MicroRNA from the blood extracellular microvesicles for minimally invasive diagnostics of lung cancer

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BACKGROUND

As of today, lung cancer remains one of the main reasons of cancer mortality worldwide, the reason of which is largely due to the disease’s frequent asymptomatic development and untimely diagnostics [1]. Despite the progress in developing new methods for lung cancer therapy, the survival rates remain low. The SEER epidemiological research (Surveillance, Epidemiology, and End Results Program) from the National Cancer Institute in the USA has demonstrated that more than half of the lung cancer patients begin their treatment at the stage when the five-year survival rate is only 8.9%, which means that early diagnostics has a fundamental value for the efficiency of treatment and for the survival time for the lung cancer patients [2].

A method for the timely detection of lung cancer among smokers is the low-dose computed tomography (LDCT). Annual screening with LDCT can reduce the risk of mortality in this group by 20%. However, this is an expensive, complex and economically inefficient method that requires modern equipment and specialists, while the rate of false-positive results ranging from 8% to 50% [2]. A separate challenge is increasing incidence of lung cancer among non-smokers. For example, in Asia non-smokers represent 45% to 70% of all lung cancer patients in recent years. The clinical prediction models based on demographic and clinical factors, such as PLCOm2012 (Prostate, Lung, Colorectal and Ovarian cancer screening trial), LLP (Liverpool Lung Project), LCDRAT (Lung Cancer Death Risk Assessment Tool) and HLCRM (Henan Lung Cancer Risk model), rely heavily on smoking history and are poorly applicable for predicting lung cancer in non-smokers [2].

Thus, currently there is an urgent need for screening diagnostics of lung cancer and for revealing the highly sensitive and specific biomarkers, suitable for timely detection of this disease at early stages. One such biomarker with high diagnostic potential is circulating nucleic acids in blood, particularly small molecules as miRNA [3, 4]. MiRNAs take part in regulating the expression of multiple genes, the impaired operation of which is observed at various stages of developing oncological diseases, including lung cancer [5, 6]. As a result of apoptosis, necrosis or active secretion by tumor cells, miRNA can reach not only the extracellular space, but also the biological fluids of the organism, including blood [7, 8]. The protection from the effects of blood RNA-hydrolyzing enzymes is provided by various miRNA-binding proteins, as well as the packaging of miRNA and their complexes into variously sized extracellular microvesicles [4, 8, 9]. Microvesicles circulate in blood for extended periods and a subset of them carry tumor-derived miRNA. Several research works have shown that microvesicles in blood plasma represent the promising source for miRNA diagnostics using “liquid biopsy” [4, 10–12].

Research Aim — to analyze the relative expression of 17 miRNAs from the blood plasma microvesicles fraction among NSCLC patients and donors.

METHODS

Research design

A controlled randomized trial was arranged.

Conformity Criteria

Inclusion criteria for patients: male sex; presence of diagnosed NSCLC.

Inclusion criteria for donors: male sex; aged older than 45 years; absence of the diseases in the lungs or oncological diseases of any location.

Non-inclusion criteria for patients and donors: female sex; presence of diagnosed oncological diseases in other location, besides the NSCLC.

Exclusion criteria for patients are not planned.

Research facilities

The research work has used the samples of blood plasma extracellular microvesicles from 27 donors and from 19 NSCLC patients, collected at the State Budgetary Healthcare Institution of the Novosibirsk oblast “Novosibirsk Regional Oncology Dispensary” (Novosibirsk) and at the Federal State Budgetary Institution “Scientific Research Institute of Pulmonology” under the Federal Medical-Biological Agency of Russia (FSBI “SRI of Pulmonology” under the FMBA of Russia, Moscow). The research was carried out within the premises of the Federal State Budgetary Institution of Science “Institute of Chemical Biology and Fundamental Medicine” under the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS, Novosibirsk).

Research Duration

The research was carried out during the time period from 2023 until 2025. Blood samples from the patients and healthy volunteers were drawn from July 2023 until February 2024.

Research Description

The expression of 17 miRNAs in the blood plasma microvesicles fraction was studied among NSCLC patients and among the healthy volunteers. The miRNA panel was previously described by us [3], while the patients and the donors are distinct from the previously examined samples. Blood plasma microvesicles were isolated using the aggregation-precipitation method; later on, from the obtained blood microvesicles fraction, miRNAs were isolated using glass fiber filters. By employing the reverse transcription polymerаse chain reaction (RT-PCR), an evaluation was carried out for the diagnostic efficiency of previously proposed miRNA pairs [3] using the method of running all the possible miRNA pairs for selecting the most effective diagnostic pairs and panels.

Other procedures included a literature analysis of the biological effects of the diagnostic miRNAs, the genes through which these effects are mediated, and their potential effects on lung cancer treatment efficiency.

Research Outcomes

The main outcome, the parameters of which were analyzed during the current research, was the relative expression of miRNA pairs in samples of blood plasma microvesicles from the donors and the NSCLC patients.

Methods for registration of outcomes

Blood samples were processed to obtain plasma microvesicles. MiRNA was then isolated from these microvesicles using a previously described method [3]. Real time RT- PCR for the evaluation of expression levels for 17 miRNAs (sequences of primers and probes are provided in Suppelment 1) were carried out as described in [13, 14] using two parallel tests (sets):

• microRNA-19b, -374a, -324, -22, -222, -133b, -144, -425;
• microRNA-205, -660, -30e, -125b, -92a, -378а, -375, -27b, -31.

As the internal control (spike-in), each sample of microvesicles before the initiation of the isolation process was supplemented with cel-miR-39 miRNA. the quantity of which was further assessed using the real time RT-PCR (the primers and the probe are provided in Suppelment 1).

Statistical analysis

All analyses were performed using the R statistical environment. for each miRNA pair analyzed within the same RT-PCR testing set, the difference in threshold cycles (dCt=Ct₁-Ct₂₎ was calculated for every donor and patient, where Ct₁ and Ct₂ represent the threshold cycle values for the first and second miRNA in the pair, respectively.).

Subsequently, data distribution was assessed for normality using the Shapiro-Wilk test. Based on this assessment, intergroup differences (patients vs. donors) were evaluated for statistical significance using either the Wilcoxon rank-sum test (for non-normal distributions) or Welch’s t-test (for normal distributions). Finally, receiver operating characteristic (ROC) analysis was conducted for each pair, and Pearson’s correlation was used to test for associations between miRNA pair expression levels and participant age.

RESULTS

Research sample (Participants)

The trial populations included only men. The age of the NSCLC patients varied from 37 to 78 (mean age 66±9.9) years; the disease stages according to the TNM classification are provided in table 1.

 

Table 1 The clinical data of the patients with non-small-cell lung cancer

TNM stage		Number of patients, %
T (tumor)	1	21
	2	37
	3	32
	4	10
N (nodus)	0	53
	1	32
	2	11
	3	4
M (metastasis)	0	95
	1	5

 

The donor group included men aged from 45 to 62 (mean age 53±5.4) years.

Primary Research Outcomes

According to the previous data on the operating ranges of real time PCR, the statistical processing included the threshold cycle values (cycle threshold, Ct) within a range from 24 to 38. All miRNA samples isolated from the microvesicles of the NSCLC patients and donors, the PCR findings were within the abovementioned range. As an additional inclusion parameter for donor samples, the value for the exogenous spike-in control (cel-miR-39), which was added before miRNA isolation, was required to be 25 ± 1.

By means of using the ROC-analysis, the efficiency of pre-formed [3] diagnostic panel of six miRNA pairs was evaluated (-133b/-374a; -30e/-660; -125b/-660; -31/-125b; -133b/-425; -133b/-222; the pairs included eight various miRNA: -133b, -374a, -30e, -660, -125b, -31, -425, -222) in the independent sample (Suppelment 2). All listed miRNA pairs showed absolute specificity, i.e. none of the donors was identified as having the NSCLC. The data provided suggest that the sensitivity of NSCLC detection among the patients for the -31/-125b miRNA pair is 42%, for the -133b/-374a miRNA pair — 10.5% and for the -133b/-425 and -133b/-222 miRNA pairs — 37%. Notably, the miR-133b/-425 and miR-133b/-222 pairs were both detected in the blood plasma microvesicles of the same subset of patients. In total, the previously proposed miRNA panel [3], detected 79% of the NSCLC patients in the independent group, of which 8 patients had a single pair, 5 — two pairs and 2 donors had three miRNA pairs (with 100% specificity).

To identify miRNA pairs with higher sensitivity and specificity, All possible pair combinations were examined, and those exhibiting the most significant intergroup differences were selected for further evaluation. Statistical significance was assessed via one-way analysis of variance (ANOVA), with results detailed in Suppelment 3.

The statistical analysis has revealed 19 aberrantly expressed miRNA pairs (Suppelment 4).

The results of ROC-analysis have demonstrated that the highest diagnostic potential for the provided group of NSCLC patients and donors was shown by the following miRNA pairs: -19b/-144, -30e/-92a, -205/-660, that were demonstrating the sensitivity exceeding 60% with 100% specificity (see Suppelment 4; 5).

After using the Pearson’s correlation coefficient, it was found that the ratios of levels among 12 miRNA pairs (-19b/-133; -19b/-144; -19b/-222; -19b/-324; -19b/-374a; -19b/-425; -22/-222; -22/-324; -22/-374a; -22/-425; -133/-324; -324/-374a) have weak correlation relation (k ≤0.4) with the age of the patient/donor and with the statistical significance being p < 0.05 (see Suppelment 4). Among the remaining miRNA pairs, no correlation relationship was detected.

Using the previously proposed approach to the compilation of marker panel and taking into consideration the standard deviation of the values along with the choice of pairs with 100% specificity [15], three various minimal panels were compiled, which allow for discriminating the NSCLC patients and the donors with 100% specificity and precision (Suppelment 6). The pairs included into the minimal panels, partially overlap. While each individual panel successfully diagnosed every patient, combining all three panels revealed that several patients were detected by only a single miRNA pair (see Suppelment 6).

DISCUSSION

In recent years, liquid biopsy has gained significant popularity in oncology diagnostics, including for lung cancer. Several studies [3, 16–18] have identified extracellular microvesicles in blood as a promising source of cancer-specific miRNA, highlighting their potential to enhance the efficiency of “liquid biopsy”. The isolation of microvesicles using the traditional method of ultracentrifugation is practically not applicable in the settings of clinical-diagnostic laboratories. The methods based on the sedimentation of microvesicles, do not present any technical challenges and do not require special equipment [10, 16, 18], but often result in the co-isolation of polymerase reaction inhibitors. To address this, we previously proposed a method based on the selective sedimentation of microvesicles with low concentration of polyethylene glycol in the presence of blue dextran, comparable to the ultracentrifugation by the efficiency [19]. While yielding microvesicle preparations free from PCR inhibitors, making it suitable for diagnostic laboratory use. Furthermore, the isolation and the analysis of miRNA from these microvesicles are technically simple procedures that can be easily implemented in the laboratories of various levels of equipment [20–23].

The attempts to develop the diagnostic systems for oncological diseases based on the detection of the concentrations of one or two circulating miRNAs, were not successful despite the number of publications in this area [8, 24–26]. Indeed, the complex regulatory network of miRNA expression, the breadth of their targets and the small dispersion in the diagnostic miRNA concentrations, all indicate that a reliable diagnosis cannot be based on the expression changes of just one or two miRNAs [27, 28]. Even when using established epigenetic regulators like DNA methylation for cancer diagnosis, analyzing a single gene or locus is generally insufficient for a definitive diagnosis [4, 26]. For instance, in the diagnostic systems approved in the USA and China (CE-IVD mark, FDA approved, Chinese market) [29], the diagnostics of lung cancer uses the analysis of methylation of SHOX2 and PTGER4 genes (prostaglandin E4 receptor gene) [30].

The procedures involved in miRNA analysis necessitate standardization, for the purpose of compiling the conditions for data inclusion/exclusion from the analysis and their normalization. As a inclusion/exclusion criterion, we spiked the samples with internal (spike-in) control (addition of cel-miR-39 directly before the isolation of miRNA from microvesicles) along with the limitations in the range/scattering of PCR data for each miRNA. We have used the method of paired normalization of data from quantitative PCR of miRNA. This approach avoids the need to normalize against stably expressed miRNA [31, 32], as their concentration in blood plasma and microvesicles is known to frequently vary [33, 34] instead, it allows for the selection of miRNA pairs exhibiting larger numerical ddCt differences that are characteristic of the studied disease. These significant differences in the ddCt outweigh the operator errors and enable the creation of the reliable diagnostic systems.

Previously we have formulated the principles of compiling the diagnostic panel based on the extracellular miRNAs, which also include the miRNAs from the blood plasma microvesicles [3, 15]. These principles, along with the criteria for the inclusion/exclusion of the samples in the analysis, include the set of diagnostic miRNA pairs while taking into consideration their ddCt modulus (more than 1.5), determining the diagnostic ranges that take into account the statistical distribution of data in such a way that can provide 100% specificity of the systems. The markers were combined to form a panel in such a way that each patient could be tested using at least two miRNA pairs, with this, the miRNA expression either must not correlate with the age of the NSCLC patients/the donors or it should have a weak correlation relationship. Besides, when compiling the panel, the miRNA targets were taken into consideration, namely the “coverage” of the largest possible number of regulatory pathways involved into the development of lung cancer. The abovementioned principles, in particular, statistical approaches and double coverage of patients with different miRNA pairs, were implemented for the reason that the expansion of groups of NSCLC patients/donors could not result in the loss of sensitivity and specificity of the diagnostic system based on employing this panel of miRNA/miRNA pairs. Based on these principles, during the pilot research [3], the panel of eight miRNAs was compiled that included six pairs.

During the present research, it was confirmed that all the miRNA pairs show 100% specificity, i.e. not diagnosing the donors as the NSCLC patients. Upon the verification of previously obtained data using the new group of patients/donors, its efficiency from the NSCLC diagnostics point of view was confirmed by six miRNAs out of eight [3], namely, the miRNA-133b, -31, -125b, -374а, -425 and -222, affecting the processes of proliferation, migration, invasion and apoptosis [3, 35, 36]. Regarding miRNA-30е and -660 whose diagnostic efficiency was not confirmed [3], the literature contains controversial data on their expression in lung cancer. This could become the reason for such findings (i.e. the loss of diagnostic value) observed for these miRNAs in the independent group of donors.

It is important to note that, among the NSCLC patients, the dependence of miRNA panel expression on the age was practically not observed (see Suppelment 4), which is confirmed by the literature data [37–39] and corresponds to previously compiled criteria for selecting the marker miRNA, being the undoubtful benefit for any test-systems upon their implementation into clinical practice.

Despite the unsatisfactory data on the miRNA-30е and -660, the proposed miRNA panel provides practically 80% sensitivity of detecting NSCLC among the patients along with 100% specificity. In order to find out if it is possible to reveal additional miRNA pairs and/or panels of pairs, allowing for differentiating the NSCLC patients from the healthy donors in the independent cohorts, a paired analysis was done for the tested miRNA. It was shown that miRNA-19b, -144, -378а, -205, -324 and -92а (see Suppelment 4), along with the previously proposed ones (-133b; -31; -125b; -374а; -425; -222), also belong to the ones that can be used for detecting NSCLC patients among the patients enrolled into the present research. Based on the obtained data, three minimal diagnostic miRNA panels can be compiled (see Suppelment 6). The determination of the minimal panels that allow for diagnosing all the patients, is especially topical, for the implementation of diagnostic tests into clinical practice requires the development of systems with the least number of components (miRNAs), resistant to multiplication during the PCR, for decreasing the final product cost for the purpose of their availability to the largest possible populations. Thus, the miRNA-125b/-378а pair is included into all the three minimal test-systems, while the miRNA-205/-660 and -324/-374а pairs — in the two test-systems for the diagnostics of NSCLC. MiRNAs from these panels can actually affect the development of NSCLC, as they regulate key processes such as proliferation, invasion, metastatic spreading of tumor cells, developing chemotherapy resistance etc. (Suppelment 7 [37, 38, 40–60]). As it was mentioned before, the reliability of diagnostic systems will be significantly higher with each patient diagnosed using at least two markers (two or more miRNA pairs). However, even after compiling the proposed three minimal panels into one, several patients could still only be diagnosed using a single miRNA pair (Suppelment 6). Undoubtedly, increasing the heterogeneity of samples with NSCLC along with increasing the number of patients and healthy individuals involved into the research, can result in the loss of efficiency for some of the markers and the decrease in the diagnostic efficiency of the systems, which was exactly confirmed by the data from the first block of our research work.

It is necessary to note that the pairs of diagnostic miRNAs proposed during the earlier research [3], including the miRNA-30е and -660, were shown to be ineffective in this research and, as it appears, they vary between the groups and do not represent the reliable markers. Besides, the part of previously proposed diagnostic pairs, which contributed to 80% of sensitivity in the earlier diagnostic panel, upon the verification of data using independent groups of patients/donors during the current research was not identified as the diagnostic ones. This shows that the methodology used generally enables the selection of diagnostic miRNA pairs, as part of the data from earlier research was reproduced. However, the obtained data require thorough verification with independent donor and patient samples. Furthermore, the most effective diagnostic miRNA pairs are not always robustly detected in independent groups of NSCLC patients and donors. This indicates that identifying miRNAs with consistent expression across populations a distinct research objective.

Therefore, the search methodology we previously proposed for the markers and for the compilation of panels using statistical approaches in the determination of diagnostic ranges for miRNA pairs, remains promising. All pairs detected previously [3] and during the current research, demonstrate 100% specificity.

The data obtained during the present research on the “re-educating” and on selecting other diagnostic miRNA pairs can likely be attributed to several factors related to the specific composition of the patient cohorts, the limited representativeness of the samples from the general population and to the individual donor variability.

In general, the confirmation of previously obtained data provides a hope for compiling the miRNA markers that are universally applicable for the general population of NSCLC patients. Modern literature proposes tens of miRNA markers for lung cancer. However, the comparative analysis of these data and the selection of potential markers for panel inclusion are significantly hindered by the absence of standardized methods for “liquid biopsy” in cases of lung cancer. Within this context, the best method for the rational search of such miRNA markers can become the genome-wide comparative research of miRNA obtained from the microvesicles of donor blood samples and their further PCR-verification.

CONCLUSION

Based on the results of verifying the diagnostic potential of miRNA contained in the microvesicles from blood plasma drawn from the NSCLC patients and from the donors with using real time RT-PCR, the four pairs of miRNAs (-31/-125b; -133b/-374a; -133b/-425; -133b/-222) have proven their efficiency with a sensitivity of 79% with 100% specificity.

Based on the analysis of miRNA expression in a group of NSCLC patients and healthy volunteers using paired normalization, regression modeling and multiple samples generations, three independent minimal panels of miRNA pairs were proposed that allow for diagnosing the NSCLC patients with 100% accuracy: (1) -19b/-425; -125b/-378a; -205/-660; (2) -324/-374a; -30e/-92a; -125b/-378a; (3) -324/-374a; -125b/-378a; -205/-660.

These findings support the potential for developing the high-accuracy diagnostic means for NSCLC based on miRNA from blood plasma microvesicles. Supplementation of miRNA panels with additional markers is required for the purpose of increasing their resistance. The rational search for such miRNA markers can be implemented by using the NGS of miRNA samples found in the microvesicles of blood plasma drawn from the NSCLC patients and the donors.

Additional information

Suppelment 1. Sequences of primers and probes used in the research work.

doi: 10.17816/clinpract688775-4389942

Suppelment 2. The diagnostic efficiency of the panel consisting of six miRNA pairs.

doi: 10.17816/clinpract688775-4392404

Suppelment 3. Relative expression of miRNA pairs in the fraction of microvesicles from blood plasma drawn from the donors and the patients with non-small-cell lung cancer.

doi: 10.17816/clinpract688775-4389943

Suppelment 4. Data on the differences of threshold cycles for miRNA pairs (ddCt), ROC-analysis of correlations to the age for the groups of patients with non-small-cell lung cancer and for the donors.

doi: 10.17816/clinpract688775-4392417

Suppelment 5. ROC-curves for the sensitivity and the specificity of the miRNA ratios among the donors and the non-small-cell lung cancer patients.

doi: 10.17816/clinpract688775-4392410

Suppelment 6. The example of using the minimal panels of miRNA for the diagnostics of patients with non-small-cell lung cancer in the tested group of patients

doi: 10.17816/clinpract688775-4392423

Suppelment 7. The effects of miRNA included into three proposed diagnostic panels on the development of lung cancer (according to the literature data)

doi: 10.17816/clinpract688775-4392429

Author contributions: O.E. Bryzgunova, M.Yu. Konoshenko, P.P. Laktionov, the concept and design of the study; E.V. Shutko, V.V. Chumakova, E.A. Murina, A.A. Ilyushchenko, Ya.M. Danilova, collection and processing of samples; M.Yu. Konoshenko, V.V. Chumakova, statistical analysis, visualization; M.Yu. Konoshenko, writing the manuscript; O.E. Bryzgunova, E.V. Shutko, P.P. Laktionov, editing of the manuscript; V.V. Kozlov, Yu.A. Lantsukhay, S.D. Gorbunkov, S.E. Krasilnikov, K.A. Zykov, methodological support, technical editing of the paper. Thereby, all authors provided approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethics committees of ICBFM SB RAS (№ 10, 2008 Dec 22) and the Pulmonology Scientific Research Institute under FMBA of Russia (ethics committee protocol № LEK 04-24 dated 2024 Jun 24). Informed consent was obtained from all subjects involved in the study. All study participants signed an informed consent form before being included in the study.

Funding source: The study was funded by the Russian state-funded project for the Pulmonology Scientific Research Institute under FMBA of Russia (grant number 388-03-2024-136) and supported by the Russian state-funded project for ICBFM SB RAS (grant number 125012900932-4).

Disclosure of interests: The authors declare that they have no competing interests.

Statement of originality: The authors did not use previously published information (text, illustrations, data) while conducting this work.

Data availability statement: The editorial policy regarding data sharing does not apply to this work, data can be published as open access.

Generative AI: Generative AI technologies were not used for this article creation.

About the authors

Olga E. Bryzgunova

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute

Author for correspondence.
Email: biobryz@yandex.ru
ORCID iD: 0000-0003-3433-7261
SPIN-code: 9752-3241

PhD

Russian Federation, Novosibirsk; Moscow

Maria Yu. Konoshenko

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute

Email: lacyjewelrymk@gmail.com
ORCID iD: 0000-0003-2925-9350
SPIN-code: 9374-8489

PhD

Russian Federation, Novosibirsk; Moscow

Ekaterina V. Shutko

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute; Novosibirsk National Research State University

Email: katshutko@gmail.com
ORCID iD: 0009-0004-3004-8969
SPIN-code: 3627-2494
Russian Federation, Novosibirsk; Moscow; Novosibirsk

Viktoria V. Chumakova

Institute of Chemical Biology and Fundamental Medicine

Email: v.chumakova@alumni.nsu.ru
ORCID iD: 0009-0008-2234-3342
Russian Federation, Novosibirsk

Ekaterina A. Murina

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute

Email: katyamurina1@gmail.com
ORCID iD: 0009-0001-3624-2357
SPIN-code: 2454-1260
Russian Federation, Novosibirsk; Moscow

Antonina A. Ilyushchenko

Pulmonology Scientific Research Institute

Email: Kdlmedwans@gmail.com
ORCID iD: 0009-0003-9068-5401
Russian Federation, Moscow

Yaroslava M. Danilova

Pulmonology Scientific Research Institute

Email: yaroslava.danilova.82@mail.ru
ORCID iD: 0009-0003-6679-9185
Russian Federation, Moscow

Stanislav D. Gorbunkov

Pulmonology Scientific Research Institute

Email: sdgorbunkov@mail.ru
ORCID iD: 0000-0002-8899-4294
SPIN-code: 7473-0530

MD, PhD, Assistant Professor

Russian Federation, Moscow

Kirill A. Zykov

Pulmonology Scientific Research Institute

Email: kirillaz@inbox.ru
ORCID iD: 0000-0003-3385-2632
SPIN-code: 6269-7990

MD, PhD, Professor member of the Russian Academy of Sciences, corresponding member of the Russian Academy of Sciences

Russian Federation, Moscow

Sergey E. Krasilnikov

Novosibirsk National Research State University; Federal Research Center of Fundamental and Translational Medicine

Email: professorkrasilnikov@rambler.ru
ORCID iD: 0000-0003-0687-0894

MD, PhD

Russian Federation, Novosibirsk; Novosibirsk

Vadim V. Kozlov

Novosibirsk State Medical University

Email: vadimkozlov80@mail.ru
ORCID iD: 0000-0003-3211-5139
SPIN-code: 8045-4286

MD, PhD, Assistant Professor

Russian Federation, Novosibirsk

Yuriy A. Lantsukhay

Federal Research Center of Fundamental and Translational Medicine

Email: Lancuh1@mail.ru
ORCID iD: 0009-0004-4174-7300
Russian Federation, Novosibirsk

Pavel P. Laktionov

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute

Email: lakt@1bio.ru
ORCID iD: 0000-0002-0866-0252
SPIN-code: 4114-3170

PhD

Russian Federation, Novosibirsk; Moscow

References

Supplementary files