Hypoxia effect on proliferative activity of cells in orthotopic xenograft of hepatocellular carcinoma of the liver in the experiment Текст научной статьи по специальности «Клиническая медицина»

South Russian Journal of Cancer. 2024. Vol. 5, No. 2. P. 35-42

4.0

https://doi.org/10.37748/2686-9039-2024-5-2-4

https://elibrary.ru/mfunss

South Russian

ORIGINAL ARTICLE

Journal of Cancer

Южно-Российский

онкологический журнал

Vol. 5

Hypoxia effect on proliferative activity of cells in orthotopic No. 2, 2024

xenograft of hepatocellular carcinoma of the liver in the

experiment

T. M. Kecheryukova1, V. S. Trifanov1,2, A. A. Shulga1, A. S. Goncharova1, S. V. Gurova1, E. P. Ulyanova1, A. Yu. Maksimov1

1 National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation 2 P. A. Hertsen Moscow Oncology Research Institute – Branch of the National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation, Moscow, Russian Federation

slip.anka96@mail.ru

ABSTRACT

Purpose of the study. The purpose of this research was to investigate the effect of in vivo hypoxic conditions on the proliferative potential of HepG2 liver cancer cells.

Materials and methods. Human liver cancer cells of the HepG2 line have been cultured. The HepG2 cell suspension was injected subcutaneously into mice in an amount of 5 × 106 to obtain a xenograft. Tumor nodes that had reached the required size were divided into fragments and transplanted into the orthotopic site. Balb/c nude mice with implanted HepG2 liver cancer xenograft were used in this experiment. The mice with tumor implanted in the liver were divided into two groups, intact and hypoxic. Mice from the second group underwent liver blood flow reduction by occlusion of the portal triad for 20 minutes.

Tumor nodes were extracted for histological and immunohistochemical staining for proliferation marker Ki-67 on the 4th day after the procedures. The proportion of positively stained cells was calculated, and the results were statistically analyzed using the Statistica 10.0 software.

Results. Orthotopic models of liver cancer in Balb/c Nude mice were obtained. Histological and immunohistochemical studies were carried out. Histological analysis showed that hepatocellular carcinoma is characterized by an average degree of differentiation. In the tissues of these xenografts, by using immunohistochemical analysis for the proliferation marker Ki-67, it was possible to identify statistically significant differences between the two groups, i. e. intact and the one with reduction of blood flow. The proportion of immunopositive cells was 65 [65–70] % and 19 [15–25] %, respectively.

Conclusion. A tendency to decreased proliferative activity of tumor cells after hepatic blood flow reduction, i. e. hypoxia exposure, was demonstrated. Our data indicate that the proliferative activity of tumor cells is directly related to the microenvironment, and to the hypoxic environment in particular. Further study of the effect of hypoxia on the processes of growth and development of malignant tumors may contribute to a deeper understanding of the biological features of tumors and their treatment.

Keywords: hypoxia, liver, HepG2, proliferation, Ki-67, in vivo For citation: Kecheryukova T. M., Trifanov V. S., Shulga A. A., Goncharova A. S., Gurova S. V., Ulyanova E. P., Maksimov A. Yu. Hypoxia effect on proliferative activity of cells in orthotopic xenograft of hepatocellular carcinoma of the liver in the experiment. South Russian Journal of Cancer.

2024; 5(2): 35-42. https://doi.org/10.37748/2686-9039-2024-5-2-4, https://elibrary.ru/mfunss For correspondence: Anna A. Shulga – junior researcher at the Testing Laboratory center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

Address: 63 14 line str., Rostov-on-Don 344037, Russian Federation E-mail: slip.anka96@mail.ru

ORCID: https://orcid.org/0009-0006-1125-2897

SPIN: 6457-4451, AuthorID: 1221869

Compliance with ethical standards: when performing this study, all manipulations with laboratory animals were carried out in compliance with the Rules and Regulations for Carrying Out Animal Research Work. The study was approved by the Ethics Committee of the National Medical Research Center for Oncology (Protocol No. 4/108 dated 02/10/2021)

Funding: this work was not funded

Conflict of interest: the authors declare that there are no obvious and potential conflicts of interest associated with the publication of this article The article was submitted 08.11.2023; approved after reviewing 08.04.2024; accepted for publication 09.05.2024

© Kecheryukova T. M., Trifanov V. S., Shulga A. A., Goncharova A. S., Gurova S. V., Ulyanova E. P., Maksimov A. Yu., 2024

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Южно-Российский онкологический журнал. 2024. Т. 5, № 2. С. 35-42

https://doi.org/10.37748/2686-9039-2024-5-2-4

https://elibrary.ru/mfunss

3.1.6. Онкология, лучевая терапия

ОРИГИНАЛЬНАЯ СТАТЬЯ

Влияние гипоксии на пролиферативную активность клеток ортотопического

ксенографта гепатоцеллюлярной карциномы печени в эксперименте

Т. М. Кечерюкова1, В. С. Трифанов1,2, А. А. Шульга1, А. С. Гончарова1, С. В. Гурова1, Е. П. Ульянова1, А. Ю. Максимов1

1 ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация

2 Московский научно-исследовательский онкологический институт им. П. А. Герцена – филиал ФГБУ «Национальный медицинский

исследовательский центр радиологии» Министерства здравоохранения Российской Федерации, г. Москва, Российская Федерация

slip.anka96@mail.ru

РЕЗЮМЕ

Цель исследования. Оценить пролиферативную активность клеток рака печени HepG2 при моделировании гипок-сических условий in vivo.

Материалы и методы. Культивировали клетки рака печени человека линии HepG2. Для получения ксенографта клеточ-ную суспензию HepG2 вводили мышам подкожно в количестве 5 × 106. Достигшие необходимого размера опухолевые

узлы делили на фрагменты и трансплантировали в ортотопический сайт. В работе использовали мышей линии Balb/c Nude, которым имплантировали ксенографт рака печени HepG2. Мышей с прижившейся опухолью в печени делили

на две группы – интактная и с гипоксией. Мышам из второй группы выполняли редукцию кровотока печени путем

окклюзии портальной триады в течение 20 мин. На 4-е сутки после проведенных манипуляций опухолевые узлы

извлекали для выполнения гистологического и иммуногистохимического окрашивания на маркер пролиферации

Ki-67. Вычисляли долю позитивно окрашенных клеток и проводили статистический анализ результатов с помощью

пакета программ Statistica 10.0.

Результаты. Были получены ортотопические модели рака печени у мышей линии Balb/c Nude. Проведены гисто-логическое и иммуногистохимическое исследования. Гистологический анализ показал, что гепатоцеллюлярная

карцинома характеризуется средней степенью дифференцировки. В тканях данных ксенографтов с помощью иммуногистохимического анализа на маркер пролиферации Ki-67 удалось выявить статистически значимые различия

между двумя группами – интактной и с редукцией кровотока. Доля иммунопозитивных клеток составила 65 [65–70]

% и 19 [15–25] % соответственно.

Заключение. Продемонстрирована тенденция к снижению пролиферативной активности опухолевых клеток после

редукции кровотока печени, то есть воздействия гипоксии. Полученные нами данные свидетельствуют о том, что

пролиферативная активность клеток опухоли напрямую связана с микроокружением, в частности, с гипоксической

средой. Дальнейшее изучение воздействия гипоксии на процессы роста и развития злокачественных образований

может способствовать более глубокому пониманию биологических характеристик опухолей и их лечения.

Ключевые слова: гипоксия, печень, HepG2, пролиферация, Ki-67, in vivo Для цитирования: Кечерюкова Т. М., Трифанов В. С., Шульга А. А., Гончарова А. С., Гурова С. В., Ульянова Е. П., Максимов А. Ю. Влияние

гипоксии на пролиферативную активность клеток ортотопического ксенографта гепатоцеллюлярной карциномы печени в эксперименте.

Южно- Российский онкологический журнал. 2024; 5(2):35-42. https://doi.org/10.37748/2686-9039-2024-5-2-4, https://elibrary.ru/mfunss Для корреспонденции: Шульга Анна Александровна – младший научный сотрудник испытательного лабораторного центра, ФГБУ

«Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на- Дону, Российская Федерация

Адрес: 344037, Российская Федерация, г. Ростов-на- Дону, ул. 14-я линия, д. 63

E-mail: slip.anka96@mail.ru

ORCID: https://orcid.org/0009-0006-1125-2897

SPIN: 6457-4451, AuthorID: 1221869

Соблюдение этических стандартов: при выполнении данного исследования все манипуляции с лабораторными животными проводились

в соответствии с «Правилами проведения работ с использованием экспериментальных животных». Исследование одобрено этическим

комитетом ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской

Федерации (протокол № 4/108 от 10.02.2021 г.)

Финансирование: финансирование данной работы не проводилось

Конфликт интересов: все авторы заявляют об отсутствии явных и потенциальных конфликтов интересов, связанных с публикацией

настоящей статьи

Статья поступила в редакцию 08.11.2023; одобрена после рецензирования 08.04.2024; принята к публикации 09.05.2024

36

Южно-Российский онкологический журнал 2024. Т. 5, № 2. С. 35-42

Кечерюкова Т. М., Трифанов В. С., Шульга А. А., Гончарова А. С., Гурова С. В., Ульянова Е. П., Максимов А. Ю. Влияние гипоксии на пролиферативную

активность клеток ортотопического ксенографта гепатоцеллюлярной карциномы печени в эксперименте

INTRODUCTION

the features of their microenvironment.

The effect of the level of oxygenation is studied

The hypoxic environment, characterized by low

using various approaches, including in vitro, it is also oxygen content, plays a crucial role in the process-possible to use methods of isolated primary tumors,

es of cell survival and reprogramming. This fact is

but they do not accurately reflect the real parame-

confirmed by numerous studies on the evolution and

ters of the tumor microenvironment [11]. An analysis

development of organisms [1, 2]. Particularly, it has

of the literature data shows that the most reliable

been established that the normal development of

and trustworthy data can be obtained using in vivo

mammals occurs under conditions of hypoxia (mod-

methods that allow more accurately, compared with

erate to severe), which regulates many aspects of

other research approaches, to model the effect of

ontogenesis and morphogenesis. In addition, it is

hypoxia on the activity of malignant neoplasms and

known that the oxygen gradient is an important reg-

their proliferative potential, which may be important

ulator of cellular processes in both physiological and

for planning further translational studies [12, 13].

many pathological conditions, including malignant

The purpose of the study was to evaluate the

diseases [3].

proliferative activity of HepG2 liver cancer cells in

Sudden and short-term effects of hypoxia (from

modeling hypoxic conditions in vivo.

several minutes up to 72 hours), resulting from fluc-

tuations in tumor perfusion, are accompanied by

MATERIALS AND METHODS

functional and structural defects in the vascular

network of the tumor. Such exposure can lead to

Laboratory animals and their maintenance

the formation of high levels of reactive oxygen spe-

For this experiment, mice with Balb/c Nude immu-

cies (ROS), which can damage cells [4]. Hypoxia can

nodeficiency ( n = 14) 10–12 weeks old and weighing also cause the growth of cancer cells to stop, slow

25–27 g have been used and obtained from the vivar-

down proliferation and, subsequently, their death.

ium National Medical Research Center for Oncology,

It has been shown that hypoxia- induced factors di-

the Russian Federation Ministry of Health. The mice

rectly affect the proliferative activity of tumor cells

were in an IVC system (individually ventilated cages),

[5]. The most widely used marker of proliferation in

food and water were provided without restrictions.

both normal and tumor cells is the Ki-67 protein. It

All work with experimental animals was carried out in

participates in the cell cycle, being involved in ribo-

accordance with the ethical principle of the European

some biogenesis, heterochromatin organization and

Convention for the Protection of Vertebrates Used

mitotic chromosome separation [6]. The Ki-67 index

for Experiments or Other Scientific Purposes (ETSN

makes it possible to assess the degree of malignan-

123, Strasbourg, March 18, 1986). This experiment

cy of the tumor and predict the course of the disease

was approved by the decision of the local bioethical

in combination with other factors. A direct correla-

committee of the National Medical Research Center

tion has been established between the number of

for Oncology.

tumor cells expressing Ki-67 and the stage of malig-

nant diseases [7, 8]. The proliferative potential and

Culture of human liver cancer cells

survival of cancer cells can be modulated by creating

Human liver cancer cells of the HepG2 line were

hypoxic conditions, which is actively used in such

cultured in accordance with a standard procedure

therapeutic procedures as transarterial embolization

using a culture medium for DMEM cells with the ad-

and transarterial chemoembolization [9]. However,

dition of veal serum (Gibco, Thermo Fisher Scientific)

it is also known that hypoxia is crucial for the sur-

at a concentration of 10 %, as well as 1 % penicil-

vival of cells resistant to low-oxygen environments,

lin and streptomycin. Cultivation was carried out in

characterized by resistance to therapeutic effects

a CO incubator (Thermo Fisher Scientific, 8000W)

2

and increased invasive ability [10]. On this matter,

at a humid atmosphere of 37 °C, 5 % CO .2

a comprehensive study of the tumor's response to

hypoxia, as well as an understanding of its positive

Creating an orthotopic model of liver cancer

and negative effects, will expand the understanding

Initially, before conducting the experiment, we cre-

of the mechanisms of interaction of cancer cells and

ated a liver cancer xenograph by subcutaneously

37

South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 35-42

Kecheryukova T. M., Trifanov V. S., Shulga A. A., Goncharova A. S., Gurova S. V., Ulyanova E. P., Maksimov A. Yu. Hypoxia effect on proliferative activity of cells in orthotopic xenograft of hepatocellular carcinoma of the liver in the experiment injecting a 5 × 106 cell suspension of HepG2 into Bal-tions were made using a rotary microtome, which

b/c Nude mice ( n = 2). When the obtained subcutane-were subsequently dewaxed according to a standard

ous xenographs reached a diameter of 1–1.5 cm, the

protocol. Hematoxylin and eosin staining was per-

mice were euthanized, the tumor nodes were extract-

formed for histological examination. IHC staining

ed and divided into fragments about 1 × 1 × 1 mm in

was performed automatically in the BenchMark UL-

size for further transplantation into the liver. Access

TRA Ventana immunohistostainer according to the

to the liver was carried out by performing laparotomy

protocols of manufacturers attached to the antibod-

on pre-anesthetized recipient animals. An incision

ies used. Antibodies Ki-67 (clone SP6), CellMarque

was made in the left lobe of the liver, after which the

were used in a 1:200 dilution. To analyze the expres-

previously obtained tumor fragments were placed

sion of Ki-67 by tumor cells, the proportion of cells

into the parenchyma of the left lobe of the liver using

with colored nuclei (percentage of the total number

anatomical tweezers. The wound was sewn up with

of tumor cells) in at least 10 random fields of view

a wound stitch after the manipulations.

was calculated.

Creating hypoxic conditions by reducing liver

Statistical analysis

blood flow

The results obtained during the experiment were

A control laparotomy was performed to measure

analyzed using the Statistica 10.0 software package.

the volume of tumor nodes 2 weeks after the tumor

The data are presented in the form of the median,

fragments were implanted into the liver of mice. To

25th and 75th percentiles. A comparative analysis

determine the size of the tumor node, the following

of the differences in Ki-67 nuclear staining between

formula was used: V = LW2/2, L for the length of the

the groups was carried out with the Mann- Whitney

tumor, W for the width of the tumor. Then the animals statistical criterion.

were divided into 2 groups ( n = 6 for each), the distribution criterion was the size of the tumor node, while

STUDY RESULTS

the values of the average volume of tumor nodes in

the groups differed with a minimum interval. The

During the experiment, orthotopic models of liver

first group is intact, the second group is with a re-

cancer in Balb/c Nude mice were obtained by implant-

duction in liver blood flow. To provide access to the

ing a fragment of the xenograft of the HepG2 cell line

liver and its blood vessels, the mice of the second

directly into the left lobe of the liver [14]. A control

group underwent laparotomy. Then, to occlude the

laparotomy made it possible to demonstrate that all

vessels of the portal triad of the liver, a needle with

animals developed tumor nodes in the left lobe of the

suture material was inserted under them and blood

liver. The measurement results showed that 2 weeks

flow was reduced for 20 minutes using the tension

after implantation of the HepG2 xenograft fragment

of the suture material. After that, the tension of the

into the liver, the size of intrahepatic tumor nodes was

suture material was removed to restore blood supply

130.27 [42.88–345.3] mm3. (Fig. 1).

to the liver and the surgical wound was sutured in

After performing the control laparotomy proce-

layers. The mice of the first group underwent a con-

dure, the animals were divided into 2 groups. In or-

trol laparotomy without blood flow reduction.

der to induce hypoxic conditions, group 2 animals

underwent reduction of liver blood flow (Fig. 2). For

Euthanasia

this, the right lobe of the liver was shifted closer to

On the 4th day after the surgical manipulations,

the diaphragm, which facilitated free access to the

the animals were euthanized to extract tumor nodes.

portal triad. Clamping the vessels of the portal tri-

Euthanasia was performed by dislocation of the cer-

ad with suture material made it possible to achieve

vical vertebrae.

a reduction in the blood flow of the liver and the tu-

mor node located in it, which was visually confirmed

Histological and immunohistochemical (IHC)

by a change in the color of the liver, as a result of

studies

insufficient blood supply, the organ became paler.

The resulting tumor material was fixed in 10 %

After the restoration of blood supply, the liver turned

formalin for 24 hours, then enclosed in paraffin, sec-

maroon again.

38

Южно-Российский онкологический журнал 2024. Т. 5, № 2. С. 35-42

Кечерюкова Т. М., Трифанов В. С., Шульга А. А., Гончарова А. С., Гурова С. В., Ульянова Е. П., Максимов А. Ю. Влияние гипоксии на пролиферативную

активность клеток ортотопического ксенографта гепатоцеллюлярной карциномы печени в эксперименте

The results of histological examination showed

itive cells was 65 [65–70] % (Fig. 4A), in the group

a focus of hepatocellular carcinoma in the liver tis-

with blood flow reduction, the number of stained nu-

sues, characterized by an average degree of differ-

clei was statistically significantly less, which amount-

entiation, represented by solid- trabecular structures,

ed to 19 [15–25] % ( p < 0.001) (Fig. 4B).

in the thickness of which the vessels are located.

Necrosis foci are locally present. The cellular com-

DISCUSSION

position is represented by large epithelial cells re-

sembling hepatocytes. Large polymorphic nuclei with

It is known that hypoxia is an important factor that

granular chromatin and well-distinguishable nucleoli

can contribute to the formation of cellular plasticity

are visible inside the cells. Mitosis figures are also

and tumor heterogeneity, affecting the phenotype

found, including atypical forms (Fig. 3).

and cell functions. However, despite the impressive

In the immunohistochemical study of the expres-

array of data presented in the scientific literature, it

sion of the Ki-67 proliferation marker in the tissues of

is possible to observe a lack of correlation between

liver cancer xenographs, the number of immunopos-

different methods of studying the effect of oxygen

Fig. 1. Measurement of a tumor in the liver of a mouse

Fig. 2. The process of performing liver blood flow reduction by occlusion of the portal triad to induce hypoxic conditions А

Б

Fig. 3. Histological specimen: morphological picture of hepatocellular carcinoma. A – without hypoxia; B – after hypoxia. Magnification × 100

39

South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 35-42

Kecheryukova T. M., Trifanov V. S., Shulga A. A., Goncharova A. S., Gurova S. V., Ulyanova E. P., Maksimov A. Yu. Hypoxia effect on proliferative activity of cells in orthotopic xenograft of hepatocellular carcinoma of the liver in the experiment levels, since they all provide information about dif-tion marker (Ki-67). In addition, it has been convinc-

ferent diseases, non-uniform time and topological

ingly demonstrated that ascites is an environment

points of sampling of tumor material, or, for exam-

with a very low level of oxygenation since the cells

ple, blood oxygenation. From this point of view, the

floating in it do not have adequate blood supply and

use of animal models allows, as far as possible, to

can survive only through glycolysis pathway. It is im-

bring uniformity to the experimental conditions and

portant to note that the authors mention that tumor

obtain reproducible results by performing serial ex-

hypoxia is a driving factor in resistance to radiation

periments. Considering the listed advantages of the

therapy and chemotherapy [16].

in vivo approach, we performed an experiment to

In this study, a low level of Ki-67 expression was

study the effect of low oxygenation on liver cancer

noted in tumor samples of animals with blood flow

cells. The results of the IHC study showed that in

reduction, however, zoning in the location of positive-

tumor samples of animals with reduced blood flow,

ly colored cells was observed in tumor tissues. Cells

a lower value of the Ki-67 proliferation marker was

expressing Ki-67 were concentrated along the tumor

observed. An analysis of the literature data showed

zones directly in contact with intact liver tissue. It is

that in the works of other authors there is a direct

known that the so-called "invasion front" of a tumor connection between hypoxia and the proliferative

is formed by cells located on its surface, and they

potential of tumor cells. For example, a study of en-

form patterns of invasion and tumor spread. Given

dometrial tumors showed that the expression level

this fact, it can be assumed that cells that have re-

of Ki-67 is inversely correlated with the expression

tained their proliferative potential, despite the effects

level of hypoxia- induced factor (HIF-1a), which in-

of hypoxic conditions resulting from blood flow re-

dicates low cell proliferative activity in conditions

duction, and located along the edge of the tumor

of oxygen deficiency. In addition, such a correlation

node, may have an increased invasive potential. In

may contribute to reducing the effect of anti-cancer

addition, the observed zonality of Ki-67 expression

drugs such as metformin [15]. Also, in the work on

may probably be related to proximity or distance

visualization of hypoxia of cancer cells in animals

from blood vessels with reduced blood flow.

and cancer patients, it was found that tumor cells

in effusions and micrometastases were in a state

CONCLUSION

of high hypoxia and low proliferation, regardless of

the type of tumor. In this work, samples of human

This study shows that liver tumors of mice sub-

and animal tumor cells were examined by IHC for

jected to the liver blood flow reduction procedure

HIF-1a, glucose transporter (GLUT-1) and prolifera-

were characterized by lower Ki-67 values. The ob-

А

Б

Fig. 4. IHC reaction of the tumor to Ki-67 antibodies (clone SP6). A – without reduction of liver blood flow; B – after reduction of liver blood flow by occlusion of the portal triad. Magnification × 200

40

Южно-Российский онкологический журнал 2024. Т. 5, № 2. С. 35-42

Кечерюкова Т. М., Трифанов В. С., Шульга А. А., Гончарова А. С., Гурова С. В., Ульянова Е. П., Максимов А. Ю. Влияние гипоксии на пролиферативную

активность клеток ортотопического ксенографта гепатоцеллюлярной карциномы печени в эксперименте

tained data indicate that the proliferative activity of

and development of malignant tumors may contrib-

tumor cells is directly related to the microenviron-

ute to a deeper understanding of the biological char-

ment, particularly to the hypoxic environment. Fur-

acteristics of tumors and the approaches to their

ther study of the effects of hypoxia on the growth

treatment.

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South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 35-42

Kecheryukova T. M., Trifanov V. S., Shulga A. A., Goncharova A. S., Gurova S. V., Ulyanova E. P., Maksimov A. Yu. Hypoxia effect on proliferative activity of cells in orthotopic xenograft of hepatocellular carcinoma of the liver in the experiment Information about authors:

Takhmina M. Kecheryukova – MD, X-ray physician, endovascular diagnostics and treatment of Abdominal Oncology Department No. 1, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-8092-6457

Vladimir S. Trifanov – Dr. Sci. (Med.), Associate Professor, Head of the Abdominal Surgery Center, surgeon, leading researcher of the branch P.

A. Hertsen Moscow Oncology Research Institute – Branch of the National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation, Moscow, Russian Federation; Leading researcher of the Thoracic-abdominal Department, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0003-1879-6978, SPIN: 3710-8052, AuthorID: 453981

Anna A. Shulga – junior researcher at the Testing Laboratory center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0009-0006-1125-2897, SPIN: 6457-4451, AuthorID: 1221869

Anna S. Goncharova – Cand. Sci. (Biol.), Head of the testing laboratory center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0003-0676-0871, SPIN: 7512-2039, AuthorID: 553424, Scopus Author ID: 57215862139

Sophia V. Gurova – junior researcher at the Testing Laboratory center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-9747-8515, SPIN: 5413-6901, AuthorID: 1147419

Elena P. Ulyanova – researcher at the Laboratory of Tumor Immunophenotyping National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0001-5226-0152, SPIN: 1243-9475, AuthorID: 759154, Scopus Author ID: 57203357998

Aleksei Yu. Maksimov – Dr. Sci. (Med.), professor, Deputy CEO for Advanced Scientific Research, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-1397-837X, SPIN: 7322-5589, AuthorID: 710705

Contribution of the authors:

Kecheryukova T. M. – conducting an experiment;

Trifanov V. S. – editing the text;

Shulga A. A. – text writing, statistical analysis;

Goncharova A. S. – search for literature data, writing a text; Gurova S. V. – conducting an experiment;

Ulyanova E. P. – histological analysis;

Maksimov A. Yu. – text editing.

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