An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters.

Autor:	Bhagwat N; Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Dulmage K; BD Technologies and Innovation, Research Triangle Park, NC, USA., Pletcher CH Jr; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Wang L; BD Technologies and Innovation, Research Triangle Park, NC, USA., DeMuth W; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Sen M; Division of Hematology and Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Balli D; Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Yee SS; Division of Hematology and Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Sa S; BD Biosciences, San Jose, CA, USA., Tong F; BD Technologies and Innovation, Research Triangle Park, NC, USA., Yu L; BD Biosciences, San Jose, CA, USA., Moore JS; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Stanger BZ; Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Dixon EP; BD Technologies and Innovation, Research Triangle Park, NC, USA., Carpenter EL; Division of Hematology and Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. erical@upenn.edu.
Jazyk:	angličtina
Zdroj:	Scientific reports [Sci Rep] 2018 Mar 22; Vol. 8 (1), pp. 5035. Date of Electronic Publication: 2018 Mar 22.
DOI:	10.1038/s41598-018-23217-5
Abstrakt:	Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200μm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu Plný text ve formátu PDF