title: Flow cytometric detection of prostate tumor cells using chemoaffinity labels.
abstract: BACKGROUND
  The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an emerging target for imaging and therapeutic applications for prostate cancer. However, the use of PSMA for detecting circulating prostate tumor cells remains under-explored. The present study focuses on the specific labeling of PSMA+ prostate cancer cells with a fluorescent PSMA inhibitor and the quantitation of PSMA+ cells in blood by flow cytometry (FC) using a gating strategy to separate labeled PSMA+ cells from peripheral blood mononuclear cells.
  
  
  METHODS
  Suspensions of PSMA+ (LNCaP) and PSMA- (DU145) cells were incubated with the fluorescent PSMA inhibitor FAMX-CTT-54. Incubation parameters (time, temperature, and label concentration) were varied to optimize cell labeling. A gating protocol based on double fluorescent labeling of CD45 and PSMA was developed for the quantitiation of LNCaP cells in the presence of white blood cells from bovine blood. Nonfluorescent beads were added to the labeled cell mixture and served as internal standard for precise cellular quantification of LNCaP cells by flow cytometry.
  
  
  RESULTS
  The fluorescent PSMA inhibitor FAMX-CTT-54 was specific for PSMA+ cells. The minimum time and concentration of FAMX-CTT-54 for effective labeling of PSMA+ cell suspensions at 37°C was 7.5 min and 35 nM, respectively; no labeling was observed on PSMA- cells. Co-incubation or pre-incubation of PSMA+ cells with the unlabeled PSMA inhibitor CTT-54 resulted in a concentration-dependent reduction in fluorescent labeling with FAMX-CTT-54 thereby confirming that the labeling was specific for PSMA. In blood samples in which LNCaP cells were added, an average of five cells were detected in a 115 µl sample of the most dilute sample examined (29 cells/ml); three cells were expected theoretically. The greater loss of labeling of PSMA+ cells with FAMX-CTT-54 when pre-incubated with CTT-54 is consistent with the irreversible mode of binding of CTT-54 to PSMA and subsequent internalization of the PSMA-inhibitor complex.
  
  
  CONCLUSIONS
  The results suggest that fluorescent PSMA inhibitors can be utilized to effectively detect and quantify PSMA+ cells by FC. These results support the use of such compounds in the application of FC to detect, quantify, and characterize circulating prostate tumor cells.
authors.affiliation: Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
authors.forename: Lisa Y
authors.initials: LY
authors.lastname: Wu
authors.forename: Tiancheng
authors.initials: T
authors.lastname: Liu
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Grimm
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
authors.forename: Clifford E
authors.initials: CE
authors.lastname: Berkman
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis*; D014408:Biomarkers, Tumor / Q000032:analysis*; D002277:Carcinoma / Q000737:chemistry / Q000175:diagnosis*; D002417:Cattle; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D043425:Glutamate Carboxypeptidase II / Q000032:analysis* / Q000037:antagonists & inhibitors; D006801:Humans; D008297:Male; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000175:diagnosis*
chemical_list: D000345:Affinity Labels; D000954:Antigens, Surface; D014408:Biomarkers, Tumor; C554453:FAMX-CTT-54; D005452:Fluoresceins; D009943:Organophosphorus Compounds; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II