RU2810558C1 - Method of selecting medicinal products for pharmacological induction of mitochondrial dysfunction in macrophages for antitumor therapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the methods of selecting and using known drugs and pharmaceutical compounds that cause mitochondrial dysfunction in macrophages for antitumor therapy. The claimed invention provides the possibility of using drugs and pharmacological compounds known as such that affect macrophages by changing their mitochondrial functions, namely, shifting mitochondrial metabolism from oxidative phosphorylation, characteristic of M2 type macrophages, to the glycolytic type, characteristic of M1 type macrophages, for treatment of oncological diseases.

EFFECT: invention is promising for the treatment of cancer, and can also be used in the future to prevent the occurrence of cancer.

1 cl, 14 dwg, 2 tbl, 7 ex

Description

The invention generally relates to medicine, and in more detail to the field of use of drugs and pharmaceutical compounds known as such that cause mitochondrial dysfunction in macrophages for the treatment of cancer. The claimed technical solution makes it possible to use drugs and pharmacological compounds known as such that act on macrophages by changing their mitochondrial functions, which become effective agents for the treatment of cancer. According to the applicant, this direction is promising for the treatment and prevention of the development of many types of cancer.

Further in the text, the applicant provides terms that are necessary to facilitate a clear understanding of the essence of the declared materials and to eliminate contradictions and/or controversial interpretations when performing a substantive examination.

Tumor associated macrophages (TAMs) are a class of immune cells present in large numbers in the microenvironment of solid tumors. They are actively involved in inflammation associated with cancer. Macrophages are known to originate from blood monocytes produced in the bone marrow (monocyte-derived macrophages) or yolk sac precursors (tissue macrophages) [https://en.wikipedia.org/wiki/].

Solid tumors are non-hematopoietic tumors, that is, tumors that do not develop from cells of the hematopoietic system. Solid tumors can be benign or malignant. In the context of this description, the applicant means by this term precisely malignant tumors [https://podari-zhizn.ru/ru/get-help/meditsinskii-spravochnik/].

Targeted therapy is a direction in the treatment of cancer using drugs that block certain molecules of tumor cells or cells of the tumor microenvironment. Due to this, cancer cells stop multiplying, their blood supply deteriorates, or other therapeutic effects are achieved. Compared to classical chemotherapy drugs, targeted drugs act more specifically and almost do not affect healthy cells [https://www.euroonco.ru/].

Reprogramming of macrophages - in the context of the present description, the applicant means by this term the process of changing the phenotype of macrophages.

Mitochondrial respiration is a set of biochemical reactions occurring in the cells of living organisms, during which the oxidation of carbohydrates, lipids and amino acids to carbon dioxide and water occurs, as well as the formation of energy [https://en.wikipedia.org/wiki/].

Chi3L1, chitinase 3-like protein 1, is a secreted glycoprotein that is approximately 40k Da in size. Expressed and secreted by various cell types, including macrophages, chondrocytes, fibroblast-like synovial cells, vascular smooth muscle cells and hepatic stellate cells [https://ru.abcdef.wiki/wiki/CHI3L1].

Classically activated macrophages (M1 polarization, M1 subtype) are macrophages that acquire a phenotype with higher activity against both pathogens and tumor cells. They also release inflammatory cytokines [https://ru.wikipedia.org/wiki/].

Alternatively activated macrophages (M2 polarization, M2 subtype) are macrophages that secrete minimal amounts of proinflammatory cytokines and have lower activity against intracellular pathogens [https://ru.wikipedia.org/wiki/].

Cell sensitization is the acquisition by the body of a specific increased sensitivity to foreign substances - allergens, increasing its sensitivity to the effects of irritants [https://ru.wikipedia.org/wiki/]

Coenzyme A is an enzyme distinguished by its role in the synthesis and oxidation of fatty acids, as well as the oxidation of pyruvate in the citric acid cycle [https://en.wikipedia.org/wiki/Coenzyme_A].

Exosomes are microscopic extracellular vesicles (bubbles) with a diameter of 30-100 nanometers, secreted into the intercellular space by cells of various tissues and organs [https://ru.wikipedia.org/wiki/].

Monoamine oxidase A is an enzyme that catabolizes neurotransmitters and hormones. Thus, this enzyme plays an important role in maintaining constant concentrations of endogenous monoamines in tissues, which is especially important for nervous tissue, and also limits their entry into the body with food and is involved in the metabolism of dangerous biologically active substances that are structurally similar to endogenous monoamines [https: //ru.wikipedia.org/wiki/].

Pharmacological induction is an absolute increase in the number and activity of metabolic enzymes due to exposure to a certain chemical compound, in particular a drug [https://ru.wikipedia.org/wiki/].

Opa1 is a multifunctional mitochondrial protein that encodes a dynamin-like GTPase. "The activity of the protein is necessary for the formation of cristae - the system of internal mitochondrial membranes. The protein controls the process of fusion and separation of mitochondria, which is critical for overcoming stressful conditions and maintaining the energy balance of the cell. [https://www.genokarta.ru/gene/OPA1]

Transfection is the process of introducing nucleic acid into eukaryotic cells by a non-viral method [https://web.archive.org/web/20081217035133/].

Small interfering RNA is a class of double-stranded RNA, 20-25 nucleotides in length. The interaction of small interfering RNA with messenger RNA (mRNA) of the target gene leads to degradation of the latter (in the process of RNA interference), preventing the translation of mRNA on ribosomes into the protein it encodes. Ultimately, the effect of small interfering RNA is identical to that of simply reducing gene expression [https://ru.wikipedia.org/wiki\].

THP-1 is a human monocytic cell line derived from a patient with acute monocytic leukemia. It is used to test leukemia cell lines in immunocytochemical analysis of protein-protein interactions and immunohistochemistry and as a model monocyte cell line [https/en.wikipedia.org/wiki/THP-1_cell_line].

Conditioned media is a nutrient medium containing various physiologically active waste products of cells pre-cultured in it [http://humbio.ru/humbio/tarantul_sl/00000b02.htm].

Extracellular receptors are receptors that are embedded in the plasma membrane of cells. They participate in cell signaling by binding to extracellular molecules. These are specialized integral membrane proteins that provide communication between the cell and the extracellular space [https/en.wikipedia.org/wiki/Cell_surface_receptor].

Oncological diseases in the world rank second in mortality after cardiovascular pathology, and despite certain advances in diagnostics and treatment methods, the rate of increase in cancer incidence is steadily increasing. Recent scientific research has shown that an effective approach to cancer treatment requires therapy, namely:

- therapy aimed at tumor cells (chemotherapy, radiotherapy, surgical treatment)

- therapy aimed at the tumor microenvironment (targeted therapy)

- therapy aimed at immune cells (immune therapy).

At the same time, it should be emphasized that tumor-associated macrophages (TAMs) are the main component of this microenvironment and play a significant role in the progression of cancer.

During tumor growth in the patient’s body, macrophages create a favorable inflammatory environment, which promotes more aggressive tumor growth by suppressing the antitumor immune response in the patient, which ultimately ensures the actual spread of cancer to other organs (metastasis) [Mantovani A. et. .al. (2022) Macrophages as tools and targets in cancer therapy, Nat Rev Drug Discov., 21(11), 799-820].

Solid cancers such as lung cancer and pancreatic cancer respond very poorly to both chemotherapy and immune therapy, mainly due to the active inflammatory process within the tumor due to the negative impact of cancer cells on the patient's macrophages.

The infiltration of macrophages from the peripheral blood into the site of tumor formation is an early process in cancer, with the initially good goals of launching an immune response and destroying cancer cells, however, taking into account the known property of tumor cells to reprogram the properties of macrophages, this process of destruction of tumor cells by macrophages is launched in the opposite direction, and It is macrophages that begin to support the growth of tumor cells and contribute to the progression of the patient’s tumor.

Thus, the applicant in the above material has identified the cause and effect of the development of oncological diseases, as a result of which it is necessary to find an effect on macrophages to stop the maintenance of the tumor growth process or to find a method to stop or destroy the tumor process in the patient’s body.

The so-called macrophages of the M1 subtype are responsible for the destruction of tumor cells, due to the fact that they have antitumor properties. However, tumor cells reprogram macrophages from the M1 to the M2 subtype due to the fact that cancer cells release substances into the microenvironment: cytokines and chemokines, thereby ensuring tumor growth and metastasis.

Thus, cancer cells, reprogramming macrophages from the M1 to the M2 subtype, help the tumor grow and, as a result, turn off the antitumor immunity of the patient’s body.

Such reprogrammed macrophages are called alternatively activated macrophages of the M2 subtype, while the state of the art has experimentally shown that the accumulation of such macrophages correlates with a poor prognosis for the development of the disease in cancer patients, especially lung cancer, adenocarcinoma and pancreatic cancer [Zhang B. et. al. (2011) M2-polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma, Clin (Sao Paulo), 66, 879-1886].

As of the date of submission of the declared materials, to minimize the indicated negative effect of the development of the tumor process, several strategies are used to influence cancer macrophages, namely:

- elimination of macrophages from the tumor,

- prevention of migration of macrophages (M2) into the tumor,

- suppression of the functions of alternatively activated macrophages of the M2 subtype

- reprogramming of M2 macrophages into the M1 subtype of macrophages

Next, the applicant carried out a detailed analysis of each of the known strategies presented above from the point of view of their effect on macrophages.

The disadvantage of eliminating macrophages is that circulating monocytes are continuously recruited into tumors, where they differentiate into mature M2 macrophages and re-involve in tumor progression [Zhu Y. et. al. (2017) Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression, Immunity., 47, 323 - 328].

At the same time, the main obstacle in preventing the migration of macrophages into the tumor may be that the depletion of macrophages coincides with the loss of tissue-resident populations of macrophages important for maintaining homeostasis, i.e. leads to a general disruption of the functioning of the body [Ries C.H. et. al. (2014) Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy, Cancer cell., 25(6), P. 846-859].

Another limitation of strategies aimed at suppressing the functions of M2 macrophages is due to the fact that if alternatively activated macrophages are completely removed from the body, the inflammatory process in the body will take a very long time to occur, since M2 macrophages contribute to tissue repair [Etzerodt A. et. al. (2019) Specific targeting of CD163 mobilizes inflammatory monocytes and promotes T cell-mediated tumor regression, J. Exp. Med., 216(10), 2394-2411].

Based on the analysis presented above, we can draw a logical conclusion that reprogramming macrophages from the M2 to the M1 subtype is the most promising strategy for the combined treatment of cancer. The first preclinical studies showed that treatment methods aimed at reprogramming macrophages are not toxic to the patient's body and are well tolerated, because This strategy leads to clinical activation of the patient's immune system as a whole, with this stabilization of the disease also observed in patients receiving intensive pretreatment with chemotherapy [[Mantovani A. et.al. (2022) Macrophages as tools and targets in cancer therapy, Nat Rev Drug Discov., 21(11), 799-820].

In the process of reprogramming M1 to M2, cardinal changes in the metabolic functions of the patient’s body occur in the patient’s body, i.e. pro-inflammatory M1 macrophages and alternatively activated M2 macrophages provide massive metabolic changes [Viola A. et. al. (2019) The metabolic signature of macrophage responses, Front. Immunol., 10, 1462], aimed at destroying cancer cells in the patient’s body.

Considering that most of the metabolic pathways required for macrophage polarization are associated with mitochondria, targeting the mitochondrial metabolism of M2 macrophages appears promising.

It is known that changes in the mitochondrial functions of macrophages are a hallmark of pro-inflammatory M1 macrophages [Jha A.K. et.al. (2015) Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization, Immunity., 42(3), 419-30], [Ramond E. et. al. (2019) Pivotal role of mitochondria in macrophage response to bacterial pathogens, Front. Immunol., 10.2461].

M1 macrophages cannot use mitochondrial respiration to produce energy and therefore switch their metabolism to glycolysis [Batista-Gonzalez A. (2020) New Insights on the Role of Lipid Metabolism in the Metabolic Reprogramming of Macrophages, Front. Immunol.].

However, M2 macrophages exhibit moderate levels of glycolysis and utilize the tricarboxylic acid cycle, which allows them to support high levels of oxidative phosphorylation and tumor growth.

In contrast, pro-inflammatory M1 macrophages exhibit very low levels of oxidative phosphorylation due to disruption of the electron transport chain (ETC).

Thus, we can draw a general conclusion that methods of redirecting mitochondrial metabolism in tumor macrophages (M2) from oxidative phosphorylation to glycolysis appears to be a promising method for reprogramming macrophages from M2 to M1 in cancer.

As of the date of submission of the claimed technical solution, the applicant conducted research into the level of technology and trends in the development of technology in this field based on scientific and patent information, and identified existing problems associated with the lack of an effective approach to cancer treatment aimed at both tumor cells and the tumor microenvironment.

Due to the above, there is a hitherto unresolved problem in identifying methods of influencing tumor macrophages, including by changing their mitochondrial functions for the treatment of cancer.

From the researched level of technology, an invention was identified under patent No. WO 2023287096 A1 (analogue No. 1) “Composition for inhibiting m2 polarization of macrophages and treating cancer, containing humanized monoclonal antibody specific to chi3l1” “Composition for inhibiting m2 polarization of macrophages and treating cancer, containing monoclonal antibody specific for chi3l1."

The essence of the known technical solution is a pharmaceutical composition containing: a monoclonal antibody that specifically binds to Chi3L1 as an active ingredient, as a method of inhibiting the polarization of M2 macrophages, for the prevention, treatment or inhibition of cancer metastasis. Composition for inhibiting M2 polarization of macrophages according to claim 1, characterized in that the monoclonal antibody contains the following areas:

(a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1; And

(b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3.

Composition for inhibiting M2 polarization of macrophages according to claim 1, characterized in that the monoclonal antibody contains the following areas:

(a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; And

(b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4.

Composition for inhibiting M2 polarization of macrophages according to claim 1, wherein the composition inhibits STAT6 phosphorylation.

A method for inhibiting the polarization of M2 macrophages, comprising contacting any composition of any one of claims 1-4 with cells expressing the Chi3L1 protein.

A pharmaceutical composition for preventing, treating or inhibiting cancer metastasis, comprising a composition of any one of claims 1 to 4 as an active ingredient.

A composition for diagnosing cancer, containing a composition of any one of claims 1 to 4 as an active ingredient.

A cancer diagnostic kit containing a composition of any one of claims 1 to 4 as an active ingredient.

Thus, briefly, the essence of the known technical solution is a pharmaceutical composition containing: a monoclonal antibody specific for Chi3L1. The composition has an excellent effect of inhibiting tumor growth and metastasis, and thus can be variously used as compositions for preventing and treating cancer and inhibiting cancer metastasis.

The disadvantage of the known invention under patent No. WO2023287096A1 is that monoclonal antibodies directed against tumor-associated macrophages quickly disintegrate in the body, and usually no more than 20% of the administered dose interacts with macrophages [Beckman. et al. (2007) Potential Role for Early Biomarker Testing as Part of a Modern, Multidisciplinary Approach to Sjögren’s Syndrome Diagnosis. Adv Ther, 34, 779-812. - 2017. V 34, 799-812].

Another disadvantage is that monoclonal antibodies can have several different modes of action on cells, and the actual mechanism of action once administered to patients is not always clear [E. Soderlind. et al. (2001) The Immune Diversity in a Test Tube - Non-Immunised Antibody Libraries and Functional Variability in Defined Protein Scaffolds. Bentham science, 4(5), 409-416]. Thus, the known technical solution is limited in the effectiveness of its intended use.

From the researched level of technology, an invention was identified under patent No. WO 2019143948 A1 (analogue No. 2) “Altering inflammatory states of immune cells in vivo by modulating cellular activation states.” The essence of the known technical solution is a method for changing the activation state of macrophages in a tumor, including: introducing into the tumor site nanoparticles containing a polyamino ester core and a polyglutamic acid coating with transcribed mRNA encoding one or more regulatory factors encapsulated in the core, and at the same time the activation state macrophages changes from the classical state to the activated state. The tumor is an ovarian cancer, a glioblastoma tumor, or a metastatic lung cancer tumor.

Thus, in brief, the essence of the known technical solution is the use of nanoparticles carrying mRNAs encoding M1-polarizing transcription factors that can reprogram TAMs into tumor cell-killing macrophages or otherwise cause the destruction of tumor cells, thereby treating cancer.

The disadvantage of the known invention under patent No. WO2019143948A1 is that targeting IKKβ to block the activity of M2-like macrophages is not an ideal approach for the treatment of cancer, according to the applicant, since these kinases have several directions of influence and can also cause unwanted side effects effects, namely deterioration of cardiac function [Bhullar K.S. et al. (2018) Kinase-targeted cancer therapies: progress, challenges and future directions. Mol Cancer. V. 17 (48)]. Thus, the known technical solution as a whole, according to the applicant, is limited in effectiveness when used for its intended purpose.

From the researched level of technology, an invention was identified under patent No. CN 101904837 A (analogue No. 3) “Use of rotenone as non-small cell lung cancer cell sensitizer.”

The essence of the known technical solution is the use of rotenone, an inhibitor of the mitochondrial respiratory chain, to prevent resistance of non-small cell lung cancer cells to targeted anticancer drugs.

Rotenone is a compound isolated from natural plants and can bind to mitochondrial respiratory chain complex I, thereby inhibiting mitochondrial oxidative phosphorylation, reducing mitochondrial membrane potential and causing cell damage. This technical solution showed that the use of rotenone compound has no obvious toxic and side effects on normal cells (HEK293), and shows that rotenone reduces the number of lung cancer tumor cells (A549 and NCI-H460) and is a new drug combination that can be used for the treatment of non-small cell lung cancer.

The disadvantage of the known invention according to patent No. CN101904837A is that rotenone can inhibit tumor cells, but the compound itself can induce apoptosis of nerve cells and cause many toxic and side effects, namely vomiting, incoordination, muscle tremors, clonic convulsions and respiratory failure, due to which may result in complications or death.

The claimed technical solution solves the problem by selecting the dose of rotenone and other drugs in such a way as to minimize its toxic effect on healthy cells and enhance its effect on tumor cells.

From the researched level of technology, an invention was identified under patent No. WO2020006199A2 (analogue No. 4) “Methods and agents for modulating inflammation.”

The essence of the known technical solution is a method of enhancing alternative activation of macrophages by introducing an agent that increases intracellular levels of coenzyme A. Increasing the amount of intracellular coenzyme A increases the alternating activation of macrophages, which leads to suppression of the immune response, which is useful in the treatment of inflammatory diseases. Reducing the intracellular level of coenzyme A reduces alternative activation of macrophages and changes the activity of tumor-associated immune cells, which can be effectively used for the treatment of cancer.

The disadvantage of the known invention under patent No. WO2020006199A2 is that it affects one of the stages/processes of mitochondrial metabolism, while the claimed technical solution proposes a range of drugs that affect various metabolic processes in mitochondria, namely:

- suppression of mitochondrial respiration,

- change in the electron transport chain,

- suppression of oxidative phosphorylation,

- suppression of carnitine, ATP production.

From the researched level of technology, an invention was identified under patent No. CA3088009A1 (analogue No. 5) “Methods and compositions for macrophage polarization”.

The essence of the known technical solution is the use of extracellular vesicles containing nucleic acid to change the polarization of tumor-associated macrophages.

An extracellular vesicle containing one or more components that, upon contact with a macrophage, selectively change the macrophage from the M2 phenotype to the M1 phenotype. The extracellular vesicle is an exosome and contains several components that inhibit one macrophage target gene. The component included in the vesicles is a nucleic acid - inhibitory RNA. Analyzed: extracellular vesicle uptake, target gene expression, cytokine release, and macrophage cell surface. Upon interaction of extracellular vesicles containing nucleic acid, the M1 subtype macrophage exhibited increased secretion of inflammatory cytokines and chemokines and decreased secretion of anti-inflammatory cytokines and chemokines, compared with the M2 subtype macrophage.

Thus, briefly, the essence of the known technical solution is a composition comprising extracellular vesicles containing genes that reprogram macrophage polarization for the treatment of cancer.

The disadvantage of the known invention according to patent No. CA3088009A1 is its complex technical implementation. Isolation and purification of exosomes using consistent and reproducible methods remains a challenge. The claimed technical solution solves the problem by using known pharmaceuticals for a new purpose.

Known patent No. WO2022087424A1 (analogue No. 6) “Monoamine oxidase blockade therapy for treating cancer through regulating tumor associated macrophages (tams)” “Monoamine oxidase blockade therapy for treating cancer by regulating tumor-associated macrophages.”

The essence of the known technical solution is a composition of a substance, including: a chemotherapeutic agent; monoamine oxidase A inhibitor; and a pharmaceutically acceptable carrier. The composition according to claim 1, characterized in that the monoamine oxidase A inhibitor contains at least one of the following: phenelzine; moclobemide; chlorgyline; pirlindol; isocarboxazid; tranylcypromide; iproniazid; caroxazone; befloxatone; brofaromine; cymoxatone; eprobemide; esupron; metroindole; or toloxatone. The composition according to claim 1, characterized in that the composition contains a lipid; and/or the composition contains a monoamine oxidase A inhibitor located within the nanoparticle. The composition of claim 1, wherein the monoamine oxidase A inhibitor is present in the composition in such amounts that the amount of monoamine oxidase A inhibitor available to tumor-associated macrophages in the individual to whom the composition is administered is sufficient to modulate the phenotype of the tumor-associated macrophage. The composition according to claim 4, characterized in that modulation of the phenotype of tumor-associated macrophages includes at least one of the following: reducing the levels of intracellular reactive oxygen species; increased tumor immunoreactivity. A method for modulating the phenotype of a tumor-associated macrophage, comprising introducing a monoamine oxidase A inhibitor into the environment in which the CD8 T cell is located; wherein the amount of monoamine oxidase A inhibitor introduced into the environment is selected to be sufficient to modulate the tumor-associated macrophage phenotype. The method according to claim 8, characterized in that the tumor-associated macrophage is located in the individual diagnosed with cancer. The method of claim 9, wherein the individual undergoes a therapeutic regimen comprising administration of a chemotherapeutic agent. The method according to claim 9, characterized in that the cancer is lymphoma or cancer of the skin, breast, ovary, prostate, colorectal cancer or lung cancer. The method according to claim 13, characterized in that the monoamine oxidase A inhibitor is located inside the nanoparticle; optionally a nanoparticle containing a lipid. The method according to claim 8 or claim 16, characterized in that the monoamine oxidase inhibitor A is in a composition containing a cross-linked multilayer liposome having an outer surface and an inner surface, wherein the inner surface forms the central cavity of the liposome, wherein the multilayer liposome includes at least the first a lipid bilayer and a second lipid bilayer, the first lipid bilayer being covalently linked to the second lipid bilayer; and a monoamine oxidase A inhibitor located within the liposome.

Briefly, the essence of the prior art is targeting tumor-associated macrophages (TAMs) as a promising strategy for altering the tumor microenvironment and improving cancer immunotherapy. Monoamine oxidase A (MAO-A) is an enzyme best known for its function in the brain; Small molecule MAO-A inhibitors are used clinically to treat neurological disorders. Induction of MAO-A in mouse and human TAMs has been previously observed. In this technical solution, it was determined that in mice with MAO-A deficiency there was a decrease in the anti-inflammatory functions of TAM, corresponding to enhanced antitumor immunity. This technical solution determined that MAO treatment induced TAM reprogramming and suppressed tumor cell growth in mouse and human tumor models. MAO-A has also been demonstrated to promote TAM polarization by upregulating oxidative stress. Together, these data identify MAO-A as a regulator of TAM polarization and potential applications for improving cancer immunotherapy.

The disadvantage of the known invention according to patent No. WO2022087424A1 is the safety problem during treatment with MAO inhibitors, namely the reaction of hypertensive crisis and serotonin syndrome. Hypertensive crises can occur as a result of pharmacodynamic drug interactions or with excessive dietary intake of tyramine. Serotonin syndrome occurs when drugs that increase serotonin availability are taken while inhibiting MAO activity [Bodkin J.A. et.al. (2019) Moving on With Monoamine Oxidase Inhibitors, Focus (Am Psychiatr Publ), 19(1), 50-52]. The claimed technical solution solves the problem by affecting mitochondrial metabolism, without side effects.

Thus, as of the date of submission of application materials, the applicant had not identified any information with clinical and preclinical studies on the use of pharmacological drugs that block the processes of oxidative phosphorylation in mitochondria for use in the treatment of cancer. Thus, the claimed technical solution is additional evidence of the effectiveness of pharmacological methods of influencing tumor macrophages by changing their mitochondrial functions and, as a result, is a promising direction for the introduction of effective drugs for the treatment of cancer.

The identified analogues coincide with the declared technical solution in certain respects, therefore the prototype has not been identified and the claims are drawn up without a restrictive part.

The technical result of the claimed technical solution isselection of suitable drugs and pharmacological compounds that provide the opportunity to shift mitochondrial metabolism from oxidative phosphorylation, characteristic of M2 type macrophages, to the glycolytic type, characteristic of M1 type macrophages. This approach, according to the applicant, is the most promising way to suppress the growth of tumor cells in patients with cancer.

The essence of the claimed technical solution is a method for selecting drugs for the pharmacological induction of mitochondrial dysfunction in macrophages for antitumor therapy , which consists in the fact that at the first stage, mouse and human tumor cells are cultured, for which mouse and human tumor cells are cultured in a DMEM nutrient medium containing 10% fetal cow serum, L-glutamine and a mixture of antibiotics penicillin-streptomycin, at a temperature of 37 ° C, in a humid atmosphere containing 5% CO ₂ ; at the second stage, the cultivation and differentiation of THP-1 cells is carried out, for which the human monocytic cell line THP1 as a model of human macrophages is cultivated in RPMI-160 medium containing 10% serum, antibiotics and 2 mM L-glutamine, while for the differentiation of myeloid cells - precursors to macrophages, add phorbol-12-myristate-13-acetate, PMA at a concentration of 2 ng/ml, incubate for 48 hours at a temperature of 37°C, in a humid atmosphere containing 5% CO ₂ ; then the cells are washed with Dulbecco's phosphate-buffered saline DPBS and RPMI-160 medium containing 10% serum, antibiotics and 2 mM L-glutamine is added and incubated at 37°C, in a humidified atmosphere containing 5% CO ₂ for two days; at the third stage, primary human macrophages are obtained from peripheral blood mononuclear cells of healthy donors, for which mononuclear cells obtained from human peripheral blood are washed with DPBS 2 times, then RPMI-160 nutrient medium with 5% human serum, 100 units/ml penicillin is added , 100 μg/ml streptomycin and 2 mM L-glutamine, for differentiation to macrophages, human MCSF is added at a concentration of 20 ng/ml and incubated for 9 days at a temperature of 37 ° C, in a humidified atmosphere containing 5% CO ₂ ; at the fourth stage, macrophages are obtained from myeloid progenitor cells of mouse bone marrow, for which monocytes obtained from mouse bone marrow are cultured in RPMI-160 medium with 10% fetal bovine serum FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at 37°C, in a humidified atmosphere containing 5% CO ₂ ; for differentiation of myeloid progenitor cells to macrophages, recombinant macrophage colony-stimulating factor M-CSF is added at a concentration of 20 ng/ml; at the fifth stage, mouse macrophages and tumor cells are co-cultivated in spheroids, for which, to prevent cell adhesion to plastic, the culture plastic is covered with a thin layer of 1% agarose, then the plate with 1% agarose is incubated at a temperature of 37°C for 20 minutes for complete polymerization of the matrix until it reaches a gel-like state; then a multicomponent cell co-culture is obtained by adding pre-prepared mouse lung cancer tumor cells and macrophages obtained from mouse bone marrow into the wells in a 1:3 ratio, located in a spheroid medium containing DMEM/F12, 0.4% bovine serum albumin BSA , L-glutamine, penicillin-streptomycin antibiotic mixture, 1 ml B-27™ supplement (50X), 20 ng/ml fibroblast growth factor FGF2 and 20 ng/ml epidermal growth factor EGF; Substances with test antitumor activity are added to the multicomponent cell co-culture at previously selected concentrations: etomoxir 0.5 µmol, cyclosporine A 0.5 µmol, dimethyl fumarate 1 µmol, stavudine 1.25 µmol, didanosine 2.5 µmol, rotenone 0.01 µmol, antimycin A 3 nanomol, oligomycin 0.1 µmol, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FCCP 0.15 µmol, BPTES 0.5 µmol; then the multicomponent cell co-culture is incubated for 5 days at a temperature of 37°C, in a saturated humid atmosphere containing 5% CO ₂ until the cells form sphere-like structures; the morphology of co-culture cells is analyzed using phase contrast microscopy; the cells are collected and centrifuged to pellet; the resulting sediment is resuspended and incubated for 3 minutes at a temperature of 37°C and the number of cells in the Goryaev chamber is counted, for the chamber the formula is used: N / 15 * 250 * 10^3, where N is the number of cells in fifteen squares of the chamber located diagonally / volume in ml; at the sixth stage, the growth of tumor cells is monitored in conditioned media from activated macrophages, for which, on the 5th day of differentiation, conditioned media from tumor cells of mouse lung cancer or pancreatic cancer in a ratio of 1:3 and drugs are added to macrophages obtained from mouse bone marrow at selected concentrations and incubated for 24 hours at 37°C, in a humid atmosphere containing 5% CO ₂ ; the next day, the cells are washed with DPBS buffer and DMEM medium containing 2% fetal blood serum, L-glutamine and a mixture of antibiotics is added and incubated for 72 hours; then the conditioned medium from activated macrophages is centrifuged at 300 g for 5 minutes and stored in a freezer at minus 20°C; mouse lung and pancreatic cancer tumor cells are cultured in DMEM with 10% serum and antibiotics; the next day, the cells are washed with DPBS buffer and conditioned medium from activated macrophages is added - 1 part, and DMEM medium containing 0.5% serum and antibiotics - 2 parts; after 9 days, tumor cells are counted; mouse lung and pancreatic cancer tumor cells are washed with DPBS buffer, 200 μl of 0.25% trypsin-EDTA is added, incubated for 3 minutes and the number of cells is counted, after which the growth or suppression of tumor cells is determined; at the seventh stage, polarization of primary macrophages obtained from peripheral blood is carried out, for which 20 ng/ml interleukin-4, IL-4 and a combination of 20 ng/ml lipopolysaccharide, LPS + 20 ng/ ml of interferon-gamma, IFNγ and drugs in selected concentrations; after 24 hours, 1 ml of Extra RNA solution is added to the cells for lysis and subsequent RNA isolation; at the eighth stage, polarization of mouse macrophages is carried out, for which 20 ng/ml interleukin-4, IL-4 and a combination of 20 ng/ml lipopolysaccharide, LPS + 20 ng/ml interferon are added to macrophages obtained from mouse bone marrow on the 5th day of differentiation - gamma, IFNγ and drugs in selected concentrations; the next day, 1 ml of Extra RNA solution is added to the cells for lysis and subsequent RNA isolation; at the ninth stage, polarization of THP-1 cells is carried out, for which the human monocytic cell line THP1 is differentiated according to the standard protocol, 20 ng/ml interleukin-4, IL-4 and a combination of 20 ng/ml lipopolysaccharide, LPS + 20 ng are added to the differentiated THP1 cells /ml interferon-gamma, IFNγ and drugs in selected concentrations; the next day, the cells are lysed in 1 ml of Extra RNA solution to isolate RNA; at the tenth stage, RNA is isolated, for which cell samples in Extra RNA solution are mixed for 1 minute and centrifuged at 12000 g for 5 minutes at room temperature, then stirred for 1 minute and centrifuged at 12000 g for 5 minutes at room temperature; the resulting supernatant is transferred to a new container and 200 μl of chloroform is added to it, mixed and incubated for 5 minutes at room temperature, shaking the sample periodically, and then centrifuged at 12000 g for 15 minutes at

room temperature, then select the upper phase and transfer it to a new container; add 500 μl of 100% isopropanol, mix and then incubate for 10 minutes at room temperature; then centrifuge at 12,000 g for 10 minutes at room temperature, then remove the supernatant and wash the precipitate in 1 ml of 75% EtOH; then centrifuge for 5 minutes at 12,000 g at room temperature, remove the supernatant and dry the sediment at room temperature; RNA is dissolved in 40 μl of water with sterile nuclease-free water and placed in a freezer at minus 80°C; at the eleventh stage, cDNA is obtained, for which the RNA concentration is first measured on Nano Drop at a concentration of ng/μl, the purity of the samples is assessed - the ratio of absorption peaks is 260 nm/280 nm - corresponding to impurities of proteins and amino acids; 260 nm/230 nm - impurities of salts and phenol; the required amount of RNA is adjusted to 15 μl with sterile nuclease-free water and 4 μl of 5x iScript Reaction Mix and 1 μl of iScript Reverse Transcriptase are added; the tubes are placed in a C1000 Touch cycler (Bio-Rad, USA), following the manufacturer’s instructions; at the twelfth stage, a real-time PCR reaction is carried out, for which a Master Mix is prepared: to 5 μl of SybrGreen, add 3.6 μl of nuclease-free water and 0.4 μl of a primer for the corresponding genes; the finished Master Mix is pipetted into a 96-well plate, 9 µl each; then add 1 µl of DNA samples, Standard and Negative Control in triplicate; the plate is covered with an optical transparent film and placed in a thermal cycler; PCR is carried out, data is obtained on the threshold values of PCR cycles, using which the level of expression of the genes of interest is calculated, while showing a shift in mitochondrial metabolism from oxidative phosphorylation, characteristic of M2 type macrophages, to the glycolytic type, characteristic of M1 type macrophages.

The claimed technical solution is illustrated in Figure 1 - Figure 14.

In FIG. Figure 1 presents an analysis of the polarization profile of wild-type and ^OPA1ΔM macrophages, where:

1A - qPCR analysis of gene expression that are markers of mouse M2 macrophages stimulated with IL-4 for 24 hours. Untreated M0 mouse macrophages are indicated as control (co).

1B - qPCR analysis of gene expression that are markers of M1 macrophages in mice stimulated with recombinant IFNγ and LPS for 24 hours.

1C - qPCR analysis of the expression of genes that are markers of M2 and M1 mouse macrophages stimulated for 24 hours with conditioned medium collected from tumor cells of the KPC line.

Values are means ± SD. n.s. = insignificant; *p<0.05; **p<0.01; ***p<0.001; n= 3.

Condition medium (CM) - conditional environment; relative expression (fold) - relative expression (sum); WT - macrophages obtained from Opa1flox/flox mice; KO - macrophages obtained from Opa1flox/flox Lyz2Cre/Cre mice; M1-like - M1 subtype of macrophages; M2-like - M2 subtype of macrophages; co - control; Arg1, Ym1, Fizz1 - primers for M2-subtype macrophages; TNF-α, iNOS, Acod1, CXCL9, ISG15, IRF1, GBP6, IL6; CXCL1 - primers for M1 - macrophage subtype; IL4 - macrophages polarized by IL-4; IFN - macrophages polarized by IFNγ and LPS; SM - conditioned environment.

In FIG. Figure 2 presents an analysis of the polarization profile of control and small interfering RNA-Opa1 (siRNA-Opa1) transfected human macrophages, where:

2A - OPA1 mRNA level in human macrophages after siRNA-OPA1 transfection.

2B - qPCR analysis of genes whose expression is a marker of human M2 macrophages stimulated with IL-4 for 24 hours.

2C - qPCR analysis of genes whose expression is a marker of M1 human macrophages stimulated with IFNγ and LPS for 24 hours. Untreated M0 mouse macrophages are indicated as control (co). Values are means ± SD. n.s. = insignificant; *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001 n = 3.

Relative expression (fold) - relative expression (sum), M2- markers - M2 markers, M1- markers - M1 markers, si-scr - small interfering RNA, si-OPA1 - small interfering RNA-Opa1; OPA1 - primers for the OPA1 protein; MRC1, CD163, CCL18 - primers for M2 subtype macrophages; TNF-α, CXCL10, IRF1, - primers for M1 - macrophage subtype; co - control; IL4 - macrophages polarized by IL-4; IFN - macrophages polarized by IFNγ and LPS.

In FIG. Figure 3 presents an analysis confirming the inability of ^OPA1ΔM macrophages to support the growth of tumor cells when co-cultured, where:

3A - Schematic of co-culture experiment.

3B - Microscopic evaluation of spheroids on day 5 after cell seeding.

3C - Number of tumor cells on the 5th day after seeding. The tumor cell culture was taken as a control; gray bars—tumor cells grown with wild-type macrophages (wtM); red bars are tumor cells grown with OPA1ΔM macrophages (koM). *p<0.05; **p<0.01; ***p<0.001; n = 3.

Tumor cells - tumor cells; mouse macrophages - mouse macrophages; Co-culture with tumor cells 1:3 (in spheroid medium) - co-culture with tumor cells 1:3 (in spheroid medium); 5 days - 5 days; spheroids - spheroids; count cells - counting cells; breast cancer E0771 - breast cancer, E0771; direct co-culture with macrophages - direct co-culture with macrophages; pancreatic cancer, cattle - pancreatic cancer; lung cancer, (KrasG12D/p53-/-) - lung cancer, (KrasG12D/p53-/-); macrophages - macrophages; control - control; + WT M0 - co-cultivation of tumor cells with wild-type macrophages; +KO M0 - co-culture of tumor cells with OPA1ΔM macrophages; cell number x10 ³ - number of cells x10 ³ ; wtM - tumor cells grown with wild-type macrophages; koM - tumor cells grown with OPA1ΔM macrophages.

In FIG. Figure 4 presents an analysis of macrophage phagocytosis using flow cytometry, where:

4A - Scheme depicting the analysis of the process of phagocytosis of cancer cells by macrophages using flow cytometry.

4B - Phagocytosis of tumor cell lines KPC and E0771 by wild type and OPA1ΔM macrophages.

4C - Analysis of phagocytosis by human macrophages. Macrophages were labeled with red dye; tumor cells - green dye. Phagocytic macrophages are double positive cells. Values are means ±SD. n.s. = insignificant; n = 3.

CellTraker™Green - green cell dye; CellTraker™Deep Red - red cell dye; tumor cells - tumor cells; mouse macrophages - mouse macrophages; 8 h - 8 hours; phagocytosis - phagocytosis; cancer cells - cancer cells; B525 FITC-A - fluorescent dye; R660 APC-A - fluorescent dye; phagocytic E0771 - phagocytosis E0771; phagocytic cattle - phagocytosis of cattle macrophages - macrophages; human macrophages - human macrophages; % of phagocytic macrophages - % of phagocytic macrophages; si-scr - small interfering RNA, si-OPA1 - small interfering RNA-Opa1; WT - macrophages obtained from Opa1flox/flox mice; KO - macrophages obtained from Opa1flox/flox Lyz2Cre/Cre mice; OPA-1 - OPA-1 protein.

- In Fig. Figure 5 shows the experimental scheme: culturing mouse tumor cells in a conditioned medium collected from activated macrophages (obtained from mouse bone marrow), pre-incubated with a conditioned medium from tumor cells, where:

5A is a diagram showing a co-culture experiment.

5B - Microscopic evaluation of day 5 spheroids cultured in conditioned medium from wild-type (WT) or OPA1ΔM macrophages (KO). The culture of tumor cells in a nutrient medium is labeled as control.

5C - Number of tumor cells on day 5 after seeding, cultured in conditioned media from macrophages. Gray bars are tumor cells grown with wild-type macrophages (wtM); red bars are tumor cells grown with OPA1ΔM macrophages (KO). *p<0.05; **p<0.01; n=3.

Tumor cells - tumor cells; CM from activated macrophages - conditioned environment from tumor cells; 3 days - 3 days; mouse macrophages - mouse macrophages; 24 hrs - 24 hours; CM from tumor cells - conditioned medium from tumor cells; growth for 5 day - growth within 5 days; spheroids 1:1 CM + media - spheroids 1:1 conditioned medium + medium; lung cancer KrasG12D;p53-/- - lung cancer KrasG12D;p53-/-; with CM from macrophages - with conditioned medium from macrophages; control - control; + WT M0 - conditioned medium from wild-type macrophages; + KO M0 - conditioned medium from OPA1ΔM macrophages; culture in conditioned medium from macrophages - culture in a conditioned medium from macrophages; pancreatic cancer, cattle - pancreatic cancer, cattle, CM from macrophages; cell number x10 ³ - number of cells x10 ³ ; wtM - tumor cells grown with wild-type macrophages; koM - tumor cells grown with OPA1ΔM macrophages.

In FIG. Table 6 provides a list of pharmaceutical compounds that affect mitochondrial function and optimized non-toxic concentrations for in vitro studies.

In FIG. Figure 7 presents optimization of concentrations of pharmaceutical compounds that cause mitochondrial dysfunction in macrophages and tumor cells .

Mouse macrophages - mouse macrophages; human macrophages - human macrophages; THP-1 - THP-1 cell line; A549 - human lung cancer tumor cells; KRS - tumor cells of the mouse pancreas; lung cancer - lung cancer; number of tumor cells - number of tumor cells; control - control; etomoxir - etomoxir; dimethyl fumarate - dimethyl fumarate; stavidine - stavudine; didanosine - didanosine; cyclosporine A (CyA) - cyclosporine A, rotenone - rotenone; antimycin A - antimycin A; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; DMSO - dimethyl sulfoxide; number of tumor cells, x10 - number of tumor cells.

In FIG. 8 shows an analysis of the activation of macrophages differentiated from mouse bone marrow and polarized into M2 macrophages with IL4 and M1 with IFNg+LPS in the presence of drugs that cause mitochondrial dysfunction, where:

8A - RT-qPCR analysis of mRNA expression for M2 marker genes in mouse macrophages differentiated with M-CSF for 5 days and treated with 20 ng/ml recombinant IL4 for 24 hours in the presence of DMSO (control) or selected drugs.

8B - RT-qPCR analysis of mRNA expression for M1 marker genes in mouse macrophages differentiated with M-CSF for 5 days and treated with 20 ng/ml IFNg and 100 ng/ml LPS for 24 hours in the presence of DMSO (control) or selected drugs. ns - not significant, *p<0.05; **p<0.01; ***p<0.001; n=3.

Relative expression (fold) - relative expression (sum), M2- genes - M2 genes, IL-4 - interleukin 4; M1- genes- M1- genes; IFNg+LPS - interferon gamma+ lipopolysaccharide; didanosine - didanosine; cyclosporine A (CyA) - cyclosporine A, rotenone - rotenone; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, DMSO - dimethyl sulfoxide; ng/ml - nanograms per milliliter; Arg1, Fizz1, Acod1, MRC1 - primers for M2 subtype macrophages; TNF-α, iNOS, ISG15 - primers for M1 - subtype of macrophages.

In FIG. 9 presents an analysis of the effect of drugs that cause mitochondrial dysfunction on the polarization of macrophages differentiated from human peripheral blood monocytes. The influence of drugs that cause mitochondrial dysfunction on the polarization of macrophages differentiated from human peripheral blood monocytes, where:

9A - RT-qPCR analysis of mRNA expression in donor monocytes differentiated for 8 days and treated with 20 ng/ml recombinant IL4 (M2 marker genes)

9B - or 20 ng/ml IFNg and 100 ng/ml LPS for 24 hours (M1 marker genes) in the presence of DMSO (control) or selected drugs. ns - not significant, *p<0.05; **p<0.01; ***p<0.001; n=3.

Relative expression (fold) - relative expression (sum); M2- genes - M2- genes; M1- genes- M1- genes; IL-4 - interleukin 4; M1- genes- M1- genes; didanosine - didanosine; cyclosporine A (CyA) - cyclosporine A; rotenone - rotenone; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; DMSO - dimethyl sulfoxide; MYLS22 - OPA-1 inhibitor; Alox15, CCL18, CD206, MMP12, CD163 - primers for M2 subtype macrophages; TNF-α, IRG1, IL12b, IL1b, GBP6 - primers for M1 - subtype of macrophages.

In FIG. 10 presents testing of pharmaceutical compounds that cause mitochondrial dysfunction in conditions of co-cultivation of lung cancer cells with macrophages. Primary mouse lung cancer cells were cultured in spheroids either alone (wt/ko only) or with macrophages (+macro) in the presence of DMSO (−) or inhibitors (+) at the indicated concentrations. The number of cancer cells on the 5th day after seeding is presented. ns - not significant; *p<0.05; **p<0.01; ***p<0.001; n=4.

10A - Testing of an expanded group of drugs.

10B - Normalized cell counts from three independent experiments are shown. ns- not significant, *p<0.05; **p<0.01; ***p<0.001; n=3.

Group - group; number of tumor cells - number of tumor cells; control - control; etomoxir - etomoxir; dimethyl fumarate - dimethyl fumarate; stavidine - stavudine; didanosine - didanosine; cyclosporine A (CyA) - cyclosporine A; rotenone - rotenone; antimycin A - antimycin A; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; DMSO - dimethyl sulfoxide; only wt/ko - only lung cancer cells; wt/ko + macro - lung cancer cells and macrophages; drug addition - adding a drug; μM - micromole; nM - nanomole; only tumor cells - only tumor cells; tumor cells + macro - tumor cells and macrophages; number of tumor cells; x50 - number of tumor cells; number of tumor cells (fold) - number of tumor cells (sum).

In FIG. 11 presents testing of pharmaceutical compounds that cause mitochondrial dysfunction in conditions of culturing mouse cancer cells in conditioned macrophage medium. Primary mouse lung (A) and pancreatic (B) cancer cells were cultured in conditioned medium collected from activated macrophages treated with mitochondrial drugs. The number of cancer cells on the 7th day after treatment with conditioned medium is presented. ns - not significant; *p<0.05; **p<0.01; ***p<0.001; n=3.

Cell number, x10 ⁴ - number of cells, x10 ⁴ ; control - control; didanosine - didanosine; rotenone - rotenone; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; lung cancer - lung cancer; KRS - tumor cells of mouse pancreatic cancer; CM from mouse macro - conditioned medium from macrophages.

In FIG. Figure 12 shows the effect of factors produced by THP1 macrophages on the growth of A549 lung cancer tumor cells. THP1 macrophages were differentiated with PMA at a concentration of 2 ng/ml and treated with conditioned medium from A549 lung cancer cells for 24 hours. After this, the medium was changed to 0.5% serum. Three days later, conditioned medium (CM) from THP1 macrophages was collected and added to A549 cells cultured in medium with 0.5% serum in medium (ratio 1 normal medium: 2 CM). The cells were cultured for another 8 days. The numbers of control untreated A549 cells (control) and cells grown in CM from THP1 were counted and plotted. ****p<0.0001;

Cell number, x10 ⁴ - number of cells; control - control; CM - conditioned environment; A549 - human lung cancer tumor cells.

In FIG. Figure 13 presents drugs that affect mitochondrial metabolism and the ability of THP1 macrophages to induce proliferation of cancer cells. Human lung cancer cells A549 (A) and NCI-H69 (B) were cultured under serum-starved conditions (0.5% serum) in either control medium (control) or conditioned medium from activated THP1 macrophages (CM from THP1), previously treated with these drugs. The number of cells grown over 8 days is presented in the graph. **p<0.01; ***p<0.001; n=2-4.

Number of tumor cells, x10 ³ - number of tumor cells, x10 ³ ; number of tumor cells, fold change - number of tumor cells, fold change; co - control; didanosine - didanosine; rotenone - rotenone; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; ; Conditioned medium - conditioned environment; A549 - human lung cancer tumor cells; NCI-H69 - human lung cancer tumor cells.

In FIG. 14 presents drugs that affect mitochondrial metabolism and the ability of primary macrophages to induce proliferation of cancer cells. Human lung cancer A549 (A) and pancreatic cancer Panc1 (B) cells were cultured under serum-starved conditions (0.5% serum) in either control medium or conditioned medium from activated primary macrophages differentiated from peripheral blood monocytes healthy donors (conditioned media from primary macrophages), pre-treated with the indicated drugs. The number of cells grown over 8 days is presented in the graph. *p<0.05; **p<0.01; ***p<0.001; n=3.

Number of cells, x10 ⁴ - number of cells, x10 ⁴ ; relative cell density (fold) - relative cell density (sum); control - control; didanosine - didanosine; cyclosporine A (CyA) - cyclosporine A, rotenone - rotenone; oligomycin - oligomycin; FCCP - carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; DMSO - dimethyl sulfoxide; MYLS22 - OPA-1 inhibitor; conditioned medium from prime macrophages - conditioned medium from activated macrophages; A549 - human lung cancer tumor cells; Panc1 - human pancreatic cancer tumor cells.

Next, the applicant provides a description of the claimed technical solution.

The claimed technical solution identifies pharmaceutical compounds that affect mitochondrial functions in macrophages. The applicant has carried out research on the selection of drugs and pharmacological compounds known from the available information on the date of submission of application materials and literature data, namely etomoxir, cyclosporine A, dimethyl fumarate, stavudine, didanosine, rotenone, antimycin A, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), BPTES, MYLS22, which have properties to change the mitochondrial functions of macrophages.

It should be emphasized that some of these drugs are already used to treat diseases such as:

- diabetes mellitus (ethomoxir),

- HIV (stavudine, didanosine),

- autoimmune diseases (cyclosporine A, dimethyl fumarate).

Thus, the applicant has conducted research on the use of known drugs and compounds for a new purpose, namely, for the treatment of cancer.

Medicines and pharmaceutical compounds presented on the basis of the claimed technical solution, after preclinical and clinical trials, can become promising drugs for the treatment of oncological diseases. It is possible to use these drugs and compounds in various combinations of treatment, including chemotherapy and/or immune therapy to enhance their therapeutic effect on the tumor. The applicant's studies tested two types of cancer - lung cancer and pancreatic cancer, which are the most aggressive types of cancer. However, there is a high probability that these drugs may also be effective in treating other types of cancer due to the fact that all types of cancer also depend on the effect of macrophages on tumor and healthy cells of the patient, for example, breast cancer or intestinal cancer and other types of cancer, with a pronounced contribution of macrophages.

Next, the applicant presents systematic data about the manufacturer and catalog numbers of the drugs used to conduct the experiments.

Russian name English Manufacturer Catalog no. Solvent Etomoxir Etomoxir sodium salt hydrate Sigma Aldrich E1905-5MG Dimethyl sulfoxide (DMSO) Cyclosporine A Cyclosporin A Sigma Aldrich C3662-5MG Dimethyl sulfoxide (DMSO) Dimethyl fumarate Dimethyl fumarate Sigma Aldrich 242926-25G Dimethyl sulfoxide (DMSO) Stavudin Stavudine Selleckchem S1398 Dimethyl sulfoxide (DMSO) Didanosine Didanosine Sigma Aldrich Y0000439 Dimethyl sulfoxide (DMSO) Rotenone Rotenone Sigma Aldrich R8875-1G Dimethyl sulfoxide (DMSO) Antimycin A Antimycin A Sigma Aldrich A8674-25MG Dimethyl sulfoxide (DMSO) Oligomycin Oligomycin Sigma Aldrich O4876-5MG Dimethyl sulfoxide (DMSO) Carbonyl cyanide-p-trifluoromethoxy-
phenylhydrazone FCCP Sigma Aldrich C2920-10MG Dimethyl sulfoxide (DMSO) BPTES
(no translation into Russian) BPTES Sigma Aldrich SML0601-5MG Dimethyl sulfoxide (DMSO) MYLS22
(no translation into Russian) MYLS22 GlpBio GC60260-10MG Dimethyl sulfoxide (DMSO)

Next, the applicant provides a detailed description of the claimed technical solution, which is carried out in twelve stages.

The first stage (cultivation of mouse and human tumor cells).

In the experiments, tumor cell cultures E0771, A549, NCI-H69, Panc1, Patu8988T were used, which were obtained from the American Type Culture Collection (ATCC). Lung cancer cell lines (Kras ^G12D and deleted p53) and pancreatic cancer (KPC line with Kras ^G12D and p53 ^R172H mutations) can be isolated from tumors of C57Black/6 mice.

Mouse and human tumor cells are cultured in DMEM nutrient medium (PanEco, Russia) containing 10% fetal cow blood serum (Biosera USA), L-glutamine (PanEco, Russia) and a mixture of penicillin-streptomycin antibiotics (PanEco, Russia) at a temperature of 37 °C, in a humid atmosphere containing 5% CO ₂ .

Second stage (cultivation and differentiation of THP-1 cells).

The study also uses the human monocytic cell line THP1 as a model of human macrophages (American Type Culture Collection (ATCC). The cells reproduce the main morphological, phenotypic and functional characteristics of monocytes, namely the ability for pro- and anti-inflammatory activation, production of cytokines, antigen- presenting activity Cells of the THP-1 line are a suspension culture that requires subculture once every 2-3 days.

The human monocytic cell line THP1 as a model of human macrophages is cultured in RPMI-160 medium (PanEco, Russia) containing 10% serum (Biosera USA), antibiotics and 2 mM L-glutamine (PanEco, Russia). In this case, to differentiate myeloid progenitor cells to macrophages, phorbol-12-myristate-13-acetate, PMA (Sigma, Germany) is added at a concentration of 2 ng/ml, incubated for 48 hours at a temperature of 37°C, in a humid atmosphere containing 5% _CO2 . Next, the cells are washed with Dulbecco's phosphate-buffered saline DPBS (PanEco, Russia) and RPMI-160 medium (PanEco, Russia) containing 10% serum, antibiotics and 2 mM L-glutamine (PanEco, Russia) is added and incubated at 37°C. in a humid atmosphere containing 5% CO ₂ for two days. After two days, the cells are ready for experiments.

The third stage (obtaining primary human macrophages from peripheral blood mononuclear cells of healthy donors).

Mononuclear cells from human blood are obtained by Ficoll density gradient sedimentation. To isolate mononuclear cells, peripheral blood from a healthy donor is used. All manipulations involving direct work with blood in order to level the risk of contamination are carried out under aseptic conditions in a laminar flow hood. Isolation of nucleated cells is carried out in 50 ml centrifuge tubes (PanEko, Russia). 25 ml of ficoll with a density of 1.077 g/ml (PanEco, Russia) is added to each tube and an equal volume of blood is carefully layered using an automatic dispenser. Next, the tube is centrifuged at 1080 g for 30 minutes, at room temperature (without braking). After centrifugation, the blood is clearly separated into 3 fractions: erythrocytes, leukocytes (mononuclear cells) and plasma. Mononuclear cells are collected into a separate 50 ml centrifuge tube (PanEco, Russia), 40 ml of DPBS buffer (PanEco, Russia) is added, the resulting mixture is resuspended by pipetting and centrifuged at 300 g for 5 minutes at room temperature. The supernatant is removed. The resulting cell sediment is resuspended in 40 ml of DPBS (PanEko, Russia) and centrifuged again at 10 g for 10 minutes at room temperature. The supernatant is removed, and the sediment is resuspended in 10 ml of DPBS (PanEko, Russia). Cells are counted in a Goryaev chamber and 2-3*10 ⁷ cells are transferred to a sterile 10 cm culture Petri dish and cultured for 30 minutes in an incubator at 37°C in a humid atmosphere with 5% CO ₂ content.

Mononuclear cells obtained from human peripheral blood are washed with DPBS (PanEco, Russia) 2 times. Next, add nutrient medium RPMI-160 (PanEco, Russia) with 5% human serum (SCI store, Russia), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (PanEco, Russia), for differentiation to macrophages are added with human MCSF at a concentration of 20 ng/ml (SCI store, Russia) and incubated for 9 days at a temperature of 37°C, in a humidified atmosphere containing 5% CO ₂ .

The fourth stage (obtaining macrophages from mouse bone marrow myeloid progenitor cells).

Monocytes are obtained from allogeneic material - mouse bone marrow. To collect bone marrow, 6-8 week old C57Black/6 mice (STESAR, Russia) undergo antiseptic treatment in the abdominal cavity and hind legs. Surgical manipulations on mice are carried out after their euthanasia with CO ₂ . Under aseptic conditions, the tibia and femur are removed, surrounding tissue is removed and collected in a sterile container with DPBS buffer (PanEco, Russia) and 100 units/ml penicillin, 100 μg/ml streptomycin (PanEco, Russia). Bone marrow was washed out from the ends of the bones using a 26 gauge needle and a 1 ml syringe filled with RPMI-1640 medium (PanEco, Russia) with antibiotics 100 units/ml penicillin, 100 μg/ml streptomycin (PanEco, Russia) and passed through 70- µm cell filter (SPL, South Korea). The cell suspension is centrifuged at 300 g for 5 min at room temperature to sediment the cells. The supernatant is poured out and the cell pellet is resuspended in 3 ml of erythrocyte lysis buffer (50 ml of distilled water, 0.449 g NH4Cl, 0.05 g KHCO3, 1.85 mg EDTA, pH = 7.3) and incubated for 3 min, and then centrifuged at 300 g for 5 min. at room temperature.

Monocytes obtained from mouse bone marrow are placed on a Petri dish, for example, a 10-centimeter, or similar, 6- or 12-well plate, calculating monocytes from one mouse per dish/plate and cultured in RPMI-medium. 160 (PanEco, Russia) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (PanEco, Russia) at a temperature of 37°C, in a humidified atmosphere containing 5 % CO ₂ . To differentiate myeloid progenitor cells into macrophages, recombinant macrophage colony-stimulating factor M-CSF is added at a concentration of 20 ng/ml (SCI store, Russia). The remaining cells are frozen in 90% fetal bovine serum (Biosera USA) and 10% DMSO (PanEco, Russia) at a rate of 2 ml per monocytes from one mouse (2 ampoules) and stored at minus 80°C overnight and then in liquid nitrogen storage.

Fifth stage (co-cultivation of mouse macrophages and tumor cells in spheroids).

To prevent cell adhesion to the plastic, the culture plastic is coated with a thin layer of 1% agarose (Carl Roth, Germany). To do this, 1% agarose is added to the wells of a 6-well plate in a volume of 1 ml/well and immediately removed to prevent premature polymerization. Next, the plate with 1% agarose is incubated at 37°C for 20 minutes to completely polymerize the matrix until a gel-like state is achieved. Next, a multicomponent cell co-culture is obtained by adding pre-prepared mouse lung cancer tumor cells and macrophages obtained from mouse bone marrow into the wells in a ratio of 1:3 (for a 6-well plate, 2 * 10 ⁴ tumor cells and 6 * 10 ⁴ macrophages) located in a spheroid medium containing DMEM/F12 (PanEco, Russia), 0.4% bovine serum albumin BSA (Diaem, Russia), L-glutamine (PanEco, Russia), a mixture of antibiotics penicillin-streptomycin (PanEco, Russia ), 1 ml B-27™ supplement (50X), 20 ng/ml fibroblast growth factor FGF2 (SCI store, Russia) and 20 ng/ml epidermal growth factor EGF (SCI store, Russia). Substances with test antitumor activity are added to the multicomponent cell co-culture at previously selected concentrations: etomoxir (0.5 µmol), cyclosporine A (0.5 µmol), dimethyl fumarate (1 µmol), stavudine (1.25 µmol), didanosine ( 2.5 µmol), rotenone (0.01 µmol), antimycin A (3 nmol), oligomycin (0.1 µmol), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (0.15 µmol), BPTES (0.5 µmol ). Next, the multicomponent cell co-culture is incubated for 5 days at a temperature of 37°C, in a saturated humid atmosphere containing 5% CO2 until the cells form sphere-like structures. The morphology of co-culture cells is analyzed using phase contrast microscopy. Cells are collected into a 2 ml microcentrifuge tube (Khimmed, Russia) and centrifuged at 300 g for 5 min at room temperature to sediment the cells. The resulting sediment is resuspended in 200 μl of 0.25% trypsin-EDTA (PanEco, Russia) and incubated for 3 minutes at 37°C and the number of cells is counted in a Goryaev chamber or a CytoSmart automatic cell counter (Corning, USA). For the chamber, use the formula: N / 15 * 250 * 10^3, where N is the number of cells in fifteen squares of the chamber, located diagonally / volume in ml.

At the same time, they show that classically activated macrophages are not able to promote the growth of tumor cells.

Sixth stage (monitoring the growth of tumor cells in conditioned media from activated macrophages).

On the 5th day of differentiation, conditioned medium from tumor cells of mouse lung cancer or pancreatic cancer is added to macrophages obtained from mouse bone marrow in a ratio of 1:3 and drugs in selected concentrations and incubated for 24 hours at a temperature of 37°C, in a humid atmosphere, containing 5% CO2. The next day, the cells are washed with DPBS buffer (PanEco, Russia) and DMEM medium (PanEco, Russia) containing 2% fetal blood serum of a cow (Biosera USA), L-glutamine (PanEco, Russia) and a mixture of antibiotics is added and incubated for 72 hours. Next, the conditioned medium from activated macrophages is collected into 1.5 ml microcentrifuge tubes (Khimmed, Russia), centrifuged at 300 g for 5 minutes and stored in a freezer at minus 20°C. Tumor cells of mouse lung and pancreatic cancer are planted in 24-well plates with a density of 500 cells per well in a medium cultured in DMEM medium (PanEko, Russia) with 10% serum and antibiotics. The next day, the cells are washed with DPBS buffer (PanEco, Russia) and conditioned medium from activated macrophages (1 part) and DMEM medium (PanEco, Russia) containing 0.5% serum and antibiotics (2 parts) are added. After 9 days, tumor cells are counted using standard/well-known methods of cell biology, for example, cell counting on a Goryaev chamber. Tumor cells of mouse lung and pancreatic cancer are washed with DPBS buffer, 200 μl of 0.25% trypsin-EDTA (PanEco, Russia) is added, incubated for 3 minutes and the number of cells is counted, after which the growth or suppression of tumor cells is determined.

The number of cells can be determined, for example, by the standard method using a Goryaev camera.).

In addition, the number of cells can be determined using an automatic cell counter CytoSmart (Corning, USA).

In addition, the number of cells can be determined using the gentian violet method. To do this, mouse lung cancer tumor cells and pancreatic cells are fixed with 350 μl of 10% formalin (Biovitrum, Russia) and incubated for 15 minutes at room temperature. Then the cells are washed with DPBS buffer and 250 μl of 0.1% gentian violet (Panreac, Spain) is added and incubated for 3 min at room temperature. Next, the cells are washed twice with MQ, selected dry, and the cell plates are dried for 30 min at room temperature. Then add 500 μl of acetic acid and mix on an orbital shaker (Biosan, Latvia) for 30 minutes until the color becomes uniform without areas of dense color at the bottom of the wells. Next, the absorbance of each well is measured at a wavelength of 570 nm using an Infinite M200 pro microplate reader (Tecan, Switzerland). The results are interpreted according to absorbance.

At the same time, they show that classically activated macrophages are not able to promote the growth of tumor cells.

Seventh stage (polarization of primary macrophages obtained from peripheral blood).

To differentiated macrophages obtained from the peripheral blood of donors, add 20 ng/ml interleukin-4, IL-4 (SCI store, Russia) and a combination of 20 ng/ml lipopolysaccharide, LPS (SCI store, Russia) + 20 ng/ml interferon- gamma, IFNγ (SCI store, Russia) and drugs in selected concentrations. After 24 hours, 1 ml of Extra RNA solution is added to the cells for lysis and subsequent RNA isolation. Samples are frozen at minus 80°C.

Eighth stage (polarization of mouse macrophages).

To macrophages obtained from mouse bone marrow, on the 5th day of differentiation, 20 ng/ml interleukin-4, IL-4 (SCI store, Russia) and a combination of 20 ng/ml lipopolysaccharide, LPS (SCI store, Russia) + 20 ng/ml are added ml of interferon-gamma, IFNγ (SCI store, Russia) and drugs in selected concentrations. The next day, 1 ml of Extra RNA solution is added to the cells for lysis and subsequent RNA isolation. Samples are frozen at minus 80°C.

Ninth stage (polarization of THP-1 cells).

The human monocytic cell line THP1 is plated on a 6-well plate at a concentration of 2 ppm per well and differentiated using the standard protocol described above. To differentiated THP1 cells, 20 ng/ml interleukin-4, IL-4 (SCI store, Russia) and a combination of 20 ng/ml lipopolysaccharide, LPS (SCI store, Russia) + 20 ng/ml interferon-gamma, IFNγ (SCI store) are added , Russia) and drugs in selected concentrations. The next day, the cells are lysed in 1 ml of Extra RNA solution (Evrogen, Russia) to isolate RNA and frozen at 80°C.

Tenth stage (RNA isolation).

Cell samples in Extra RNA solution are mixed by vortex for 1 minute and centrifuged at 12000 g, 5 minutes, at room temperature. Afterwards, the contents of the tubes are mixed by vortex for 1 minute and centrifuged at 12000 g, 5 minutes, at room temperature. The resulting supernatant is transferred to a new tube and 200 μl of chloroform is added to it, mixed and incubated for 5 minutes at room temperature, shaking the sample periodically. After this, centrifuge at 12000 g, 15 minutes, at room temperature, then carefully select the upper phase and transfer it to a new tube. Add 500 µl 100% isopropanol, vortex and then incubate for 10 minutes at room temperature. The tubes are centrifuged at 12,000 g for 10 minutes at room temperature, then the supernatant is carefully removed and the sediment is washed with 1 ml of 75% EtOH. Centrifuge again at 12,000 g for 5 minutes at room temperature, remove the supernatant and dry the sediment at room temperature. RNA is dissolved in 40 μl of water with sterile nuclease-free water (Evrogen, Russia) and placed in a freezer at minus 80°C.

Eleventh stage (obtaining cDNA).

To synthesize cDNA, the RNA concentration is first measured on a Nano Drop (Thermo scientific, USA) at a concentration of ng/µl, and the purity of the samples is also assessed - the ratio of absorption peaks is 260 nm/280 nm - corresponding to impurities of proteins and amino acids; 260 nm/230 nm - impurities of salts and phenol. The required amount of RNA is adjusted to 15 μl with sterile nuclease-free water (BIO-RAD, USA) and 4 μl of 5x iScript Reaction Mix (BIO-RAD, USA) and 1 μl of iScript Reverse Transcriptase (BIO-RAD, USA) are added. The tubes are placed in a C1000 Touch cycler (Bio-Rad, USA), following the manufacturer's instructions. After completion, the samples are left in the freezer at a temperature of minus 20°C.

Twelfth stage (real-time PCR reaction).

To carry out real-time PCR, prepare a Master Mix: to 5 μl of SybrGreen (Evrogen, Russia) add 3.6 μl of nuclease-free water (Evrogen, Russia) and 0.4 μl of primer (Evrogen, Russia) for the corresponding genes (table ). The finished Master Mix is dispensed into a 96-well plate, 9 µL each. Next, 1 μl of DNA samples, Standard and Negative Control samples were dispensed in triplicate. The plate is covered with an optical transparent film and placed in a thermal cycler.

PCR is carried out, for example, with the following parameters: 1) preliminary denaturation for 3 minutes at 95°C, 2) denaturation for 10 seconds at a temperature of 95°C, 3) annealing for 30 seconds, the temperature of which varies depending on the primer , 4) melting at a temperature of 72°C, lasting 30 seconds on a Real-Time CFX96 Touch thermal cycler (Bio-Rad, USA). This cycle is repeated 40 times, after which data on the threshold values of PCR cycles is obtained, using which the expression level of the genes of interest is calculated.

At the same time, they show a shift in mitochondrial metabolism from oxidative phosphorylation, characteristic of M2 type macrophages, to the glycolytic type, characteristic of M1 type macrophages.

Below, the applicant provides examples of the implementation of the claimed technical solution.

The applicant conducted the following experiments in the laboratory, which prove the effectiveness of the claimed method for the treatment of cancer in general:

Example 1. Mitochondrial dysfunction was analyzed in mouse macrophages and macrophages obtained from donor peripheral blood mononuclear cells caused by the absence of the Ora1 gene, and it was shown that the polarization of macrophages was impaired in the M2 subtype.

Example 2. An experiment was performed confirming this assumption, namely, the inability of M1 macrophages (OPA1ΔM) to stimulate the growth of tumor cells when macrophages are co-cultured with tumor cells.

Example 3. A search was made for the most effective pharmacological drugs that cause mitochondrial dysfunction.

Example 4. Studies have been carried out confirming the fact that the pharmaceutical drugs selected at the previous stage actually suppress the polarization of mouse macrophages and human macrophages, and as a consequence of this lead to changes in mitochondrial metabolism.

Example 5. Studies have been carried out confirming the fact that the pharmaceutical drugs selected by the applicant that change mitochondrial metabolism actually suppress the macrophage function of murine macrophages of the M2 subtype, which support the growth of tumor cells.

Example 6. Studies have been carried out confirming the fact that selected pharmaceutical agents affecting mitochondrial metabolism block the oncogenic properties of the macrophage-like human cell line THP1.

Example 7 Studies were performed to confirm that selected pharmaceutical agents that induce mitochondrial dysfunction in primary human macrophages reduce tumor cell proliferation.

Next, the applicant provides more detailed descriptions of the experiments confirming the sequence of experiments performed presented above.

Example 1. Performing an analysis of mitochondrial dysfunction in murine macrophages and macrophages derived from donor peripheral blood mononuclear cells caused by the absence of the Ora1 gene.

In order to test whether mitochondrial rearrangements can cause metabolic reprogramming in tumor-associated macrophages (TAMs), a fourth step is carried out - obtaining macrophages from mouse bone marrow myeloid progenitor cells according to the algorithm described above. The Applicant used a mouse model in which macrophages have mitochondrial dysfunction. To do this, the Optic Atrophy 1 (Opa1) gene was specifically inactivated in mice, and this deletion manifests itself only in macrophages. As a result of crossing, the applicant obtained mice carrying the Opa1 flox allele in which, due to the deletion of exons 2 and 3, aberrant transcription of exon1-exon4 occurs. The resulting Opa1flox/flox Lyz2Cre/Cre mice were designated Opa1ΔM (or Opa1-KO) mice and macrophages derived from these mice as OPA1ΔM macrophages, respectively. In all experiments performed, OPA1ΔM macrophages (referred to in the text as Opa1-KO) were compared with macrophages derived from Opa1flox/flox mice (hereafter referred to in the text as control or WT macrophages). The applicant suggested that macrophages deficient in OPA1 may exhibit mitochondrial aberrations that radically rearrange their metabolism, reproducing the metabolic programs of M1 macrophages, which, in turn, are able to effectively fight cancer cells.

Initially, in order to assess the functional state of polarization in ^OPA1ΔM macrophages, the applicant decided to evaluate the expression of known polarization markers. The expression of these markers often correlates with the functional ability of macrophages to polarize. To achieve this, the applicant completed the eighth stage - polarization of mouse marophages; tenth stage - RNA isolation; the eleventh stage - obtaining cDNA and the twelfth stage - carrying out a polymerase chain reaction in real time according to the algorithm described above.

The Applicant tested by qPCR the expression of M1 markers induced by IFNg and LPS and M2 markers induced by IL-4. We found that both M2 markers (Arg1, Ym1, Fizz1) and multiple M1 inflammatory markers were not induced in OPA1-deficient macrophages to the same extent as in wild-type cells (Figure 1A and Figure 1B). Interestingly, the absence of OPA1 in macrophages also prevented the expression of M2 and M1 subtype genes in response to conditioned media from tumor cells (Figure 1). Overall, these data demonstrated that mitochondrial dysfunction caused by deletion of the Opa1 gene in macrophages significantly affects the programming of macrophages into the protumor M2 subtype. It has previously been reported that M2 macrophage features correlate with worse prognosis in cancer patients. Therefore, significant suppression of M2 subtype features in tumor-associated macrophages may be an advantage for patients to mount an effective antitumor response.

As a result, the stated technical result was achieved - mitochondrial dysfunction caused by deletion of the Opa1 gene in macrophages significantly affects the programming of macrophages into the protumor M2 subtype.

Further, the applicant tested the role of Opa1 in human macrophages. For this purpose, the third stage was carried out - obtaining primary human macrophages and peripheral blood mononuclear cells from healthy donors. Peripheral blood mononuclear cells were plated on plastic and cultured for 9 days in 5% human serum. For knockdown, macrophages were transfected with siRNA targeting OPA1, which provided effective knockdown (Figure 2 A).

To analyze the polarization status of human macrophages, the seventh stage was performed. To achieve this, primary human macrophages were stimulated with IL-4 and IFN-γ and LPS. Next, the tenth stage was carried out - RNA isolation; the eleventh stage - obtaining cDNA and the twelfth stage - carrying out a polymerase chain reaction in real time according to the algorithm described above. Similar to murine macrophages upon activation by IL-4, M2 genes also showed reduced expression in human OPA1 knockout macrophages (Fig. 2B). However, M1 genes were in turn strongly upregulated by IFN-γ and LPS in OPA1-deficient macrophages ( Fig. 2C ). These data also support the finding that OPA1 is required for protumor activation of macrophages.

As a result, the stated technical result was achieved - a violation of the polarization of macrophages in the M2 subtype was shown.

Example 2. An experiment was performed confirming this assumption, namely the inability of M1 macrophages (OPA1ΔM) to stimulate the growth of tumor cells when macrophages are co-cultured with tumor cells .

The applicant further analyzed how macrophages with ^OPA1ΔM can influence the growth of tumor cells. To achieve this, the applicant carried out the first stage - culturing mouse tumor cells; the fourth stage is the production of macrophages from myeloid progenitor cells of mouse bone marrow and the fifth stage is the co-cultivation of mouse macrophages and tumor cells in spheroids according to the algorithm described above (Fig. 3A). Cultivation of tumor cells together with differentiated macrophages under spheroid growth conditions makes it possible to evaluate the effect of direct contact between tumor cells and macrophages and to analyze the influence of the macrophage secretome on tumor cell proliferation. Notably, under direct coculture conditions, ^OPA1ΔM macrophages were unable to stimulate proliferation of the three mouse tumor cell lines. This effect resulted in the formation of smaller spheroids (Figure 3B) and a significant reduction in cell number (Figure 3C). These data clearly confirm that ^OPA1ΔM macrophages are unable to promote tumor cell growth. This implies that OPA1 and normal mitochondrial function are critical for protumor reprogramming of macrophages. These results also correlate with qPCR data from activated macrophages. Therefore, drugs that cause mitochondrial dysfunction in macrophages may become promising candidates for anticancer therapy. The Applicant believes that it is likely that ^OPA1ΔM macrophages do not produce sufficient factors/metabolites that stimulate tumor cell proliferation or release certain cytotoxic molecules.

There are several possible reasons to explain why ^OPA1ΔM macrophages are less supportive of tumor cell growth: either their metabolic dysfunction results in altered production of secreted factors and metabolites required to stimulate tumor cell proliferation; or ^OPA1ΔM macrophages are more cytotoxic in killing tumor cells. The applicant first decided to analyze the phagocytic ability of wild-type and ^OPA1ΔM macrophages. To do this, tumor cells were labeled with a green dye and plated on top of macrophages labeled with a red dye. The percentage of double-positive cells representing macrophages with tumor cells was measured by flow cytometry 8 hours after plating (Fig. 4A). As a result, we found no significant difference between wild-type and OPA1-deficient macrophages in their ability to phagocytose tumor cells among all three cell lines tested (Figure 4B).

As a result, the stated technical result was achieved - OPA1 ^ΔM macrophages do not promote the growth of tumor cells.

The applicant further analyzed how macrophages obtained from the peripheral blood of healthy donors with OPA1 ^ΔM can influence the growth of human tumor cells. To achieve this, the applicant carried out the first stage - culturing human tumor cells; the third stage is the production of primary human macrophages from peripheral blood mononuclear cells of healthy donors and the fifth stage is the co-cultivation of macrophages and tumor cells in spheroids according to the algorithm described above. The applicant obtained similar results for human macrophages with siRNA knockout of OPA1 (Fig. 4C).

As a result, the stated technical result was achieved - OPA1 ^ΔM macrophages do not produce a sufficient amount of factors/metabolites that stimulate the proliferation of tumor cells.

To test whether mitochondrial dysfunction and OPA1 regulate the production of macrophage-secreted factors necessary for tumor growth, the applicant used the fourth step - obtaining macrophages from mouse bone marrow myeloid progenitor cells; the first stage is the cultivation of mouse tumor cells; fifth stage - co-cultivation of mouse macrophages and tumor cells in spheroids; the sixth stage is monitoring the growth of tumor cells in conditioned media from activated macrophages according to the algorithm described above. The applicant obtained murine macrophages and further treated them with conditioned medium collected from tumor cells. After this, the macrophages were washed and grown for three days in spheroid medium to enrich it with macrophage secretions. Tumor cells were then plated into either spheroid medium or macrophage-collected medium (Figure 5 A). After 5 days, the applicant took photographs of the spheroids and counted the cells of the spheroids. Notably, we found that conditioned media from macrophages significantly stimulated tumor cell proliferation, but factors produced by activated ^OPA1ΔM macrophages were insufficient to support tumor cell growth (Figure 5B and Figure 5C).

As a result, the stated technical result was achieved - mitochondrial metabolism regulates the production of secreted factors by macrophages necessary for tumor growth.

Example 3. A search was made for the most effective pharmacological drugs that cause mitochondrial dysfunction.

Further, the applicant has compiled a list of pharmaceuticals (Fig. 6) that could affect mitochondrial function in macrophages with a physiological effect similar to knockout of the Opa1 gene. The applicant selected several pharmacological compounds based on our previous observations and literature search: etomoxir, cyclosporine A, dimethyl fumarate, stavudine, didanosine, rotenone, antimycin A, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, BPTES and the new Ora1 inhibitor MYLS22. The applicant completed the first stage - culturing mouse and human tumor cells; the second stage is the cultivation and differentiation of THP-1 cells; the third stage is the production of primary human macrophages from peripheral blood mononuclear cells of healthy donors and the fourth stage is the production of macrophages from mouse bone marrow myeloid progenitor cells. The Applicant has tested these compounds on murine macrophages, macrophages differentiated from the peripheral blood of healthy donors, the marophagal cell line THP1 and several murine cancer cells (primary Kras ^G12D /p53-/- lung cancer, pancreatic cancer KPC - Kras ^G12D and mutant p53 ^R172H , and breast cancer cell line E0771; human tumor cell lines (A549, PANC1, NCI-H69).

As a result, the stated technical result was achieved - a concentration of the drug was identified that is not toxic to cells (Fig. 7).

Example 4. Studies have been carried out confirming the fact that the pharmaceutical drugs selected at the previous stage actually suppress the polarization of mouse macrophages and human macrophages, and as a consequence of this lead to changes in mitochondrial metabolism.

To explain exactly how drugs targeting mitochondrial metabolism affect the functions of TAMs, the applicant performed the third stage - obtaining primary macrophages; the fourth stage is the production of macrophages from mouse bone marrow myeloid progenitor cells; their effect on the polarization of macrophages obtained from mouse bone marrow and human macrophages differentiated from peripheral blood monocytes was tested; seventh stage - polarization of primary macrophages obtained from peripheral blood; eighth stage - polarization of mouse macrophages; tenth stage - RNA isolation; eleventh stage - obtaining cDNA; the twelfth stage is a real-time PCR reaction according to the algorithm described above. Treatment with the cytokine IL4 was used to polarize macrophages into the M2 subtype (pro-tumorigenic macrophages), and a combination of interferon gamma and lipopolysaccharides (IFNg-γ and LPS) was used to polarize into the antitumor M1 subtype. Thus, the applicant showed that all selected drugs that cause mitochondrial dysfunction prevented the activation of several M2-like markers in both mouse macrophages (Fig. 8A) and human macrophages (Fig. 9A). Importantly, the expression of M1 polarization markers in mouse and human macrophages was either increased ( TNFa, IL12, GBP6 ) or unchanged ( Isg15, IRF1 ) in response to pharmacological compounds that induce mitochondrial dysfunction (Figures 8B and 9B). However, some differences between the drugs were noted. However, oligomycin, rotenone and didanosine appear to be the most promising drugs in inhibiting the functions of tumor-associated macrophages. Our data indicate the importance of mitochondrial functions for the activation of pro-tumorigenic TAMs and provide evidence for the antitumor effects of selected mitochondrial drugs. Thus, oligomycin, rotenone, didanosine, FCCP and the new synthetic Opa1 inhibitor MYLS22 inhibit the pro-oncogenic M2 polarization of TAMs and do not affect the antitumor activation of M1 macrophages, and some substances even enhance the expression of the inflammatory genes TNFa and iNOS2, which are involved in tumor suppression mechanisms.

As a result, the stated technical result was achieved - the selected drugs that cause mitochondrial dysfunction prevented the activation of several M2-like markers in both mouse and human macrophages.

Example 5. Studies have been carried out confirming the fact that the pharmaceutical drugs selected by the applicant that change mitochondrial metabolism actually suppress the macrophage function of murine macrophages of the M2 subtype, which support the growth of tumor cells .

Further, the applicant tested the drugs to block the function of macrophages and stimulate the proliferation of tumor cells in vitro . The applicant used the first stage - a method of culturing mouse lung cancer cells with mouse macrophages differentiated from bone marrow cells; the fourth stage is obtaining macrophages from mouse bone marrow myeloid progenitor cells; the fifth stage is the co-cultivation of mouse macrophages and tumor cells in spheroids according to the algorithm described above. The results showed that cyclosporine A, rotenone and oligomycin, didanosine, antimycin A and FCCP were quite effective in inhibiting the growth of tumor cells when cultured with macrophages (Fig. 10A), but did not particularly affect the growth of tumor cells cultured without macrophages. Four drugs (etomoxir, dimethyl fumarate, stavudine and BPTES) did not demonstrate such an effect and were excluded from further studies. The drug concentrations used in the co-culture experiments were not toxic to mouse bone marrow-derived macrophages, with the exception of cyclosporine A. Therefore, the applicant continued to test the following pharmaceutical compounds: rotenone, oligomycin; didanosine, antimycin A and FCCP and repeated the experiment of co-culturing tumor cells with macrophages in the presence of selected drugs at least three times (Fig. 10B).

As a result, the stated technical result was achieved - the selected drugs that cause mitochondrial dysfunction inhibited the growth of tumor cells under culture conditions with macrophages.

In addition to direct cultivation of macrophages with tumor cells, the applicant tested the growth of tumor cells in the presence of conditioned medium collected from activated mouse macrophages pre-treated with drugs that cause mitochondrial dysfunction. To achieve this, the applicant performed the first stage - culturing mouse tumor cells; the fourth stage is the production of macrophages from mouse bone marrow myeloid progenitor cells; the sixth stage is monitoring the growth of tumor cells in conditioned media from activated macrophages according to the algorithm described above. The Applicant included two mouse cell lines in the analysis: lung cancer and pancreatic cancer. The results were similar to experiments with direct cultivation of macrophages and cancer cells, with the exception of the substance FCCP, which did not show inhibition of tumor cell growth (Fig. 11). Thus, the drugs didanosine, rotenone and oligomycin are the most promising potential antitumor drugs that affect the functions of macrophages.

As a result, the stated technical result was achieved - selected drugs that cause mitochondrial dysfunction inhibited the growth of tumor cells in the presence of a conditioned medium collected from activated mouse macrophages pre-treated with the drugs.

Example 6. Studies have been carried out confirming the fact that selected pharmaceutical agents affecting mitochondrial metabolism block the oncogenic properties of the macrophage-like human cell line THP1.

To translate our results to human cells, the applicant used the monocytic cell line THP1 as a model of human macrophages. The applicant completed the first stage - culturing human tumor cells; the second stage is the cultivation and differentiation of THP-1 cells; the sixth stage is monitoring the growth of tumor cells in conditioned media from activated macrophages according to the algorithm described above. The sixth stage - co-cultivation of macrophages and tumor cells in spheroids is not suitable for our purposes due to the high percentage of phagocytosis in THP1, which will interfere with accurate interpretation of the data.

As a result, the stated technical result was achieved - factors produced by THP1 macrophages differentiated after PMA treatment increased the proliferation of A549 cells in a low serum medium (Fig. 12).

The applicant used the first stage - culturing human tumor cells; the second stage is the cultivation and differentiation of THP-1 cells; sixth stage - monitoring the growth of tumor cells in conditioned media from activated macrophages; the ninth step is the polarization of THP-1 cells according to the algorithm described above to monitor how factors secreted by macrophages stimulate the proliferation of human cancer cells. The applicant has tested a set of mitochondrial drugs that the applicant has already tested in a mouse system. It is important to note that all four drugs - rotenone, didanosine, oligomycin and FCCP, which inhibited the proliferation of mouse cancer cells, were also effective in blocking the pro-tumorigenic function of human THP1 macrophages (Fig. 13) in relation to the growth of human lung cancer cell line A549 (Fig. 13A) and NCI-H69 line (Fig. 13B).

As a result, the stated technical result was achieved - drugs that cause mitochondrial dysfunction in macrophages inhibited the proliferation of cancer cells.

Example 7. Studies have been carried out confirming the fact that selected pharmaceutical agents that induce mitochondrial dysfunction in primary human macrophages reduce the proliferation of tumor cells.

Having received convincing data on the THP1 cell line, the applicant switched to primary human macrophages differentiated from peripheral blood monocytes of healthy donors. The applicant completed the first stage - cultured human tumor cells; the third stage - obtaining primary macrophages from peripheral blood mononuclear cells of healthy donors; the sixth stage is monitoring the growth of tumor cells in conditioned media from activated macrophages according to the algorithm described above. Macrophages differentiated for nine days, after which they were activated with conditioned medium from the corresponding cancer cells (A549 lung cancer and Panc1 pancreatic cancer) in the presence of a DMSO solvent as a control, or drugs that cause mitochondrial dysfunction. As in experiments with mouse macrophages and the THP1 line, the drugs didanosine, oligomycin, rotenone, FCCP, cyclosporine A, as well as the Ora1 inhibitor MYLS22 significantly suppressed the function of primary human macrophages in maintaining the growth of tumor cells, both in A549 and Panc1 cells (Fig. . 14).

As a result, the stated technical result was achieved - drugs that cause mitochondrial dysfunction in macrophages inhibited the proliferation of cancer cells.

As of the filing date of this application, the applicant has not identified sources that would demonstrate similar effects of the above drugs on the functions of tumor-associated macrophages. The role of Ora1 in tumor-associated macrophages has also not been identified. At the same time, it was revealed in vitro that drugs that cause mitochondrial dysfunction directly in tumor cells, when used in high doses, prevent their growth [Zamberlan M. et. al. (2022) Inhibition of the mitochondrial protein Opa1 curtails breast cancer growth, Exp Clin Cancer Res., 41(1), 95], [Herkenne S. et. al. (2020) Developmental and Tumor Angiogenesis Requires the Mitochondria-Shaping Protein Opa1, Cell Metab., 31(5), 987-1003]. These data suggest that when combining the action of drugs in vivo, an increase in their effect on macrophages (studies by the applicant) and on tumor cells (publications) can be observed.

Thus, based on the given experimental data, it is possible to draw conclusions that the applicant has achieved the stated technical result, namely: the selection of suitable drugs and pharmacological compounds has been carried out, which provide the opportunity to shift mitochondrial metabolism from oxidative phosphorylation, characteristic of M2 type macrophages, to the glycolytic type, characteristic of M1 type macrophages. This approach, according to the applicant, is the most promising way to suppress the growth of tumor cells in patients with cancer.

It has been shown that the metabolism of mitochondria in macrophages regulates their tumor properties associated with changes in extracellular receptors (contacts) and the production of secreted factors necessary for enhanced growth of tumor cells. Pharmaceutical compounds that cause mitochondrial dysfunction of TAMs can be used as potential anticancer drugs. The applicant has identified and tested drugs and pharmacological substances didanosine, oligomycin, rotenone, FCCP, cyclosporine A, as well as the Ora1 inhibitor MYLS22, which block the electron transport chain in macrophages and significantly reduce their ability to induce tumor cell proliferation. The applicant believes that these drugs can be used in preclinical trials as anticancer agents and substances affecting tumor-associated macrophages. It is possible that these drugs and substances could be used in combination with chemotherapy or immune therapy to enhance their therapeutic effect on the tumor. The claimed technical solution tested two types of cancer - lung cancer and pancreatic cancer. However, according to the applicant, these drugs and substances may also be effective for other types of cancer that also depend on macrophage function, such as breast cancer or colorectal cancer.

The claimed technical solution complies with the “novelty” patentability condition imposed on inventions, since as of the date of submission of the application materials by the applicant, no sources were identified from the researched level of technology that have a set of features identical to the set of features of the claimed technical solution.

The claimed technical solution meets the “inventive step” patentability requirement for inventions, since it is not obvious to a specialist in the field of technology being analyzed.

The claimed technical solution meets the patentability condition of “industrial applicability” applied to inventions, because can be carried out using standard equipment and known techniques.

Claims (13)

A method for selecting drugs for the implementation of pharmacological induction of mitochondrial dysfunction in macrophages for antitumor therapy, which consists in the fact that at the first stage, the cultivation of mouse and human tumor cells is carried out, for which mouse and human tumor cells are cultured in the DMEM nutrient medium containing 10% fetal blood serum of a cow, L-glutamine and a mixture of penicillin-streptomycin antibiotics, at a temperature of 37 ° C, in a humid atmosphere containing 5% CO ₂ ; at the second stage, the cultivation and differentiation of THP-1 cells is carried out, for which the human monocytic cell line THP1 as a model of human macrophages is cultivated in RPMI-160 medium containing 10% serum, antibiotics and 2 mM L-glutamine, while for the differentiation of myeloid cells - precursors to macrophages, add phorbol-12-myristate-13-acetate, PMA at a concentration of 2 ng/ml, incubate for 48 hours at a temperature of 37 °C, in a humid atmosphere containing 5% CO ₂ ; then the cells are washed with Dulbecco's phosphate-buffered saline DPBS and RPMI-160 medium containing 10% serum, antibiotics and 2 mM L-glutamine is added and incubated at 37 °C, in a humidified atmosphere containing 5% CO ₂ for two days; at the third stage, primary human macrophages are obtained from peripheral blood mononuclear cells of healthy donors, for which mononuclear cells obtained from human peripheral blood are washed with DPBS 2 times, then RPMI-160 nutrient medium with 5% human serum, 100 units/ml is added penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine, for differentiation to macrophages, human MCSF is added at a concentration of 20 ng/ml and incubated for 9 days at 37 °C, in a humidified atmosphere containing 5% CO ₂ ; at the fourth stage, macrophages are obtained from myeloid progenitor cells of mouse bone marrow, for which monocytes obtained from mouse bone marrow are cultured in RPMI-160 medium with 10% fetal bovine serum FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at a temperature of 37 ° C, in a humid atmosphere containing 5% CO ₂ ; to differentiate myeloid progenitor cells to macrophages, recombinant macrophage colony-stimulating factor M-CSF is added at a concentration of 20 ng/ml; at the fifth stage, mouse macrophages and tumor cells are co-cultivated in spheroids, for which, to prevent cell adhesion to plastic, the culture plastic is coated with a thin layer of 1% agarose, then the plate with 1% agarose is incubated at a temperature of 37 °C for 20 minutes for complete polymerization matrix until it reaches a gel-like state; then a multicomponent cell co-culture is obtained by adding pre-prepared mouse lung cancer tumor cells and macrophages obtained from mouse bone marrow in a 1:3 ratio, located in a spheroid medium containing DMEM/F12, 0.4% bovine serum albumin BSA, into the wells , L-glutamine, penicillin-streptomycin antibiotic mixture, 1 ml B-27™ supplement (50X), 20 ng/ml fibroblast growth factor FGF2 and 20 ng/ml epidermal growth factor EGF; Substances with test antitumor activity are added to the multicomponent cell co-culture at previously selected concentrations: etomoxir 0.5 µmol, cyclosporine A 0.5 µmol, dimethyl fumarate 1 µmol, stavudine 1.25 µmol, didanosine 2.5 µmol, rotenone 0.01 µmol, antimycin A 3 nanomol, oligomycin 0.1 µmol, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FCCP 0.15 µmol, BPTES 0.5 µmol; then the multicomponent cell co-culture is incubated for 5 days at a temperature of 37 °C, in a saturated humid atmosphere containing 5% CO ₂ , until the cells form sphere-like structures; the morphology of co-culture cells is analyzed using phase contrast microscopy; the cells are collected and centrifuged to pellet; the resulting sediment is resuspended and incubated for 3 minutes at a temperature of 37 °C and the number of cells in the Goryaev chamber is counted, for the chamber the formula is used: N / 15 ⋅ 250 ⋅ 10^3, where N is the number of cells in fifteen squares of the chamber located diagonally / volume , ml; at the sixth stage, the growth of tumor cells is monitored in conditioned media from activated macrophages, for which, on the 5th day of differentiation, conditioned media from tumor cells of mouse lung cancer or pancreatic cancer in a ratio of 1:3 and drugs are added to macrophages obtained from mouse bone marrow at selected concentrations and incubated for 24 hours at 37 °C, in a humid atmosphere containing 5% CO ₂ ; the next day, the cells are washed with DPBS buffer and DMEM medium containing 2% fetal blood serum, L-glutamine and a mixture of antibiotics is added and incubated for 72 hours; then the conditioned medium from activated macrophages is centrifuged at 300 g for 5 minutes and stored in a freezer at minus 20 ° C; mouse lung and pancreatic cancer tumor cells are cultured in DMEM with 10% serum and antibiotics; the next day, the cells are washed with DPBS buffer and conditioned medium from activated macrophages is added - 1 part, and DMEM medium containing 0.5% serum and antibiotics - 2 parts; after 9 days, tumor cells are counted; mouse lung and pancreatic cancer tumor cells are washed with DPBS buffer, 200 μl of 0.25% trypsin-EDTA is added, incubated for 3 minutes and the number of cells is counted, after which the growth or suppression of tumor cells is determined; at the seventh stage, polarization of primary macrophages obtained from peripheral blood is carried out, for which 20 ng/ml interleukin-4, IL-4 and a combination of 20 ng/ml lipopolysaccharide, LPS + 20 ng/ ml of interferon-gamma, IFNγ and drugs in selected concentrations; after 24 hours, 1 ml of Extra RNA solution is added to the cells for lysis and subsequent RNA isolation; at the eighth stage, polarization of mouse macrophages is carried out, for which 20 ng/ml interleukin-4, IL-4 and a combination of 20 ng/ml lipopolysaccharide, LPS + 20 ng/ml interferon are added to macrophages obtained from mouse bone marrow on the 5th day of differentiation -gamma, IFNγ and drugs in selected concentrations; the next day, 1 ml of Extra RNA solution is added to the cells for lysis and subsequent RNA isolation; at the ninth stage, polarization of THP-1 cells is carried out, for which the human monocytic cell line THP1 is differentiated according to the standard protocol, 20 ng/ml interleukin-4, IL-4 and a combination of 20 ng/ml lipopolysaccharide, LPS + 20 ng are added to the differentiated THP1 cells /ml interferon-gamma, IFNγ and drugs in selected concentrations; the next day, the cells are lysed in 1 ml of Extra RNA solution to isolate RNA; at the tenth stage, RNA is isolated, for which cell samples in Extra RNA solution are stirred for 1 min and centrifuged at 12,000 g for 5 min at room temperature, then stirred for 1 min and centrifuged at 12,000 g for 5 min at room temperature; the resulting supernatant is transferred to a new container and 200 μl of chloroform is added to it, mixed and incubated for 5 minutes at room temperature, shaking the sample periodically, then centrifuged at 12,000 g for 15 minutes at room temperature, then the upper phase is selected and transferred to a new one capacity; add 500 μl of 100% isopropanol, mix and then incubate for 10 minutes at room temperature; then centrifuge at 12000 g for 10 min at room temperature, then remove the supernatant and wash the precipitate in 1 ml of 75% EtOH; then centrifuge for 5 minutes at 12,000 g at room temperature, remove the supernatant and dry the sediment at room temperature; RNA is dissolved in 40 μl of water with sterile nuclease-free water and placed in a freezer at minus 80 °C; at the eleventh stage, cDNA is obtained, for which the RNA concentration is first measured on Nano Drop at a concentration of ng/μl, the purity of the samples is assessed - the ratio of absorption peaks is 260 nm/280 nm - corresponding to impurities of proteins and amino acids; 260 nm/230 nm – impurities of salts and phenol; the required amount of RNA is adjusted to 15 μl with sterile nuclease-free water and 4 μl of 5x iScript Reaction Mix and 1 μl of iScript Reverse Transcriptase are added; the tubes are placed in a C1000 Touch cycler (Bio-Rad, USA), following the manufacturer’s instructions; at the twelfth stage, a real-time PCR reaction is carried out, for which a Master Mix is prepared: 3.6 μl of nuclease-free water and 0.4 μl of a primer for the corresponding genes are added to 5 μl of SybrGreen; the finished Master Mix is pipetted into a 96-well plate, 9 µl each; then add 1 µl of DNA samples, Standard and Negative Control in triplicate; the plate is covered with an optical transparent film and placed in a thermal cycler; carry out PCR, obtain data on the threshold values of PCR cycles, using which the level of expression of the genes of interest is calculated: markers of mouse macrophages M1 type TNFa, iNOS, Acod1, CXCL9, ISG15, IRF1, GBP6, IL6, CXCL1 and mouse macrophages M2 type Arg1, Fizz1, MRC1, Ym1, Acod1; markers of human macrophages M1 type IRF1, GBP6, TNFa, IL12, IL1b and M2 type ALOX15, CCL18, CD206, MMP12, CD169; The data obtained show suppression of the polarization of macrophages in the M2 type and increased polarization in the M1 type, which confirms a shift in mitochondrial metabolism from the oxidative phosphorylation characteristic of M2 type macrophages to the glycolytic type characteristic of M1 type macrophages, and differences between the expression of these genes when adding drugs allow in addition to data on the proliferation of tumor cells with a conditioned environment from macrophages, determine which drug is suitable for realizing the pharmacological induction of mitochondrial dysfunction in macrophages for antitumor therapy.