title: Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining.
abstract: In tissue biopsies formalin fixed paraffin embedded cancer blocks are micro-sectioned producing multiple semi-identical specimens which are analyzed and subtyped proteomically, and genomically, with numerous biomarkers. In blood based biopsies (BBBs), blood is purified for circulating tumor cells (CTCs) and clinical utility is typically limited to cell enumeration, as only 2-3 positive fluorescent markers and 1 negative marker can be used. As such, increasing the number of subtyping biomarkers on each individual CTC could dramatically enhance the clinical utility of BBBs, allowing in depth interrogation of clinically relevant CTCs. We describe a simple and inexpensive method for quenching the specific fluors of fluorescently stained CTCs followed by sequential restaining with additional biomarkers. As proof of principle a CTC panel, immunosuppression panel and stem cell panel were used to sequentially subtype individual fluorescently stained patient CTCs, suggesting a simple and universal technique to analyze multiple clinically applicable immunomarkers from BBBs.
authors.affiliation: Creatv MicroTech, Inc., 1 Deer Park Dr. Monmouth Junction, NJ 08850, USA.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Adams
authors.affiliation: Fox Chase Cancer Center, Protocol Support Laboratory, 333 Cottman Ave., Philadelphia, PA 19111, USA.
authors.forename: R Katherine
authors.initials: RK
authors.lastname: Alpaugh
authors.affiliation: The Medical College of Wisconsin Milwaukee, Milwaukee, WI 53226, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Tsai
authors.affiliation: Creatv MicroTech, Inc. 9900 Belward Campus Dr., Rockville, MD 20850, USA.
authors.forename: Cha-Mei
authors.initials: CM
authors.lastname: Tang
authors.affiliation: HeMemics Biotechnologies Inc., 12111 Parklawn Drive Rockville, MD 20852, USA.
authors.forename: Steingrimur
authors.initials: S
authors.lastname: Stefansson
mesh_terms: D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D058750:Epithelial-Mesenchymal Transition; D005453:Fluorescence; D006801:Humans; D009360:Neoplastic Cells, Circulating / Q000145:classification* / Q000473:pathology; D013194:Staining and Labeling