Chen Yunxi, Jin Eva, Abdouh Mohamed, Bonneil Éric, Jimenez Cruz Daniel Alexander, Tsering Thupten, Zhou Qianqian, Fuentes-Rodriguez Aurélie, Bartolomucci Alexandra, Goyeneche Alicia, Landreville Solange, Burnier Miguel N, Burnier Julia V
Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.
Department of Pathology, McGill University, Montreal, QC, Canada.
BMC Biol. 2025 Jan 21;23(1):16. doi: 10.1186/s12915-025-02118-w.
Uveal melanoma (UM) is the most common intraocular tumor in adults, arises either de novo from normal choroidal melanocytes (NCMs) or from pre-existing nevi that stem from NCMs and are thought to harbor UM-initiating mutations, most commonly in GNAQ or GNA11. However, there are no commercially available NCM cell lines, nor is there a detailed protocol for developing an oncogene-mutated CM line (MutCM) to study UM development. This study aimed to establish and characterize premalignant CM models from human donor eyes to recapitulate the cell populations at the origin of UM.
Given the precious value of human donor eyes for studying multiple ocular cell types, we validated a co-isolation protocol of both human NCMs and retinal pigment epithelial (RPE) cells from a single eye. To this end, NCMs and RPE cells were sequentially isolated from 20 donors, with success rates of 95% and 75%, respectively. MutCMs were generated from 10 donors using GNAQ-carried lentivirus with high mutant copies (up to 98.8% of total GNAQ copies being mutant). NCM growth and behavior were characterized under different culture conditions (i.e., supplementation with serum and 12-O-tetradecanoylphorbol-13-acetate) to determine optimized protocols. Particularly, Matrigel™ coating induced spheroid growth under certain coating thickness and cell seeding density but did not improve NCM metabolic activity. Current methodologies in NCM isolation, culture, and research applications were summarized. Proteomic profiling of 4 NCMs, 1 MutCM, and 3 UMs allowed to discover significant differences in UMs including a downregulation of proteins linked to melanocyte differentiation and an upregulation of proteins involved in RNA metabolism. RNA sequencing revealed enriched pathways related to cancer, notably PI3K-Akt and MAPK signaling pathways, in MutCMs and UM cells compared to NCMs, providing insights into molecular changes in GNAQ-mutated pre-cancer cell models and UM cells.
We successfully isolated and established NCM, RPE, and MutCM cell lines. We describe efficient methods for the isolation and growth of NCMs and report their phenotypic, proteomic, and transcriptomic characteristics, which will facilitate the investigation of UM development and progression. The co-isolated RPE cells could benefit research on other ocular pathologies, such as age-related macular degeneration.
葡萄膜黑色素瘤（UM）是成人中最常见的眼内肿瘤，它要么起源于正常脉络膜黑色素细胞（NCM），要么起源于源自NCM的既有痣，这些痣被认为携带UM起始突变，最常见于GNAQ或GNA11。然而，目前尚无市售的NCM细胞系，也没有详细的方案来构建携带致癌基因突变的脉络膜黑色素细胞系（MutCM）以研究UM的发生发展。本研究旨在从人类供体眼中建立并表征癌前脉络膜黑色素细胞模型，以重现UM起源处的细胞群体。
鉴于人类供体眼对于研究多种眼细胞类型的珍贵价值，我们验证了从单眼中共同分离人类NCM和视网膜色素上皮（RPE）细胞的方案。为此，从20名供体中依次分离出NCM和RPE细胞，成功率分别为95%和75%。使用携带GNAQ的慢病毒从10名供体中生成了MutCM，其突变拷贝数较高（高达98.8%的总GNAQ拷贝为突变型）。在不同培养条件下（即补充血清和12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯）对NCM的生长和行为进行了表征，以确定优化方案。特别地，基质胶™包被在一定包被厚度和细胞接种密度下诱导了球体生长，但并未提高NCM的代谢活性。总结了目前NCM分离、培养和研究应用中的方法。对4个NCM、1个MutCM和3个UM进行蛋白质组分析，发现UM之间存在显著差异，包括与黑色素细胞分化相关的蛋白质下调以及参与RNA代谢的蛋白质上调。RNA测序显示，与NCM相比，MutCM和UM细胞中与癌症相关的富集通路，特别是PI3K - Akt和MAPK信号通路，这为GNAQ突变的癌前细胞模型和UM细胞中的分子变化提供了见解。
我们成功分离并建立了NCM、RPE和MutCM细胞系。我们描述了NCM分离和生长的有效方法，并报告了它们的表型、蛋白质组和转录组特征，这将有助于研究UM的发生发展过程。共同分离出的RPE细胞可能有益于其他眼部疾病的研究，如年龄相关性黄斑变性。
