title: Multiparameter Evaluation of the Heterogeneity of Circulating Tumor Cells Using Integrated RNAIn SituHybridization and Immunocytochemical Analysis.
abstract: Circulating tumor cells (CTCs) are routinely identified as cytokeratin (CK)-positive, epithelial cell adhesion molecule (EpCAM)-positive, and CD45-negative and are enriched based on EpCAM. However, there are a number of methodological challenges regarding both isolation and characterization of these rare CTCs including downregulation or absence of EpCAM in a variety of solid tumors leading to the omission of subpopulations of CTCs, difficulties in analyzing RNA and protein targets in CTCs due to the rarity of these cells, and low levels of targets and technological limitations of visualizing the targets of interest on each individual cell. Building on our previous CTC research on CD45-based negative magnetic separation and four-color fluorescent immunocytochemical (ICC) staining, RNAin situhybridization (ISH) was applied to fluorescently target mRNA sequences corresponding to tumor-related genes at the single CTC level. Multiple categories of markers are targeted including CK, human epidermal growth factor receptor family markers, Hedgehog pathway markers, human papillomavirus markers, and protein arginine methyltransferase 5. In addition, an integrated method of RNA ISH and fluorescent ICC staining was developed to visualize CTCs on both mRNA and protein levels. The robustness of the integrated co-ICC and RNA ISH staining was demonstrated by a series of tests on representative tumor markers of different categories. The integrated staining can incorporate the advantages of both RNA ISH and fluorescent ICC staining and provide more intense signals and more specific bindings. With this integrated staining methodology, distinct staining patterns were applied in this report to facilitate the searching and characterization of rare subgroups of CTCs. These results support the existence of diverse groups of CTCs at both protein and mRNA transcript levels and provide an analytical tool for the research on CTCs of rare subgroups.
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University , Columbus, OH , USA.
authors.forename: Yongqi
authors.initials: Y
authors.lastname: Wu
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University , Columbus, OH , USA.
authors.forename: Kyoung-Joo Jenny
authors.initials: KJ
authors.lastname: Park
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University , Columbus, OH , USA.
authors.forename: Clayton
authors.initials: C
authors.lastname: Deighan
authors.affiliation: Department of Biomedical Engineering, The Ohio State University , Columbus, OH , USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Amaya
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University , Columbus, OH , USA.
authors.forename: Brandon
authors.initials: B
authors.lastname: Miller
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, The James Cancer Hospital and Solove Research Institute, Wexner Medical Center at The Ohio State University , Columbus, OH , USA.
authors.forename: Quintin
authors.initials: Q
authors.lastname: Pan
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic , Cleveland, OH , USA.
authors.forename: Maciej
authors.initials: M
authors.lastname: Zborowski
authors.affiliation: Breast Medical Oncology, Department of Internal Medicine, James Cancer Hospital, Ohio State University Comprehensive Cancer Center , Columbus, OH , USA.
authors.forename: Maryam
authors.initials: M
authors.lastname: Lustberg
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA; Analytical Cytometry Shared Resource, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Jeffery
authors.initials: J
authors.lastname: Chalmers
keywords: circulating tumor cells; fluorescent labeling; integrated RNA ISH and ICC staining; multiparameter analysis; negative magnetic enrichment