Identification of Circulating Tumor Cells in Patients with Lung Cancer using DNA Aptamers

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Abstract

BACKGROUND: Lung cancer (LC) is one of the most prevalent types of cancer, with mortality rates reaching 25% of all cancer-related deaths. The majority of patients are classified as stage IV at the time of diagnosis, a stage at which the probability of successful treatment is considerably low. Conventional diagnostic methods for lung cancer are expensive, labor-intensive, and highly invasive if a biopsy is performed. Consequently, liquid biopsy, which involves the extraction of circulating tumor cells from blood samples, has emerged as a pivotal area of research in oncology.

AIM: To develop a method for the isolation and identification of circulating tumor cells in the peripheral blood of patients with lung cancer using DNA aptamers.

MATERIALS AND METHODS: The study objects are lung cancer tissue, blood of patients with lung cancer and primary lung cancer cell culture. Gold-decorated magnetic nanoparticles and thiolated aptamers LC-17, LC-183 and LC-224 were used to isolate proteins. A hybrid of a thiol primer and a non-specific DNA sequence composed of two AG nucleotide repeats was used as a control. Mass spectrometry was performed using UltiMate 3000 nano-UHPLC system coupled with Orbitrap Fusion mass spectrometer (Thermo Scientific, USA). Circulating tumor cell counts were measured on CytoFLEX flow cytometer (Beckman Coulter, USA) after triple staining. Fluorescence microscopy was used for CSC visualization.

RESULTS: The potential protein targets of the aptamers LC-17, LC-183, and LC-224 were identified. These aptamers were then used to isolate circulating tumor cells from the blood of patients with lung cancer. The identification of circulating tumor cells was performed using flow cytometry and fluorescence microscopy.

CONCLUSIONS: The proposed method for the identification of circulating tumor cells through magnetic separation and flow cytometry provides a quantitative analysis of the target analyte, facilitated by the use of cell-specific lung cancer aptamers, including LC-17, LC-183, and LC-224, which exhibit high-affinity binding properties.

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About the authors

Aleksey V. Krat

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky

Email: alexkrat@mail.ru
ORCID iD: 0009-0006-5357-2637
SPIN-code: 2846-8592

MD, Cand. Sci. (Medicine)

Russian Federation, Krasnoyarsk; Krasnoyarsk

Daria A. Kirichenko

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: astheno@mail.ru
ORCID iD: 0000-0003-4087-731X
SPIN-code: 2401-6465

Cand. Sci. (Biology)

Russian Federation, Krasnoyarsk

Galina S. Zamay

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: galina.zamay@gmail.com
ORCID iD: 0000-0002-2567-6918
SPIN-code: 6501-0371

Cand. Sci. (Biology)

Russian Federation, Krasnoyarsk; Krasnoyarsk

Ruslan A. Zukov

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky

Email: zukov_rus@mail.ru
ORCID iD: 0000-0002-7210-3020
SPIN-code: 3632-8415

MD, Cand. Sci. (Medicine)

Russian Federation, Krasnoyarsk; Krasnoyarsk

Olga S. Kolovskaya

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: olga.kolovskaya@gmail.com
ORCID iD: 0000-0002-2494-2313
SPIN-code: 2254-5474

Dr. Sci. (Biology)

Russian Federation, Krasnoyarsk; Krasnoyarsk

Tatiana N. Zamay

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: tzamay@yandex.ru
ORCID iD: 0000-0002-7493-8742
SPIN-code: 8799-8497

Dr. Sci. (Biology)

Russian Federation, Krasnoyarsk; Krasnoyarsk

Viktoria D. Fedotovskaya

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: viktoriia.fedotovskaia@gmail.com
ORCID iD: 0000-0002-6472-0782
SPIN-code: 4500-4728
Russian Federation, Krasnoyarsk; Krasnoyarsk

Anastasia A. Koshmanova

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: koshmanova.1998@mail.ru
ORCID iD: 0000-0001-7339-8660
SPIN-code: 2217-2229
Russian Federation, Krasnoyarsk

Natalia A. Luzan

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: laskimo@mail.ru
ORCID iD: 0009-0001-8983-5017
SPIN-code: 1280-0005
Russian Federation, Krasnoyarsk

Semen A. Sidorov

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky

Email: sidorov.syoma2014@yandex.ru
ORCID iD: 0000-0001-6676-6656
SPIN-code: 8740-5259
Russian Federation, Krasnoyarsk; Krasnoyarsk

Kirill A. Lukyanenko

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: k.a.lukyanenko@yandex.ru
ORCID iD: 0000-0002-1115-6735
SPIN-code: 2154-2046
Russian Federation, Krasnoyarsk; Krasnoyarsk

Yury E. Glazyrin

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: yury.glazyrin@gmail.com
ORCID iD: 0000-0002-2826-5751
SPIN-code: 2079-1786

Cand. Sci. (Biology)

Russian Federation, Krasnoyarsk; Krasnoyarsk

Yuri S. Pats

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: y.patz@mail.ru

MD, Cand. Sci. (Medicine)

Russian Federation, Krasnoyarsk

Olga V. Kryukova

Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: marta913@mail.ru
ORCID iD: 0000-0001-5241-5409
SPIN-code: 5882-0170

Cand. Sci. (Biology)

Russian Federation, Krasnoyarsk

Anna S. Kichkailo

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences

Email: annazamay@yandex.ru
ORCID iD: 0000-0003-0690-7837
SPIN-code: 5387-9071

Dr. Sci. (Biology)

Russian Federation, Krasnoyarsk; Krasnoyarsk

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Supplementary files

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2. Fig. 1. General chromatogram of the mass spectrometric experiment for sample LC-17 (a), primary mass spectrum obtained at 52.62 minutes of the experiment (b) and second-order mass spectrum containing characteristic fragments of the WQEEMELYR peptide unique to the apolipoprotein A-I protein (m/z 642.29) (c).

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3. Fig. 2. Analyte fractions determined by flow cytometry. From left to right: magnetic particles; leukocytes isolated from the blood of a patient with non-small cell lung cancer; non-small cell lung cancer cells isolated from a post-operative tissue sample of the patient; circulating tumor cells isolated from the blood of a patient with non-small cell lung cancer using magnetic particles.

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4. Fig. 3. An example of a flow cytometry data processing sequence. From left to right: top — histogram of cells bound to aptamers (P1); histogram of cells bound to CD45 antibodies (P5); histogram of cells with nuclei stained with DAPI (P6).

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5. Fig. 4. Cell samples isolated from blood samples of patients with lung cancer. From left to right — phase contrast; cells stained with CD45 antibodies, DAPI and aptamers; cells stained with CD45 antibodies. In the middle strip of the figure, in the center, there is a dead circulating tumor cell without a nucleus, and at the bottom, a multinucleated a circulating tumor cell.

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