Doxycycline Modulates Uveal-Melanoma-Associated Marker Expression in BAP1-Repressed Human Ocular Organoids

Abstract

Uveal Melanoma (UM) is the most common eye cancer, with a metastatic mortality rate of 80%. Only 1-3% of patients have detectable UM at metastasis, and UM exhibits punctuated early growth. Doxycycline has recently been shown to inhibit metabolic processes exploited by cancer cells and reduce cancer cell growth in models of liver cancer. We hypothesized doxycycline may also be effective in UM and therefore tested doxycycline treatment in an eye organoid model of uveal melanoma. Using a stem cell line whereby BAP1 can be knocked down with a tetracycline-inducible system, we differentiated this line into a whole eye organoid model termed self-formed ectodermal autonomous multi-zone of ocular cells (SEAM). We found an enhanced proliferation in neural crest cells within the SEAM colonies. To identify the neural crest cells, we conducted single-cell RNA sequencing (scRNA-seq) analysis utilizing the Seurat R toolkit to pinpoint genes within neural crest clusters. To confirm the results of the in silico scRNA-seq analysis, genes with notable functions and differential expression in the neural crest cluster in relation to UM proliferation, angiogenesis, and oxidative phosphorylation were analyzed through immunofluorescence and RT-qPCR. Based on the scRNA-seq analysis, immunofluorescence, and RT-qPCR, the novel BAP1 KD (UM phenotype) model was found to replicate UM-relevant gene and protein expressions effectively, so the BAP1 KD (UM phenotype) was then treated with doxycycline to evaluate its effect on UM metastasis. Subsequent analysis found that doxycycline significantly inhibited UM growth, angiogenesis, and oxidative phosphorylation in the BAP1 KD (UM phenotype) model more than that of the control model, perhaps due to doxycycline targeting higher regions with more mitochondrial activity, indicating doxycyclines therapeutic potential in treating UM.