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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 469 | 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)
Figures

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 3059 | 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 ">
Open AccessReview
Targeted Magnetic Nanotheranostics of Cancer
Molecules 2017, 22(6), 975; https://doi.org/10.3390/molecules22060975
Received: 19 April 2017 / Revised: 2 June 2017 / Accepted: 6 June 2017 / Published: 12 June 2017
Cited by 3 | Viewed by 2402 | PDF Full-text (2713 KB) | HTML Full-text | XML Full-text
Abstract
Current advances in targeted magnetic nanotheranostics are summarized in this review. Unique structural, optical, electronic and thermal properties of magnetic materials in nanometer scale are attractive in the field of biomedicine. Magnetic nanoparticles functionalized with therapeutic molecules, ligands for targeted delivery, fluorescent and [...] Read more.
Current advances in targeted magnetic nanotheranostics are summarized in this review. Unique structural, optical, electronic and thermal properties of magnetic materials in nanometer scale are attractive in the field of biomedicine. Magnetic nanoparticles functionalized with therapeutic molecules, ligands for targeted delivery, fluorescent and other chemical agents can be used for cancer diagnostic and therapeutic purposes. High selectivity, small size, and low immunogenicity of synthetic nucleic acid aptamers make them attractive delivery agents for therapeutic purposes. Properties, production and functionalization of magnetic nanoparticles and aptamers as ligands for targeted delivery are discussed herein. In recent years, magnetic nanoparticles have been widely used in diagnostic methods, such as scintigraphy, single photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), and Raman spectroscopy. Therapeutic purposes of magnetic nanoconstructions are also promising. They are used for effective drug delivery, magnetic mediated hypertermia, and megnetodynamic triggering of apoptosis. Thus, magnetic nanotheranostics opens a new venue for complex differential diagnostics, and therapy of metastatic cancer. Full article
Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Illustration of superparamagnetic and ferromagnetic particles in the presence and absence of a magnetic field (MF), and after exposure to a MF. In the presence of an alternating magnetic field, the magnetic moment of both superparamagnetic and ferromagnetic nanoparticles are aligned. Upon removal of the magnetic field, the nanoparticles maintain the net magnetization.</p>
Full article ">Figure 2
<p>Size scale of MNS as compared to biomolecules.</p>
Full article ">Figure 3
<p>Schematic illustration of a multifunctional magnetic nanoparticle structure with different types of coatings, target ligands and imaging agents. Therapeutic drugs can be embedded in the coating, or conjugated on the surface.</p>
Full article ">Figure 4
<p>Modes of tumor-targeting magnetic nanoparticles. (<bold>A</bold>) Passive targeting (enhanced permeability and retention (EPR) effect) of magnetic nanoparticles. Nanoparticles reach tumor cells selectively through the leaky vasculature surrounding the tumors; (<bold>B</bold>) Active (molecular targeting). Ligands (aptamers, antibodies, peptides, small molecules, etc.) linked with magnetic nanoparticles that bind to receptors overexpressed by tumor cells; (<bold>C</bold>) Magnetic targeting.</p>
Full article ">Figure 5
<p>Schematic representation of the two mechanisms of controlled drug delivery using a magnetic field based hyperthermia. (<bold>a</bold>) Magnetic hyperthermia-based controlled drug delivery through enhanced permeability; (<bold>b</bold>) Magnetic hyperthermia-based controlled drug delivery through bond breaking (linkers).</p>
Full article ">Figure 6
<p>Principle of magnetic mediated hyperthermia. Targeted magnetic nanoparticles delivered to tumor cells are exposed to an alternating magnetic field (AMF). Afterword, AMF energy is converted into heat by the magnetic nanoparticles, which leads to local heating of tumor cells between 41 and 47 °C.</p>
Full article ">Figure 7
<p>The concept of targeted magnetomechanical cancer-cell destruction using magnetic nanoparticles with different shape.</p>
Full article ">

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