancy in adults, and a life-threatening disease due to metastasis. Understanding the biology of UM is essential to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools to model disease behavior. In this study, we aim to characterize the behavior of UM cells under different 3D culture settings as a suitable model of therapeutic assessment.
Methods :
Six well characterized UM cell lines were tested in 2D (used as control) and 3D culture methods. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, on hanging drops (HD), or +/methylcellulose as scaffold. Cultures were analyzed for multicellular tumor structures (MCTS) by phase contrast and confocal imaging, and cell behavior (viability, membrane integrity and DNA synthesis) using the Live/Dead, CCK-8, and Click-it assays. Vascular endothelial growth factor (VEGF) production in the different environments was evaluated under hypoxic conditions (1% O2) for cell function analysis.
Results :
UM cells cultured following AF methods developed spherical MCTS with different level of compaction. Regardless of size and degree of compaction, these MCTS displayed a ring of viable cells with active DNA synthesis, and a cytotoxic core with less proliferating cells. In contrast, UM cells maintained under AD methods (i) remained isolated and rounded, (ii) formed irregular aggregates of different sizes, or (iii) adopted a 2D-like flat appearance. Basically, cells conserved a melanocytic phenotype (i.e., expression of Melan A/Mart-1 and HMB45) and were functionally active as they secrete the proangiogenic factor VEGF in response to hypoxia.
Conclusions :
UM cells maintained under AF environment form MCTS shaped as spheres with some attributes of a solid tumor in vivo. UM cell performance in AD manner is more heterogeneous, yet cells keep their melanocytic phenotype and function. This study provides a 3D cell culture platform to test new UM therapeutics (i.e., anti-angiogenic interventions), and co-culture methods (i.e., immune modulation by co-culturing with immune cells).
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.