Purchase this article with an account.
Luna Djirackor, Haleh Shahidipour, Sarah E Coupland, Helen Kalirai; A 3D spheroid model of Uveal Melanoma (UM). Invest. Ophthalmol. Vis. Sci. 2019;60(9):730.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cell based screening assays to identify effective drugs for the treatment of uveal melanoma (UM) have historically been undertaken using cells grown in 2D monolayer cultures. It is clear, however that these inadequately reflect the physiological conditions seen in patient tumours due to lack of oxygen and nutrient gradients or cell-cell and cell-matrix interactions. The use of three-dimensional (3D) cultures has been developed to overcome these hurdles and offers a high throughput way of drug screening. In this study, we investigated the ability of UM cell lines, primary UM (PUM) and normal choroidal melanocytes (NCM) to form spheroids and then characterised them in detail.
The spheroid forming ability of 5 cell lines (92.1, OMM1, Mel 270, Omm2.3 and Omm2.5) was tested using hanging drop and ultra-low attachment (ULA) plates. The ULA cultures were more successful. Subsequently, 21 PUMs and 5 NCM were cultured as spheroids using this method. Differing cell densities and length of culture were initially tested in 11 PUMs. The 3D spheroids and matching 2D monolayers were profiled by immunohistochemistry (IHC) and multiplex ligation dependent amplification to determine the expression of protein markers and chromosomal alterations respectively.
Spheroids from the hanging drop assay were of varying sizes and compactness. They did not retain their integrity when manipulated and most disintegrated when collected. The spheroids formed in the ULA plates, however, were more compact and retained their structure during collection and processing for IHC. Following assessment of spheroid growth and necrosis, an optimal culture regimen of 8000 UM cells/well cultured for 3 to 6 days was identified. IHC showed that using these conditions, PUM spheroids retained protein expression and were histologically similar to the patient tumour. Similarly, the genetic profile matched that of the patient tumour at passage 1. Comparison between the NCM and PUM spheroids showed that nestin is upregulated while α-SMA is downregulated in the tumours.
In this study, we have developed culture conditions that enable us to create 3D spheroids from the majority of patient tumours and NCM. These spheroids resembled the original tumour. This opens many possibilities for UM studies, including testing for therapeutic agents and advances personalised medicine.We acknowledge Prof. Goran Petrovski for his co-supervision in the PhD studentship of Luna Djirackor.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only