Method for determining presence of distant metastases from rectal adenocarcinoma

Abstract

FIELD: oncology.SUBSTANCE: used to determine the presence of distant metastases of rectal adenocarcinoma. The perimeter of mitochondria isolated from the tumor and conditionally healthy tissue of the rectum of patients is measured along the line of adenocarcinoma resection. When containing mitochondria with a perimeter of 2μ m in conditionally healthy tissue at the same level as in the tumor, the presence of distant metastases is determined, and with a decrease in the content of mitochondria with a perimeter of 2μ m in conditionally healthy tissue compared to the content in the tumor the absence of distant metastases is determined.EFFECT: differentiated assessment of the development of distant metastases.1 cl, 4 dwg, 1 tbl, 4 ex

Description

[0001] The invention relates to medicine, namely to oncology, and can be used to determine the presence of distant metastases of rectal adenocarcinoma.

[0002] The high mortality rate of colon cancer is mainly due to distant metastasis, and the grade of colorectal cancer (CRC) and histopathological type are all factors influencing the prognosis of patients (see Gupta P, Chiang SF, Sahoo PK, Mohapatra SK, You JF, Onthoni DD, et al. Prediction of colon cancer stages and survival period with a machine learning approach. Cancers (Basel). 2019;11(12):2007. doi: 10.3390 / cancers11122007; see Recio-Boiles A, Cagir B. Colon Cancer. StatPearls. Treasure Island (FL): StatPearls Publishing; 2022).

[0003] Considering recent advances in colon cancer screening, diagnosis, and treatment, the long-term prognosis of patients with colon cancer remains poor (see Liu Y., Kang R., Zheng H., Wang P., Jiang W., Xiong B., et al. Female colon cancer metastasis pattern and prognosis: a seer-based study. BioMed research international. 2022; 3865601. doi: 10.1155 / 2022 / 3865601. et al., 2022). Although modern research has clarified the pathogenesis of CRC and provided effective screening strategies, its prevalence is still increasing. A better understanding of the occurrence, progression, and metastasis of CRC can help develop molecular markers for early detection and risk stratification methods to improve the clinical care of patients with CRC.

[0004] Liu Y. et al. (2022) made a detailed comparison of distant metastasis patterns in male and female CRC patients using the SEER database to understand the differences in survival between patients. More than three-quarters of the patients had single-site metastasis. After tumor metastasis, patient survival decreased, and the decrease was further reduced with the increase in metastatic sites. Similar results were obtained for other tumors (see Wang J. e., Li S., Liu Y., Zhang C., Li H., Lai B. Metastatic patterns and survival outcomes in patients with stage IV colon cancer: a population-based analysis. Cancer Medicine . 2020;9(1):361-373. doi: 10.1002 / cam4.2673.t al., 2020). It is currently accepted that metastatic dissemination is characterized by the separation of cells from the primary tumor mass, further migration through the blood and lymphatic vessels and colonization of various tissues.The metastatic cascade can be divided into various stages, including local invasion, intravasation, survival in the bloodstream, extravasation, survival at a second target site, and finally proliferation at that target site.

[0005] Epithelial-mesenchymal transition (EMT) is known to be a biological phenomenon that occurs during embryonic development, but is also associated with cancer metastasis (see Chaffer KL, Weinberg RA, A look at cancer cell metastasis / / Science. 2011;331:1559-1564. doi:10.1126 / science.1203543).

[0006] A high level of mitochondrial self-organization and the ability to actively move determines the processes of tumor formation and spread. In this case, various mechanisms are used, including the regulation of the redox status, regulatory signaling, and cell death pathways (see Monzel, AS, Enriquez, JA & Picard, ., Enríquez, JA, Picard, M. Multifaceted mitochondria: moving mitochondrial science beyond function and dysfunction. Nat. Meta. 2023;5:546-562. DOI: 10.1038 / s42255-023-00783-1).

[0007] Recent publications have highlighted the existence of a complex interaction between the functions and spatial state of mitochondria - their separation or fusion to form network structures (see Denisenko TV, Gorbunova AS, Zhivotovsky B. Mitochondrial Involvement in Migration, Invasion and Metastasis / / Front Cell Dev Biol. 2019;7:355. doi: 10.3389 / fcell.2019.00355 2019; see Conroy G. Cells are swapping their mitochondria. What does this mean for our health? Nature. 2025;640(8058):302-304. DOI: 10.1038 / d41586-025-01064-5). In turn, based on experimental material, a connection was established between the therapeutic effect of intact mitochondria and their ability to tissue movement (see Kit OI, Shikhlyarova AI, Frantsiyants EM, Mitochondrial transplantation: new challenges for cancer. South Russian Journal of Cancer. 2024;5(1):60-70. DOI: 10.37748 / 2686-9039-2024-5-1-7. (in Russ.).Mitochondrial dynamics appear to be deeply involved in the regulation of various mitochondrial functions associated with cancer cell invasion and metastasis, however, this issue remains highly controversial, and the mechanisms linking mitochondrial dynamics with metastasis development remain poorly understood.

[0008] The authors of the work (see Nagase H. Watanabe T, Koshikawa N. et al. Mitochondria: Endosymbiont bacteria DNA sequence as a target against cancer / / Cancer Sci. 2021;112(12):4834-4843. doi: 10.1111 / cas.15143) investigated the effect of mitochondria on the metastatic potential in the malignant process and found that the process of nuclear and cytoplasmic exchange between highly metastatic mitochondria with the G13997A mutation and mitochondria of low-metastatic cancer tumors with wild-type mtDNA can lead to mitochondrial replacement. This process of replacing mitochondria in low-metastatic cells with mitochondria from highly metastatic cells leads to an increase in metastatic potential.

[0009] Conversely, no evidence of metastasis was observed in highly metastatic cells that incorporated mitochondria from low-metastatic cells. It has been shown that mitochondria can be transferred from bone marrow stromal cells to leukemic blasts to enhance their proliferation in vitro and in vivo through a mechanism that increases OXPHOS in leukemic blasts (see Burt R., Dey A., Aref S. et al., et al. Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress / / Blood. 2019; 134(17):1415-1429. doi: 10.1182 / blood.2019001398).

[0010] The ability for intercellular translocation is possessed by both whole mitochondria and the mitochondrial genome or other mitochondrial components (see Singh B., Modica-Napolitano JS, Singh KK. Defining the momiome: Promiscuous information transfer by mobile mitochondria and the mitochondrial genome. Semin Cancer Biol. 2017; 47: 1-17. DOI: 10.1016 / j.semcancer.2017.05.004). Mitochondrial transport can be carried out using tunneling nanotubes (TNT), gap junctions (GJC), and extracellular vesicles (MVs) from 100 nm to 1 μm, which are capable of enclosing whole mitochondria, genomic DNA, and mitochondrial DNA (see Singh B., Modica-Napolitano JS, Singh KK. Defining the momiome: Promiscuous information transfer by mobile mitochondria and the mitochondrial genome. Semin Cancer Biol. 2017; 47: 1-17. DOI: 10.1016 / j.semcancer.2017.05.004).

[0011] However, the mitochondria themselves, as active organelles, are transported along the cytoskeleton and can take various forms, for example, merging into long or interconnected tubules or dividing into small spheroids, which is regulated by the opposing processes of fusion and division (see Burt R., Dey A., Aref S. et al., et al. Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress / / Blood. 2019;134(17):1415-1429. doi: 10.1182 / blood.2019001398). Continuous processes of mitochondrial membrane fusion and fission help regulate the morphology and number of mitochondria, ensuring their uniformity and efficient functioning (see Davis CH, Kim KY, Bushong EA, Mills EA, Boassa D, Shih T et al. Transcellular degradation of axonal mitochondria. Proc Natl Acad Sci USA. 2014; 111(26): 9633-8. DOI: 10.1073 / pnas.1404651111).Furthermore, unbalanced mitochondrial fusion and fission during the cell cycle appears to be associated with mitochondria-dependent metabolic reprogramming processes, promoting cancer cells to enter mitosis, thereby providing a proliferation and survival advantage (see Conroy G. Cells are swapping their mitochondria. What does this mean for our health? Nature. 2025;640(8058):302-304. DOI: 10.1038 / d41586-025-01064-5).

[0012] Mitochondrial fusion is defined as the complete fusion of two mitochondria by end-to-end collision (see Chan DK Mitochondrial dynamics and its involvement in disease. Annu Rev Pathol. 2020; 15: 235-59. DOI: 10.1146 / annurev-pathmechdis-012419-032711). Mitochondria are composed of two membranes: the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Fusion begins with fusion of the outer membrane, followed by fusion of the inner membrane, which occurs in close proximity. The IMM contains the mitochondrial lumen (matrix), an inner bordering membrane parallel to the OMM, and a deeply convoluted polymorphic invagination known as the crista. The crista increases the surface area of ​​the inner membrane and contains components necessary for mitochondrial respiration. When the four lipid bilayers fuse, the contents mix and the matrix components diffuse, forming a single, fused mitochondrion (see Chen W., Zhao H., Li Y.Mitochondrial dynamics in health and disease: mechanisms and potential targets. Signal Transl Purpose Tam. 2023; 8(1): 333. DOI: 10.1038 / s41392-023-01547).

[0013] In addition to complete fusion, there is a so-called “kiss-and-run” transient fusion pattern. Unlike complete fusion, transient fusion occurs when two mitochondria join, partially exchange intact membrane proteins, and separate, thereby maintaining their original topology. This type of fusion enhances mitochondrial functional stability and plasticity and is required to maintain mitochondrial metabolism (see Wang S., Xiao W., Shan S., Jiang S., Chen M., Zhang W. et al. Multistep dynamics of mitochondrial fission and fusion in living cells. PLoS One. 2012; 7(5): e19879. DOI: 10.1371 / journal.pone.0019879). While moderate fusion protects intestinal epithelial cells from oxidative stress-induced mitochondrial damage and prevents CRC, abnormal mitochondrial fusion leads to ATP overproduction and abnormal tumor proliferation.

[0014] It is important to emphasize that the principle of the inextricable link between structure and function, long developed in Russian science by Academician D.S. Sarkisov, a pioneer of morphofunctional unity, has now achieved full understanding in the global community, and recognition of the equivalence of previously considered polar concepts at the fine ultrastructural level of mitochondria. Major global publishers such as Nature Review Cancer, Nat. Metabolism, Redox Biology, and many others, hundreds of specialized articles and literature reviews have been published over the past 10 years, including 2025, that present a new paradigm for the multifaceted nature of mitochondria and claim to move mitochondrial science beyond function and dysfunction (see Wallace DC A Mitochondrial Paradigm of Metabolic and Degenerative Diseases, Aging, and Cancer: A Dawn for Evolutionary Medicine. Annu. Rev. Genet. 2005;39:359-407. doi: 10.1146 / annurev.genet.39.110304.095751; see Monzel, AS, Enriquez, JA & Picard, ., Enríquez, J.A., Picard, M. Multifaceted mitochondria: moving mitochondrial science beyond function and dysfunction. Nat. Meta. 2023;5:546-562. DOI: 10.1038 / s42255-023-00783-1; см. Conroy G. Cells are swapping their mitochondria. What does this mean for our health? Nature. 2025;640(8058):302-304. DOI: 10.1038 / d41586-025-01064-5).

[0015] The close relationship between mitochondrial structure and function has led to the concept of mitochondrial “morphofunctionality” (see Benard G, Rossignol R. Mitochondrial ultrastructure and its impact on function and bioenergetics. Antioxid Redox Signal. 2008;10(8):1313-42. DOI: 10.1089 / ars.2007.2000), and included “mitochondrial morphofunctional analysis” as “the simultaneous quantification of mitochondrial morphological and functional parameters.” The authors (see Renault TT, Floros KV, Elkholi R, Corrigan KA, Kushnareva Y, Wieder SY et al. Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis. Mol Cell. 2015 Jan 8;57(1):69-82. DOI: 10.1016 / j.molcel.2014.10.028) showed that both mitochondrial hyperfusion and hyperfragmentation protected cells from BAX-mediated apoptosis, suggesting that BAX requires a specific mitochondrial shape to induce mitochondrial outer membrane permeabilization.In addition, it was found that in response to low ADP concentrations, the IMM structure changed: the mitochondrial matrix contracted and compacted (“condensed state”), and then became less dense and more stretched (“orthodox state”), which was associated with a more compact arrangement of the cristae (see Hackenbrock CR. Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol. 1966 Aug;30(2):269-97. DOI: 10.1083 / jcb.30.2.269). The study by Wang Y. et al. (2017) showed that mitochondrial fragmentation allows the release of Ca2+ from the endoplasmic reticulum into the cytosol, which promotes phagocytosis by stimulating Ca2+-mediated vesicle transport (see Wang Y., Subramanian M., Yurdagul A. Jr., Barbosa-Lorenzi VK, Tsai B., de Juan-Saens H. et al. Mitochondrial fission promotes further macrophage clearance of apoptotic cells.Cell. 2017;171(2):331-345.e22. DOI: 10.1016 / j.cell.2017.08.041 Wang Y., Subramanian M., Yurdagul A. Jr., Barbosa-Lorenzi VK, Tsai B., de Juan-Saens H. et al. Mitochondrial fission promotes further macrophage clearance of apoptotic cells. Cell. 2017;171(2):331-345.e22. DOI: 10.1016 / j.cell.2017.08.041). It has been shown that the structure of mitochondria influences the phase of the cell cycle, and vice versa (see Horbay R, Bily R. Mitochondrial dynamics during the cell cycle. Apoptosis. 2016; 21(12):1327-1335. DOI: 10.1007 / s10495-016-1295-5). For example, CDK5 inhibits DRP1 through phosphorylation (see Cho B, Cho HM, Kim HJ, Jeong J, Park SK, Hwang EM et al. CDK5-dependent inhibitory phosphorylation of Drp1 during neuronal maturation. Exp Mol Med. 2014;46(7):e105. doi: 10.1038 / emm.2014.36), and mitochondria form a hyperfusion network during the G1-to-S-phase transition associated with cyclin E accumulation (see Mitra K, Vander S, Roysam B, Lin G, Lippincott-Schwartz J.A hyperfused mitochondrial state achieved in G1-S regulates cyclin E accumulation and entry into S phase. Proc Natl Acad Sci US A. 2009;106(29):11960-5. DOI: 10.1073 / pnas.0904875106). The latter study also showed that membrane depolarization Δψ at the beginning of G1 prevents cell cycle progression into S phase.

[0016] Meanwhile, the microenvironment in which tumors grow is extremely unfavorable for mitochondria, since unstable oxygen concentrations and oxidative radicals can disrupt organelle integrity, disintegrate the regulation of multiple mitochondrial functions, and activate cell death (see Damaghi M, West J, Robertson-Tessi M, Xu L, Ferrall-Fairbanks MC, Stewart PA, et al. The harsh microenvironment in early breast cancer selects for a Warburg phenotype. Proc Natl Acad Sci USA. 2021;118(3):e2011342118. DOI: 10.1073 / pnas.2011342118). The questions of how mitochondria cope with the loss of their “functional form” and what is the influence of poor-quality or damaged mitochondria on the state of the tumor, including the transfer of malignant information, remain unclear and poorly studied (see Humpton TJ, Alagesan B., DeNicola GM, Lu D., Yordanov GN, Leonhardt CS, et al., Oncogenic KRAS induces NIX-mediated mitophagy to promote pancreatic cancer.CancerDiscov.2019; 9(9):1268-87. DOI: 10.1158 / 2159-8290).

[0017] In early 2025, the “Recommendations for mitochondria transfer nomenclature and characterization” were published, developed by more than 30 scientists from around the world (see Brestoff JR, Singh KK, Aquilano K, Becker LB, Berridge MV, Boilard E, Caicedo A, Crewe C, Enríquez JA, Gao J, Gustafsson ÅB, Hayakawa K, Khoury M, Lee YS, Lettieri-Barbato D, Luz-Crawford P, McBride HM, McCully JD, Nakai R, Neuzil J, Picard M, Rabchevsky AG, Rodriguez AM, Sengupta S, Sercel AJ, Suda T, Teitell MA, Thierry AR, Tian R, Walker M, Zheng M. Recommendations for mitochondria transfer and transplantation nomenclature and characterization. Nat Metab. 2025 Jan;7(1):53-67. doi: 10.1038 / s42255-024-01200-x. Epub 2025 Jan 16. PMID: 39820558).

[0018] These guidelines significantly eased the complex task of characterizing all forms of mitochondria—whether structurally damaged, intact, or fragmented. They emphasized the need for mitochondria to be characterized, at least in terms of size, as this is a physical characteristic that can be measured using a variety of methods.

[0019] Thus, based on the generally accepted regulatory metabolic, proliferative, and migratory functions of mitochondria, it is hypothesized that in malignant tumors themselves, the development of mitochondrial dysfunction forms a trigger mechanism for pro-oncogenic action, suggesting that mitochondria are one of the target carriers of malignant information. If this hypothesis is valid, the possibility of such malignant transfer can be studied using isolated mitochondrial ultrastructures from tumors, rather than tumor cells themselves.

[0020] Research on tissue-isolated mitochondria can not only help expand our understanding of the relationship between functional and morphological aspects, but also determine the prognostic significance of mitochondrial size in tumor progression, particularly in distant metastasis processes.

[0021] The technical result of the present invention is the creation of a method for determining the presence of distant metastases of rectal adenocarcinoma by measuring the perimeter of mitochondria isolated from the tumor and conditionally healthy tissue along the tumor resection line in men and women.

[0022] The stated goal is achieved by recording the perimeter of mitochondria on mitochondrial preparations isolated from the tumor and conditionally healthy tissue along the line of rectal resection in images obtained by electron microscopy in patients of both sexes and comparing them with clinical diagnostic data on the presence or absence of distant metastases, the coincidence with which determines the validity of the prognosis.

[0023] The invention "Method for determining the presence of distant metastases of rectal adenocarcinoma" is new, since it is unknown in the field of clinical research in oncology about the presence of a relationship between the size of the perimeter of mitochondria isolated from conditionally healthy tissue along the resection line of a rectal tumor and the development of distant metastases.

[0024] The novelty of the invention lies in the use of mitochondria isolated from conditionally healthy tissue along the resection line of rectal adenocarcinoma, with morphologically significant perimeter parameters, to determine the presence of distant metastasis. Comparison of mitochondrial morphometric data with clinical metastasis diagnostic data demonstrates a direct correlation between a statistically significant increase in the number of large mitochondria and the presence of distant metastases in both men and women.

[0025] The invention "Method for determining the presence of distant metastases of rectal adenocarcinoma" is industrially applicable, since it can be used in oncology research institutions in the morphological study of the size of mitochondria isolated from conditionally healthy tissue along the resection line of rectal adenocarcinoma in men and women, as a criterion for assessing the development of distant metastases.

[0026] For a better understanding of the method, we provide figures.

[0027] Figure 1. Markup map with the applied contours of mitochondria isolated from a tumor (a) and conditionally healthy tissue along the resection line of a rectal tumor (b) in patient N. with the presence of distant metastases. Magnification x 50,000.

[0028] Figure 2. Marking map with applied contours of mitochondria isolated from a tumor (a) and conditionally healthy tissue along the resection line of a rectal tumor (b) in patient D. with the presence of distant metastases. Magnification x 50,000.

[0029] Figure 3. Markup map with the applied contours of mitochondria isolated from the tumor (a) and conditionally healthy tissue along the resection line of the rectal tumor (b) in patient K. without distant metastases. Magnification x 50,000.

[0030] Figure 4. Marking map with applied contours of mitochondria isolated from a tumor (a) and conditionally healthy tissue along the resection line of a rectal tumor (b) in patient Sh. without distant metastases. Magnification x 50,000.

[0031] The method for determining the presence of distant metastases of rectal adenocarcinoma is as follows.

[0032] During a laparotomy operation, a malignant tumor is removed from a patient. The tumor portion is 1 cm. 3 quickly placed in a sterile cold solution containing 0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4.

[0033] Mitochondria are isolated using differential centrifugation on a high-speed refrigerated centrifuge Avanti JE, BECMAN COULTER, USA according to the method of Egorova M.V. and Afanasyev S.A. (see Egorova M.V., Afanasyev S.A., Isolation of mitochondria from animal and human cells and tissues: Modern methodological techniques / / Siberian Medical Journal. 2011; 26 (1-1): 22-28; see Gureev A.P., Kokina A.V., Syromyatnikova M.Yu., Popov V.N. Optimization of methods for isolating mitochondria from different mouse tissues / / VSU Bulletin, series: chemistry, biology, pharmacy. 2015; 4: 61-65).

[0034] To destroy intercellular connections, cell walls and plasma membranes, mechanical processing of tissues was used, including grinding with scissors and homogenization in a glass homogenizer with a Teflon pestle (Potter-Elveheim homogenizer).

[0035] For each gram of tissue, 10 ml of sterile isolation medium (0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4) were added. The tissues were homogenized and centrifuged for the first time for 10 min at 1000 g, temperature 0-2°C, the second and third centrifugations were performed at 20,000 g, 20 min, temperature 0-2°C.

[0036] Between centrifugations, the mitochondrial sediment was resuspended in the isolation medium.

[0037] Mitochondria were further purified from lysosomes, peroxisomes, melanosomes, etc., by centrifugation in a 23% Percoll gradient. The subcellular structure suspension was layered on the Percoll gradient and centrifuged for 15 min at 21,000 g. Separation into three phases was then observed. The lower mitochondrial layer was discarded and resuspended in isolation medium. The mitochondria were further "washed" by centrifugation for 10 min at 15,000 g at 0-2°C.

[0038] A portion of the mitochondria suspension isolated from tumor and conditionally healthy rectal tissue is immediately placed in a fixing solution containing formaldehyde and glutaraldehyde.

[0039] After preliminary preparation, the tissue sample was embedded in pure Epon-812 resin (SPI Inc., USA) and cured for 72 h at 70°C. Ultrathin 90-nm sections were obtained using an ultramicrotome equipped with a diamond knife.

[0040] The sections were mounted on copper slit grids and contrasted with 2% aqueous uranyl acetate for 40 min and lead citrate for 2 min. The sections were examined and photographed using a Jeol JEM-1011 electron microscope (JEOL Inc., Japan).

[0041] Mitochondrial perimeter determination in electron microscopy images was performed using FiJi software. Before analysis, the image scale was manually adjusted using the scale bar present in each micrograph. Mitochondria were selected using the Freehand Selection tool.

[0042] Contrast correction and image smoothing were also performed where necessary to improve visualization of mitochondrial boundaries. After outlining each mitochondrion, the result was saved as an ROI, and the resulting contours were added to ROI Manager. Based on these selections, the program automatically calculated the perimeter of each mitochondrion.

[0043] To improve the reproducibility and transparency of the analysis, a markup map was created for each image: files with the plotted mitochondria contours were saved separately, which allowed for subsequent verification of the accuracy of object identification.

[0044] Statistical analysis of the results was performed using the Statistica 10.0 software package. Normality distribution was assessed using the Shapiro-Wilk test (for small samples).

[0045] Comparison of quantitative data in groups (independent samples) was performed using the Student's t-test and the Mann-Whitney U-test. A p-value of <0.05 was used as the threshold for statistical significance. The data in the tables are presented as M±m, where M is the arithmetic mean and m is the standard error of the mean (see Table 1).

[0046] Table 1.

[0047] Perimeter of mitochondria isolated from tumor and conditionally healthy tissue of the rectum of male and female patients with or without metastases.

[0048] Patient groups tumor conditionally healthy tissue 0-1 μm 1-2 μm 1-2 μm 0-1 μm 1-2 μm 1-2 μm women with MTS n=7 65,5±5,1% 0,63±0,009 n=272 22.9±2,0% 1,46±0.03 n=95 11,6±1,4% 2,43±0,05 n=48 50,6±4,9% 0,69±0,01 n=167 35,2±2,9% 1,45±0,03 n=116 14,2±1,7% 2,41±0,06 n=47 women without MTS n=7 49,6±4,4% 0,69±0,01 n=130 36,3±3,1% 1,45±0,03 n=95 14,1±1,6% 2,43±0,06 n=37 61,5±5,4% 0,68±0,01 n=220 34,6±2,7% 1,32±0,02 n=124 3,9±0,5% * 2,32±0,06 n=14 men with MTS n=7 58,1±5,3% 0,68±0,01 n=122 27,1±2,2% 1,41±0,04 n=57 14,8±1,9% 2,65±0,12 n=31 42,5±3,9% 0,69±0,02 n=119 42,5±3,7% 1,41±0,03 n=119 15,0±1,6% 2,63±0,09 n=42 men without MTS n=7 55,9±4,9% 0,7±0,02 n=85 32,3±2,7% 1,4±0,04 n=49 11,8±1,5% 2,5±0,08 n=18 64,5±5,1% 0,76±0,01 n=82 32,4±2,5% 1,37±0,03 n=71 3,1±0,2% * 2,48±0,16 n=19

[0049] Legendary designations: % - frequency of detection of mitochondria of each cluster dimension; mean values ​​and standard error of the mean perimeter of mitochondria isolated from tumor and conditionally healthy tissue; * - the difference between the frequency of detection of the maximum mitochondrial dimension in conditionally healthy tissue of patients with and without metastases is statistically significant (p< 0.05).

[0050] These statistical data indicate that the maintenance of mitochondrial counts with a perimeter of -2 μm, i.e., the largest size in relatively healthy rectal tissue, at the same level as in tumors, is characteristic of patients with distant metastases. Conversely, a statistically significant decrease in the frequency of detection of mitochondria with a perimeter of -2 μm is characteristic of patients without distant metastases.

[0051] We present examples of the method’s implementation in patients with distant metastases (No. 1, 2) and without distant metastases (No. 3, 4).

[0052] Example 1.

[0053] Patient characteristics. Patient N., born in 1978.

[0054] Upon admission on October 13, 2023, to the Federal State Budgetary Institution "National Medical Research Center of Oncology" of the Ministry of Health of the Russian Federation, the condition is moderate. The main disease is C20. (C20) Rectal cancer St.IV, pT4bN0M1 (HEP, PUL), the condition is after abdominoperineal extirpation of the rectum with extended lymphadenectomy, resection of the posterior vaginal wall, bisegmentectomy S2-S3 of the liver (16.10.2023) cl.gr.2. Concomitant diseases (N80.0) adenomyosis. Myocardial dystrophy CHF 1 st. FC 2.

[0055] Results of pathological and anatomical studies: Protocol of the PAO dated 10.22.23: in the rectal wall, moderately differentiated low-grade / Grade 2 adenocarcinoma with comedo-necrosis, with growth of all layers of the colon wall with perineural and angiolymphvascular invasion (pPn1, pLV1) with invasion into the mesorectal tissue and the adjacent vaginal wall up to the mucosa, with metastases of carcinoma to the liver tissue. Fourteen regional lymph nodes were examined - without tumor growth, sinus histiocytosis. In the resection lines of the rectum and liver, tumor growth is not determined pT4bN0M1 (hep).

[0056] To obtain mitochondria from tumor and conditionally healthy tissue, the material was quickly placed in a sterile cold solution containing 0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4. Mitochondria were isolated using differential centrifugation on a high-speed refrigerated centrifuge Avanti JE, BECMAN COULTER, USA according to the method of Egorova M.V. and Afanasyev S.A. (see Egorova M.V., Afanasyev S.A., Isolation of mitochondria from animal and human cells and tissues: Modern methodological techniques / / Siberian Medical Journal. 2011;26(1-1):22-28; see Gureev A.P., Kokina A.V., Syromyatnikova M.Yu., Popov V.N. Optimization of methods for isolating mitochondria from different mouse tissues / / VSU Bulletin, series: chemistry, biology, pharmacy. 2015;4:61-65).To disrupt intercellular connections, the cell wall, and plasma membranes, the tissues were mechanically processed by chopping with scissors and homogenizing in a glass homogenizer with a Teflon pestle (Potter-Elveheim homogenizer). For each gram of tissue, 10 ml of sterile isolation medium (0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4) were added. The tissues were homogenized and centrifuged for the first time for 10 min at 1000 g, temperature 0-2 °C, the second and third centrifugations were carried out at 20,000 g, 20 min, temperature 0-2 °C. Between centrifugations, the mitochondrial pellet was resuspended in the isolation medium. Mitochondria were additionally purified from lysosomes, peroxisomes, melanosomes, etc., by centrifugation in a 23% Percoll gradient.The subcellular structure suspension was layered on a Percoll gradient, centrifuged for 15 min at 21,000 g, and then separated into three phases. The lower mitochondrial layer was retained and resuspended in the isolation medium. The mitochondria were then "washed" by centrifugation for 10 min at 15,000 g at 0-2°C. A portion of the mitochondrial suspension, isolated from tumor and otherwise healthy rectal tissue, was immediately placed in a fixative solution containing formaldehyde and glutaraldehyde. After preliminary preparation, the tissue sample was embedded in pure Epon-812 resin (SPI Inc., USA) and cured for 72 h at 70°C. Ultrathin 90-nm sections were obtained using an ultramicrotome equipped with a diamond knife. The sections were mounted on copper slit grids and contrasted with a 2% aqueous uranyl acetate solution for 40 min and lead citrate for 2 min. The sections were examined and photographed using a Jeol JEM-1011 electron microscope (JEOL Inc., Japan).

[0057] Mitochondrial perimeters were determined in electron microscopy images using FiJi software. Before analysis, the image scale was manually set using the scale bar included in each micrograph. Mitochondria were isolated using the Freehand Selection tool. Contrast correction and image smoothing were performed to improve visualization of mitochondrial boundaries. After outlining each mitochondrion, the result was saved as an ROI, and the resulting contours were added to ROI Manager. A markup map was created for each image: files with the applied mitochondrial contours were saved separately, which allowed for subsequent verification of the accuracy of object identification (see Fig. 1).

[0058] Based on these isolations, the FiJi program automatically calculated the perimeter of each mitochondria. A total of 315 mitochondrial perimeter measurements were taken on four slides isolated from the tumor and four slides (310 measurements) from apparently healthy rectal tissue. No difference was found in the frequency of detection of mitochondria with critically large perimeters in the tumor and apparently healthy tissue along the tumor resection line in patient N.

[0059] Example 2.

[0060] Patient characteristics. Patient D., born in 1960.

[0061] Upon admission on September 26, 2023, to the Federal State Budgetary Institution "National Medical Research Center of Oncology" of the Ministry of Health of the Russian Federation, the patient's condition is satisfactory. The main disease is C20. (C20) Cancer of the upper ampullary part of the rectum, pT4N1M1 (HEP, PUL), with the formation of an inflammatory infiltrate involving the distal appendix, St. IVB, metastasis to the liver and lung, cl.group 2. Concomitant diseases: myocardial dystrophy CHF stage 1. FC 2. (150)

[0062] On September 28, 2023, the patient underwent surgery - A16.19.021 rectal resection with extended lymphadenectomy, preventive ileostomy, and appendectomy. Postoperatively, the patient received intensive antibacterial, anti-inflammatory, and detoxifying infusion therapy.

[0063] Pathological examination results: low-grade / G2 colon adenocarcinoma with ulceration of adjacent adipose tissue, invasion of all layers, including the serous membrane, perineural and lymphovascular invasion. Nine of 14 lymph nodes contain adenocarcinoma metastases with invasion of adjacent adipose tissue. Histological analysis: no signs of tumor growth were detected in the separately delivered tissue from the resection line. Histological analysis: follicular hyperplasia of the lymphoid tissue of the lamina propria of the mucosa, fibrosis of adjacent adipose tissue in the appendix tissue. pT4aN2b.

[0064] To obtain mitochondria from the tumor and conditionally healthy tissue, the material was quickly placed in a sterile cold solution containing 0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4. Mitochondria were isolated using differential centrifugation in a high-speed refrigerated centrifuge Avanti JE, BECMAN COULTER, USA according to the method of Egorova M.V. and Afanasyev S.A. (see Egorova M.V., Afanasyev S.A., Isolation of mitochondria from animal and human cells and tissues: Modern methodological techniques / / Siberian Medical Journal. 2011; 26 (1-1): 22-28). To disrupt intercellular connections, cell walls, and plasma membranes, tissues were mechanically processed by mincing with scissors and homogenizing in a glass homogenizer with a Teflon pestle (Potter-Elveheim homogenizer). For each gram of tissue, 10 ml of sterile isolation medium (0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4) were added.The tissues were homogenized and centrifuged for the first time for 10 min at 1000 g, temperature 0-2 °C, the second and third centrifugations were carried out at 20,000 g, 20 min, temperature 0-2 °C. Between centrifugations, the mitochondrial pellet was resuspended in the isolation medium. Mitochondria were additionally purified from lysosomes, peroxisomes, melanosomes, etc., by centrifugation in a 23% Percoll gradient. The suspension of subcellular structures was layered on a Percoll gradient, centrifuged for 15 min at 21,000 g, after which separation into three phases was observed, the lower mitochondrial layer was preserved and resuspended in the isolation medium. The next "wash" of mitochondria was carried out by centrifugation for 10 min at 15,000 g, temperature 0-2 °C. A portion of the mitochondrial suspension isolated from tumor and apparently healthy rectal tissue was immediately placed in a fixative solution containing formaldehyde and glutaraldehyde. After preliminary preparation, the tissue sample was embedded in pure Epon-812 resin (SPI Inc.), USA) and cured for 72 h at 70°C. Ultrathin 90-nm sections were obtained using an ultramicrotome equipped with a diamond knife. The sections were mounted on copper slit grids and counterstained with 2% aqueous uranyl acetate for 40 min and lead citrate for 2 min. The sections were examined and photographed using a Jeol JEM-1011 electron microscope (JEOL Inc., Japan). Mitochondrial perimeter was determined in electron microscopy images using FiJi software. Before analysis, the image scale was set manually using the scale bar present in each micrograph. Mitochondria were selected using the Freehand Selection tool. Contrast correction and image smoothing were performed to improve visualization of mitochondrial boundaries. After completing the outlining of each mitochondria, the result was saved as an ROI, and the resulting contours were added to the ROI Manager.For each image, a markup map was created: files with the plotted mitochondria contours were saved separately, which made it possible to subsequently check the accuracy of object identification (see Fig. 2).

[0065] Based on these isolations, the FiJi program automatically calculated the perimeter of each mitochondria. A total of 276 perimeter measurements of mitochondria isolated from the tumor and 293 from apparently healthy rectal tissue were taken on four slides. No difference was found in the frequency of detection of mitochondria with critically large perimeters in the tumor and apparently healthy tissue along the tumor resection line in patient D.

[0066] Example 3. Patient characteristics. Patient K., born in 1956.

[0067] Upon admission on October 9, 2023, to the National Medical Research Center of Oncology of the Russian Ministry of Health, the patient's condition was moderate. The main disease was C20. (C20) Rectal cancer with transition to the rectosigmoid region pT2N0M0, St.I, condition after surgical treatment (10.10.2023), cl.group 3. Concomitant diseases: (C50) Right breast cancer pT2N1M0, St.IIB, condition after complex treatment in 2017. Cl.group 3.

[0068] On October 10, 2023, the patient underwent laparoscopically assisted rectal resection with extended lymphadenectomy and preventive ileostomy. Postoperatively, the patient received post-syndromic detoxification, hemostatic, anti-inflammatory, antibacterial infusion therapy, and thromboprophylaxis.

[0069] Pathological examination results: PAO protocol dated October 21, 2023: low-grade colon wall adenocarcinoma with ulceration, submucosal and muscularis propria invasion, without lymphovascular or perineural invasion, lymphoid infiltration along the tumor periphery. Sinus histiocytosis and follicular hyperplasia are found in 8 lymph nodes. Resection lines show no signs of tumor growth, pT2N0M0.

[0070] To obtain mitochondria from tumor and conditionally healthy tissue, the material was quickly placed in a sterile cold solution containing 0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4. Mitochondria were isolated using differential centrifugation on a high-speed refrigerated centrifuge Avanti JE, BECMAN COULTER, USA according to the method of Egorova M.V. and Afanasyev S.A. (see Egorova M.V., Afanasyev S.A., Isolation of mitochondria from animal and human cells and tissues: Modern methodological techniques / / Siberian Medical Journal. 2011;26(1-1):22-28; see Gureev A.P., Kokina A.V., Syromyatnikova M.Yu., Popov V.N. Optimization of methods for isolating mitochondria from different mouse tissues / / VSU Bulletin, series: chemistry, biology, pharmacy. 2015;4:61-65).To disrupt intercellular connections, the cell wall, and plasma membranes, the tissues were mechanically processed by chopping with scissors and homogenizing in a glass homogenizer with a Teflon pestle (Potter-Elveheim homogenizer). For each gram of tissue, 10 ml of sterile isolation medium (0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4) were added. The tissues were homogenized and centrifuged for the first time for 10 min at 1000 g, temperature 0-2 °C, the second and third centrifugations were carried out at 20,000 g, 20 min, temperature 0-2 °C. Between centrifugations, the mitochondrial pellet was resuspended in the isolation medium. Mitochondria were additionally purified from lysosomes, peroxisomes, melanosomes, etc., by centrifugation in a 23% Percoll gradient.The subcellular structure suspension was layered on a Percoll gradient, centrifuged for 15 min at 21,000 g, and then separated into three phases. The lower mitochondrial layer was retained and resuspended in the isolation medium. The mitochondria were then "washed" by centrifugation for 10 min at 15,000 g at 0-2°C. A portion of the mitochondrial suspension, isolated from tumor and otherwise healthy rectal tissue, was immediately placed in a fixative solution containing formaldehyde and glutaraldehyde. After preliminary preparation, the tissue sample was embedded in pure Epon-812 resin (SPI Inc., USA) and cured for 72 h at 70°C. Ultrathin 90-nm sections were obtained using an ultramicrotome equipped with a diamond knife. The sections were mounted on copper slit grids and contrasted with a 2% aqueous uranyl acetate solution for 40 min and lead citrate for 2 min. The sections were examined and photographed using a Jeol JEM-1011 electron microscope (JEOL Inc., Japan).

[0071] Mitochondrial perimeters were determined in electron microscopy images using FiJi software. Before analysis, the image scale was manually set using the scale bar included in each micrograph. Mitochondria were isolated using the Freehand Selection tool. Contrast correction and image smoothing were performed to improve visualization of mitochondrial boundaries. After outlining each mitochondrion, the result was saved as an ROI, and the resulting contours were added to ROI Manager. A markup map was created for each image: files with the applied mitochondrial contours were saved separately, which allowed for subsequent verification of the accuracy of object identification (see Fig. 3).

[0072] Based on these isolations, the FiJi program automatically calculated the perimeter of each mitochondria. A total of 326 perimeter measurements of mitochondria isolated from the tumor were made on four preparations, and 313 measurements were made on four preparations from apparently healthy rectal tissue. The detection rate of mitochondria with critically large perimeters in patient K.'s tumor exceeded that in apparently healthy tissue along the tumor resection line by 3.5 times (14% versus 4%), corresponding to a statistically significant difference of p < 0.05.

[0073] Example 4. Patient characteristics. Patient Sh., born in 1969.

[0074] Upon admission on May 20, 2024, to the Federal State Budgetary Institution "National Medical Research Center of Oncology" of the Ministry of Health of the Russian Federation, the patient's condition is moderate. The underlying disease is C20. (C20) Rectal cancer T4aN2bM0, St.IIIc, the condition after surgical treatment in the volume is: anterior-superior resection of the rectum with extended lymphadenectomy, preventive ileostomy (May 21, 2024), class group 2. Complications of the underlying disease: leakage of the colonic anastomosis.

[0075] Postoperatively, the patient underwent detoxification, hemostatic, anti-inflammatory, and antibacterial infusion therapy. Adjuvant antitumor drug therapy for 6 months using the XELOX or FOLFOX-6 regimen was indicated.

[0076] Results of pathological examination: Protocol of the PAO dated 06 / 03 / 2024: colon tumor - poorly differentiated (high grade / G3) adenocarcinoma with a mucinous component (5%) with invasion of all layers of the intestinal wall, ingrowth of the visceral peritoneum, foci of necrosis, moderate chronic inflammation, there are signs of lymphovascular and perineural invasion.

[0077] Histological analysis: in all 11 examined lymph nodes of the adipose tissue there are metastases of adenocarcinoma, and in one of the listed ones there are mucinous structure, in the rest of the area there are tumor deposits in the adipose tissue.

[0078] Histological analysis: no tumor growth was detected in the tissue samples collected along the resection lines. pT4aN2b, ICD code for the test results: C20. Malignant neoplasm of the rectum.

[0079] Operation: 05 / 21 / 2024: anterior-superior rectal resection with extended lymphadenectomy, preventive ileostomy.

[0080] To obtain mitochondria from tumor and conditionally healthy tissue, the material was quickly placed in a sterile cold solution containing 0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4. Mitochondria were isolated using differential centrifugation on a high-speed refrigerated centrifuge Avanti JE, BECMAN COULTER, USA according to the method of Egorova M.V. and Afanasyev S.A. (see Egorova M.V., Afanasyev S.A., Isolation of mitochondria from animal and human cells and tissues: Modern methodological techniques / / Siberian Medical Journal. 2011;26(1-1):22-28; see Gureev A.P., Kokina A.V., Syromyatnikova M.Yu., Popov V.N. Optimization of methods for isolating mitochondria from different mouse tissues / / VSU Bulletin, series: chemistry, biology, pharmacy. 2015;4:61-65).To disrupt intercellular connections, the cell wall, and plasma membranes, the tissues were mechanically processed by chopping with scissors and homogenizing in a glass homogenizer with a Teflon pestle (Potter-Elveheim homogenizer). For each gram of tissue, 10 ml of sterile isolation medium (0.22 M mannitol, 0.3 M sucrose, 1 mM EDTA, 2 mM TRIS-HCL, 10 mM HEPES, pH 7.4) were added. The tissues were homogenized and centrifuged for the first time for 10 min at 1000 g, temperature 0-2 °C, the second and third centrifugations were carried out at 20,000 g, 20 min, temperature 0-2 °C. Between centrifugations, the mitochondrial pellet was resuspended in the isolation medium. Mitochondria were additionally purified from lysosomes, peroxisomes, melanosomes, etc., by centrifugation in a 23% Percoll gradient.The subcellular structure suspension was layered on a Percoll gradient, centrifuged for 15 min at 21,000 g, and then separated into three phases. The lower mitochondrial layer was retained and resuspended in the isolation medium. The mitochondria were then "washed" by centrifugation for 10 min at 15,000 g at 0-2°C. A portion of the mitochondrial suspension, isolated from tumor and otherwise healthy rectal tissue, was immediately placed in a fixative solution containing formaldehyde and glutaraldehyde. After preliminary preparation, the tissue sample was embedded in pure Epon-812 resin (SPI Inc., USA) and cured for 72 h at 70°C. Ultrathin 90-nm sections were obtained using an ultramicrotome equipped with a diamond knife. The sections were mounted on copper slit grids and contrasted with a 2% aqueous uranyl acetate solution for 40 min and lead citrate for 2 min. The sections were examined and photographed using a Jeol JEM-1011 electron microscope (JEOL Inc., Japan).

[0081] Mitochondrial perimeters were determined in electron microscopy images using FiJi software. Before analysis, the image scale was manually set using the scale bar included in each micrograph. Mitochondria were isolated using the Freehand Selection tool. Contrast correction and image smoothing were performed to improve visualization of mitochondrial boundaries. After outlining each mitochondrion, the result was saved as an ROI, and the resulting contours were added to ROI Manager. A markup map was created for each image: files with the applied mitochondrial contours were saved separately, which allowed for subsequent verification of the accuracy of object identification (see Fig. 4).

[0082] Based on these isolations, the FiJi program automatically calculated the perimeter of each mitochondria. A total of 298 perimeter measurements were taken on 4 preparations of mitochondria isolated from the tumor, and 311 measurements were taken on 4 preparations of apparently healthy rectal tissue. The detection rate of mitochondria with critically large perimeters in patient Sh.'s tumor was four times higher than that in apparently healthy tissue along the tumor resection line, namely 12% versus 3%, corresponding to a statistically significant difference of p < 0.05.

[0083] Thus, this study morphologically and statistically confirms the possibility of determining the presence or absence of distant metastases of rectal adenocarcinoma in men and women based on the size of the perimeter of mitochondria isolated from conditionally healthy rectal tissue along the resection line, which allows for the application of timely approaches to antimetastatic treatment.

[0084] The technical and economic effectiveness of this method for detecting distant metastases from rectal adenocarcinoma lies in the fact that measuring the perimeter of mitochondria isolated from the tumor and relatively healthy rectal tissue allows for a differentiated assessment of the development of distant metastases. When large, abnormal mitochondria with high carcinolytic potential are detected in relatively healthy tissue along the resection line of the adenocarcinoma, just like in the tumor, this serves as an unfavorable indicator of the presence of distant metastases (liver, lungs), while a significant reduction in their detection rate indicates the suppression of distant metastasis processes, which is clinically important, as it demonstrates a new, previously unknown mechanism of colorectal cancer dissemination and the possibility of alternative treatment strategies. The method is cost-effective and easily implemented.

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