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Flow cytometric detection of prostate tumor cells using chemoaffinity labels
Author(s) -
Wu Lisa Y.,
Liu Tiancheng,
Grimm Amanda L.,
Davis William C.,
Berkman Clifford E.
Publication year - 2010
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/pros.21221
Subject(s) - lncap , flow cytometry , glutamate carboxypeptidase ii , du145 , prostate cancer , peripheral blood mononuclear cell , circulating tumor cell , microbiology and biotechnology , chemistry , cancer research , in vitro , medicine , cancer , biology , biochemistry , metastasis
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. Prostate 71: 52–61, 2011. © 2010 Wiley‐Liss, Inc.