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. 2019 Mar 12;11(3):351.
doi: 10.3390/cancers11030351.

Development of DNA Aptamers to Native EpCAM for Isolation of Lung Circulating Tumor Cells from Human Blood

Galina S Zamay  1   2 , Olga S Kolovskaya  3   4 , Tatiana I Ivanchenko  5 , Tatiana N Zamay  6   7 , Dmitry V Veprintsev  8 , Valentina L Grigorieva  9 , Irina I Garanzha  10 , Alexey V Krat  11   12 , Yury E Glazyrin  13   14 , Ana Gargaun  15 , Ivan N Lapin  16 , Valery A Svetlichnyi  17 , Maxim V Berezovski  18 , Anna S Kichkailo  19   20
Affiliations

Development of DNA Aptamers to Native EpCAM for Isolation of Lung Circulating Tumor Cells from Human Blood

Galina S Zamay et al. Cancers (Basel). .

Abstract

We selected DNA aptamers to the epithelial cell adhesion molecule (EpCAM) expressed on primary lung cancer cells isolated from the tumors of patients with non-small cell lung cancer using competitive displacement of aptamers from EpCAM by a corresponding antibody. The resulting aptamers clones showed good nanomolar affinity to EpCAM-positive lung cancer cells. Confocal microscopy imaging and spectral profiling of lung cancer tissues confirmed the same protein target for the aptamers and anti-EpCAM antibodies. Furthermore, the resulted aptamers were successfully applied for isolation and detection of circulating tumor cells in clinical samples of peripheral blood of lung cancer patients.

Keywords: EpCAM; SELEX (Systematic evolution of ligands by exponential enrichment); aptamers; blood; circulating tumor cells; non-small-cell lung cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The scheme of DNA aptamer selection using aptamer displacement via antibody. The first several rounds include only positive selection and start with the incubation of the ssDNA library or aptamer pools with receptor positive cells, followed by partitioning unbound DNA, and amplifying bound DNA with symmetric and asymmetric polymerase chain reaction (PCR). In the next rounds, positive rounds alternate with antibody displacement steps and include the incubation of aptamers with the receptor positive cells, washing, the displacement of the bound aptamers by antibodies (Ab), and the following amplification of free aptamers.
Figure 2
Figure 2
Binding evaluation of aptamer pools. Flow cytometry of lung cancer (LC) cells incubated with pools of 6–9th rounds of aptamer selection against EpCAM at the first step and displaced by EpCAM antibodies at the second step in comparison with LC cells alone.
Figure 3
Figure 3
Competitive displacement of aptamers with antibodies. (A) Flow cytometry of LC cells and LC cells preincubated with Cy-3 labeled anti-EpCAM or anti-α-Tubulin antibodies. (B) Flow cytometry of LC cells (red), LC cells preincubated with 6-carboxyfluorescein (FAM)-labeled EPCAM-APT-01, EPCAM-APT-02 or oligonucleotide (AG)40 before (green) and after (blue) replacement by Cy-3 labeled anti-EpCAM or anti-α-Tubulin antibodies.
Figure 4
Figure 4
Aptamer affinity curves. The percentage of bound LC cells measured by flow cytometry versus concentrations of EPCAM-APT-01 or EPCAM-APT-02.
Figure 5
Figure 5
Co-staining aptamers and antibodies. Confocal microscopy of different regions of two squamous LC tissue sections stained with Alexa-Fluor 405-labeled anti-EpCAM antibodies and Cy-5-labeled aptamers EPCAM-APT-01 (A) and EPCAM-APT-02 (B). (A1,A5,B1,B5)—fluorescence of Cy-5-labeled aptamers, (A2,A6,B2,B6)—fluorescence of Alexa 405-labeled anti-EpCAM antibodies, (A3,A7,B3,B7)—overlays, (A4,A8,B4,B8)—overlaid fluorescence intensity spectra from the marked (A3,A7,B3,B7)—regions.
Figure 5
Figure 5
Co-staining aptamers and antibodies. Confocal microscopy of different regions of two squamous LC tissue sections stained with Alexa-Fluor 405-labeled anti-EpCAM antibodies and Cy-5-labeled aptamers EPCAM-APT-01 (A) and EPCAM-APT-02 (B). (A1,A5,B1,B5)—fluorescence of Cy-5-labeled aptamers, (A2,A6,B2,B6)—fluorescence of Alexa 405-labeled anti-EpCAM antibodies, (A3,A7,B3,B7)—overlays, (A4,A8,B4,B8)—overlaid fluorescence intensity spectra from the marked (A3,A7,B3,B7)—regions.
Figure 6
Figure 6
Aptamer-facilitated isolation of circulating tumor cells (CTCs). CTCs were isolated from the blood of two LC patients: ID#101 (A1–A3) and ID#113 (B1–B3,C1–C3), using biotinylated aptamers EPCAM-APT-01 and EPCAM-APT-02 and then stained with the same fluorescent aptamers.
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