Document Detail


Flow cytometric detection of prostate tumor cells using chemoaffinity labels.
MedLine Citation:
PMID:  20632319     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Lisa Y Wu; Tiancheng Liu; Amanda L Grimm; William C Davis; Clifford E Berkman
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural    
Journal Detail:
Title:  The Prostate     Volume:  71     ISSN:  1097-0045     ISO Abbreviation:  Prostate     Publication Date:  2011 Jan 
Date Detail:
Created Date:  2010-11-24     Completed Date:  2010-12-28     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8101368     Medline TA:  Prostate     Country:  United States    
Other Details:
Languages:  eng     Pagination:  52-61     Citation Subset:  IM    
Copyright Information:
© 2010 Wiley-Liss, Inc.
Affiliation:
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
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MeSH Terms
Descriptor/Qualifier:
Affinity Labels / chemistry*
Animals
Antigens, Surface / analysis*
Carcinoma / chemistry,  diagnosis*
Cattle
Cell Line, Tumor
Cell Separation / methods
Flow Cytometry / methods*
Fluoresceins / chemistry*
Glutamate Carboxypeptidase II / analysis*,  antagonists & inhibitors
Humans
Male
Organophosphorus Compounds / chemistry*
Prostatic Neoplasms / chemistry,  diagnosis*
Tumor Markers, Biological / analysis*
Grant Support
ID/Acronym/Agency:
1R21CA135463-01/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Affinity Labels; 0/Antigens, Surface; 0/FAMX-CTT-54; 0/Fluoresceins; 0/Organophosphorus Compounds; 0/Tumor Markers, Biological; EC 3.4.17.21/Glutamate Carboxypeptidase II; EC 3.4.17.21/glutamate carboxypeptidase II, human

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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