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Open AccessArticle Development of DNA Aptamers to Native EpCAM for Isolation of Lung Circulating Tumor Cells from Human Blood
Cancers 2019, 11(3), 351; https://doi.org/10.3390/cancers11030351
Received: 6 February 2019 / Revised: 7 March 2019 / Accepted: 8 March 2019 / Published: 12 March 2019
Viewed by 396 | PDF Full-text (6308 KB) | HTML Full-text | XML Full-text | Supplementary Files
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue New Biomarkers in Cancers)
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Figure 1

Figure 1
<p>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.</p>
Full article ">Figure 2
<p>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.</p>
Full article ">Figure 3
<p>Competitive displacement of aptamers with antibodies. (<bold>A</bold>) Flow cytometry of LC cells and LC cells preincubated with Cy-3 labeled anti-EpCAM or anti-α-Tubulin antibodies. (<bold>B</bold>) Flow cytometry of LC cells (red), LC cells preincubated with 6-carboxyfluorescein (FAM)-labeled EPCAM-APT-01, EPCAM-APT-02 or oligonucleotide (AG)<sub>40</sub> before (green) and after (blue) replacement by Cy-3 labeled anti-EpCAM or anti-α-Tubulin antibodies.</p>
Full article ">Figure 4
<p>Aptamer affinity curves. The percentage of bound LC cells measured by flow cytometry versus concentrations of EPCAM-APT-01 or EPCAM-APT-02.</p>
Full article ">Figure 5
<p>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 (<bold>A</bold>) and EPCAM-APT-02 (<bold>B</bold>). (<bold>A1</bold>,<bold>A5</bold>,<bold>B1</bold>,<bold>B5</bold>)—fluorescence of Cy-5-labeled aptamers, (<bold>A2</bold>,<bold>A6</bold>,<bold>B2</bold>,<bold>B6</bold>)—fluorescence of Alexa 405-labeled anti-EpCAM antibodies, (<bold>A3</bold>,<bold>A7</bold>,<bold>B3</bold>,<bold>B7</bold>)—overlays, (<bold>A4</bold>,<bold>A8</bold>,<bold>B4</bold>,<bold>B8</bold>)—overlaid fluorescence intensity spectra from the marked (<bold>A3</bold>,<bold>A7</bold>,<bold>B3</bold>,<bold>B7</bold>)—regions.</p>
Full article ">Figure 5 Cont.
<p>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 (<bold>A</bold>) and EPCAM-APT-02 (<bold>B</bold>). (<bold>A1</bold>,<bold>A5</bold>,<bold>B1</bold>,<bold>B5</bold>)—fluorescence of Cy-5-labeled aptamers, (<bold>A2</bold>,<bold>A6</bold>,<bold>B2</bold>,<bold>B6</bold>)—fluorescence of Alexa 405-labeled anti-EpCAM antibodies, (<bold>A3</bold>,<bold>A7</bold>,<bold>B3</bold>,<bold>B7</bold>)—overlays, (<bold>A4</bold>,<bold>A8</bold>,<bold>B4</bold>,<bold>B8</bold>)—overlaid fluorescence intensity spectra from the marked (<bold>A3</bold>,<bold>A7</bold>,<bold>B3</bold>,<bold>B7</bold>)—regions.</p>
Full article ">Figure 6
<p>Aptamer-facilitated isolation of circulating tumor cells (CTCs). CTCs were isolated from the blood of two LC patients: ID#101 (<bold>A1</bold>–<bold>A3</bold>) and ID#113 (<bold>B1</bold>–<bold>B3</bold>,<bold>C1</bold>–<bold>C3</bold>), using biotinylated aptamers EPCAM-APT-01 and EPCAM-APT-02 and then stained with the same fluorescent aptamers.</p>
Full article ">
Open AccessReview Current and Prospective Protein Biomarkers of Lung Cancer
Cancers 2017, 9(11), 155; https://doi.org/10.3390/cancers9110155
Received: 12 October 2017 / Revised: 2 November 2017 / Accepted: 6 November 2017 / Published: 13 November 2017
Cited by 13 | Viewed by 2949 | PDF Full-text (1671 KB) | HTML Full-text | XML Full-text
Abstract
Lung cancer is a malignant lung tumor with various histological variants that arise from different cell types, such as bronchial epithelium, bronchioles, alveoli, or bronchial mucous glands. The clinical course and treatment efficacy of lung cancer depends on the histological variant of the [...] Read more.
Lung cancer is a malignant lung tumor with various histological variants that arise from different cell types, such as bronchial epithelium, bronchioles, alveoli, or bronchial mucous glands. The clinical course and treatment efficacy of lung cancer depends on the histological variant of the tumor. Therefore, accurate identification of the histological type of cancer and respective protein biomarkers is crucial for adequate therapy. Due to the great diversity in the molecular-biological features of lung cancer histological types, detection is impossible without knowledge of the nature and origin of malignant cells, which release certain protein biomarkers into the bloodstream. To date, different panels of biomarkers are used for screening. Unfortunately, a uniform serum biomarker composition capable of distinguishing lung cancer types is yet to be discovered. As such, histological analyses of tumor biopsies and immunohistochemistry are the most frequently used methods for establishing correct diagnoses. Here, we discuss the recent advances in conventional and prospective aptamer based strategies for biomarker discovery. Aptamers like artificial antibodies can serve as molecular recognition elements for isolation detection and search of novel tumor-associated markers. Here we will describe how these small synthetic single stranded oligonucleotides can be used for lung cancer biomarker discovery and utilized for accurate diagnosis and targeted therapy. Furthermore, we describe the most frequently used in-clinic and novel lung cancer biomarkers, which suggest to have the ability of differentiating between histological types of lung cancer and defining metastasis rate. Full article
(This article belongs to the Special Issue Aptamers: Promising Tools for Cancer Diagnosis and Therapy)
Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The new World Health Organization (WHO) classification of lung cancer histological types. The various types of lung cancer have different origins and histological features (<xref ref-type="fig" rid="cancers-09-00155-f002">Figure 2</xref>). Small-cell lung carcinoma (SCLC) is characterized by small size cells, absence of differentiation, fast tumor growth, metastasis at early stages, and release of specific biomarkers and hormones. At present, there are two points of view on SCLC histogenesis. According to the first hypothesis, SCLC arises from cells of the diffuse endocrine system, i.e., the amine precursor uptake decarboxylation (APUD)-system (<xref ref-type="fig" rid="cancers-09-00155-f002">Figure 2</xref>); the second suggests this type of lung cancer originates from the endodermbronchial lining layer [<xref ref-type="bibr" rid="B10-cancers-09-00155">10</xref>]. CA: carcinoma.</p>
Full article ">Figure 2
<p>Histogenesis of histological types of lung cancer. SM—Smooth Muscle; M—Macrophage; L—Lymphocyte; NC—Neuroendocrine Cell; EC—Epithelial Cell; SC—Secretory Cell.</p>
Full article ">Figure 3
<p>Schematic representation of aptamer based biomarker discovery. Affinity purification of aptamer protein targets: (<bold>a</bold>) from whole cells; (<bold>b</bold>) form cell lysates.</p>
Full article ">Figure 4
<p>Schematic representation of aptamer-based lung cancer diagnostic tools. (<bold>a</bold>) analyses of blood plasma oncomarkers using electrochemical detection; (<bold>b</bold>) circulating tumor cells capture and fluorescence detection ; (<bold>c</bold>) aptamer based immunohistochemistry-like characterization of lung cancer histological structure.</p>
Full article ">Figure 5
<p>Biomarkers of Small Cell Lung Cancer (<bold>a</bold>), Squamous Lung Cancer (<bold>b</bold>), Lung Adenocarcinoma (<bold>c</bold>), Large Cell Lung Cancer (<bold>d</bold>). GRP: gastrin-releasing peptide ; CEA: carcinoembryonic antigen ; NSE: neuron specific enolase; SCCA: squamous cell carcinoma antigen ; CYFRA 21-1: cytokeratins ; Sid5 : Systemic RNA interference defective protein 5 ; Psf1-Psf3: GINS complex subunits 1-3.</p>
Full article ">

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