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. 2008 Oct 15;123(8):1968-73.
doi: 10.1002/ijc.23717.

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands

Wei He  1 , Sumith A Kularatne, Kimberly R Kalli, Franklyn G Prendergast, Robert J Amato, George G Klee, Lynn C Hartmann, Philip S Low
Affiliations

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands

Wei He et al. Int J Cancer. .

Abstract

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.

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Figures

Figure 1
Figure 1
Comparison of KB cell labeling intensity with folate-FITC versus various anti-FR antibodies. KB cells were labeled with (i) folate-FITC (green), (ii) affinity-purified polyclonal anti-FR antibody directly labeled with FITC (PU17-FITC, orange), (iii) monoclonal anti-FR antibody (mAb343, cyan) followed by FITC-labeled goat anti-mouse IgG or (iv) affinity-purified polyclonal anti-FR antibody (PU17, navy) followed by FITC-labeled rat anti-rabbit IgG prior to analysis by flow cytometry. Unlabeled KB cells (black) and KB cells incubated with 10 μM folic acid plus 100 nM folate-FITC (yellow, totally competed) served as negative controls.
Figure 2
Figure 2
CTC quantitation in blood samples from ovarian and prostate cancer patients. Dots in (a) and (b) represent mean values from multiple independent measurements of CTCs in blood samples from individual healthy subjects, or ovarian or prostate cancer patients. Bars in (a) and (b) indicate the average number of CTCs in the collection of blood samples from healthy donors, or ovarian or prostate cancer patients. Threshold values of 1.5 CTCs/ml for ovarian cancer samples and 3 CTC/ml for prostate cancer samples are indicated by dashed lines in (a) and (b).
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