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Carlos Rogerio De Figueiredo, Helen Kalirai, Joseph Sacco, Judy Coulson, Sarah E Coupland; Using Mass-Cytometry approach for the characterisation of the microenvironment of primary uveal melanoma.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):748.
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© ARVO (1962-2015); The Authors (2016-present)
Immunotherapy using immune checkpoint inhibitors (ICIs) induces durable responses in an increasing number of metastatic cancers. However, some tumours are resistant to ICIs or develop escape mechanisms. Metastatic uveal melanoma (mUM) is one of the most refractory cancers to ICIs, with an average survival less than 12 month. Understanding how immunoediting progresses towards immune escape in primary uveal melanoma (pUM), at both protein and single-cell level, is critical to elucidate mechanisms of immune tolerance, which may correlate with metastatic disease progression. To proceed with these studies, we sought to apply a high-resolution mass cytometry (CyTOF) approach that will allow the interrogation of 40-50 parameters at single-cell level.
Fresh pUM enucleations from consented patients were used for these trials. Briefly, single-cell suspensions were prepared by mechanical and enzymatic disruption using collagenase-based digestion and analysed by CyTOF as described. Live/dead cells were identified using the DNA marker cisplatin. Subsequent immune stainings were performed using a bespoke panel of metal-chelated antibodies, including multiple phenotyping markers. Cell-ID Intercalator-Ir (Fluidigm) was used to identify nucleated cells. Immune cell populations were gated using positive/negative selection and further quantified using viSNE in Cytobank software.
Five successful single-cell staining preparations of pUM enucleations revealed that most CD45+ infiltrated cells were monocytes (CD45+CD14+) and lymphocytes (CD45+CD3+), with both frequencies varying from 20% to 60%. We found a predominant ratio of 4:1 between CD8+ and CD4+ T lymphocytes. In addition, the majority of monocytes showed macrophage specific markers. We also detected B cells (CD45+CD19+, ~5%), but neutrophils (CD45+CD662bhi) and natural killer cells (CD45+CD16+CD56+) were nearly absent, 0.2% and 0.01%, respectively.
We have validated a methodology to evaluate the immune infiltrates of fresh pUM by CyTOF. Further development of the bespoke antibody panel will enable us to dissect the activation and functional level of the detected immune cells, as well as their frequency throughout a large cohort of pUM and mUM.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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