Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype
Circulating tumor cells entering epithelial-mesenchymal transition can lose the surface epithelial markers that capture-based methods depend on; this AACR 2018 poster reports a 5-parameter immunofluorescence assay that identifies CTCs without surface-marker capture and applies it to 5 prostate and 3 breast cancer patients.
Presented by RareCyte and the University of Washington.
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- A 5-parameter immunofluorescence assay tells epithelial CTCs apart from those carrying mesenchymal markers. Sytox orange stains nuclei, a cytokeratin and EpCAM cocktail marks epithelial cells, vimentin marks mesenchymal differentiation, a CD45, CD11b and CD105 cocktail excludes white blood cells, and EGFR is read as an investigational biomarker; cells are spread onto slides with AccuCyte and imaged on CyteFinder rather than captured on their surface markers.
- In 5 prostate and 3 breast cancer patients, CTC counts ranged from 1 to 164 per sample. Epithelial CTCs averaged 64% of the CTCs found in a patient and epithelial-mesenchymal CTCs 36%, each spanning the full 0 to 100% range, and EGFR-positive cells appeared in both classes; assay specificity was established first against COLO 205 and SKBR-3, which stayed vimentin-negative, and MDA-MB-231 and A549, which stained vimentin-positive.
- Single-cell sequencing confirmed that the epithelial-mesenchymal cells were malignant. In a prostate cancer patient whose tumor carried a TP53 G244S mutation, individual cells retrieved with the CytePicker module were whole-genome amplified and sequenced: the mutation appeared in 2 of 2 epithelial CTCs and 6 of 7 epithelial-mesenchymal CTCs, and in 0 of 6 control white blood cells.
