A flexible enrichment strategy for the isolation and single-cell characterization of disease-specific circulating tumor cells

Background: Circulating tumor cells (CTCs) are cells that have broken off from a primary solid tumor, entered the blood stream, and have the potential to metastasize to nearby tissues. Thus, CTCs represent an important biomarker to monitor cancer progression and patient outcome. The isolation, identification and characterization of CTCs are critical for this goal. We previously developed a method for the detection and isolation of melanoma CTCs from peripheral blood through negative and positive antibody enrichment. Methods: Here we show that this protocol is easily applied to other cancers; in this case we expand and validate this method to include prostate cancer. Results: We show that this combined negative and positive enrichment protocol successfully isolates CTCs for characterization using single cell RNA sequencing and have updated the protocol to include a newer version of single cell library preparation, Smart-seq3. In our analysis, cells prepared for sequencing with Smart-seq3, which uses unique molecular identifiers (UMIs), showed no considerable difference compared to those prepared using the previous iteration of the protocol, Smart-seq2. Conclusion: This study demonstrates a robust and flexible method for the isolation and characterization of CTCs from various cancers for potential use as biomarkers for disease management and for characterization with single cell RNA-seq.