The tumor microenvironment (TME) plays an essential role in tumor progression and in modulating tumor response to anticancer therapy. Cellular senescence leads to a switch in the cell secretome, characterized by the senescence-associated secretory phenotype (SASP), which may regulate tumorigenesis. Senolytic therapy is considered a novel anticancer strategy that eliminates the deleterious effects of senescent cells in the TME. Here, we show that two different types of senolytic drugs, despite efficiently depleting senescent cells, have opposite effects on cancer-associated fibroblasts (CAFs) and their ability to regulate epithelial–mesenchymal transition (EMT). We found that senolytic drugs, navitoclax and the combination of dasatinib/quercetin, reduced the number of spontaneously senescent and TNF-induced senescent CAFs. Despite the depletion of senescent cells, the combination of dasatinib/quercetin versus navitoclax increased the secretion of the SASP pro-inflammatory cytokine IL-6. This differential effect correlated with the promotion of enhanced migration and EMT in MC38 colorectal cancer cells. Our results demonstrate that some senolytics may have side effects unrelated to their senolytic activity and may promote tumorigenesis. We argue for more careful and extensive studies of the effects of senolytics on various aspects of tumor progression and tumor resistance to therapy before the senolytic strategy is implemented in the clinic.

Introduction The store-operated calcium entry (SOCE) is a key mechanism for maintaining STIM-ORAI signaling and Ca2+ balance, which is necessary for cell proliferation, including tumor stem cells. The role of SOCE in myelofibrosis (MF) pathogenesis and response to treatment is not well understood. Material and Methods Whole blood samples were obtained from healthy donors (HD; n=19) and patients with MF associated with JAK2v617f or CALRI (n=15) after local ethical approvement and subjects’ informed consent. Monocytes were isolated from peripheral blood mononuclear cells by 24h-plastic adherence. Fluorescent measurements of calcium were performed on Zeiss Observer.Z1 using 2 μM Fluo-4AM and were normalized to basal intensity. For endoplasmic reticulum (ER) depletion, 1 μM thapsigargin was used. The amplitudes of ER depletion and SOCE were analyzed in MF- compared to HDmonocytes and according to treatment: hydrea (HU) or ruxolitinib (RUX). Expression profile of SOCE components was analyzed by flow cytometry with anti-TRPC1-AF594, anti-Orai1-AF647 and anti-STIM1-AF680 antibodies. Statistical analysis was performed using GraphPad Prism 10 software. Results The SOCE was 1.7- to 2.2-fold decreased in both treated JAK2- and CALR-MF (p<0.001) and, conversely, was 1.7-fold increased in untreated CALR-MF monocytes (p=0.04), respectively. Simultaneously, ER depletion amplitude had a 2.5- to 3.1-fold increase in HU-treated JAK2-MF and both RUX-treated and untreated CALR-MF monocytes (p<0.0001). The overexpression of Orai1 and STIM1 was observed on both treated JAK2- and CALR-MF monocytes (p<0.05). The kinetics of SOCE activity was tracked in CALR-MF monocytes before and during 16 mo of RUX treatment. The improvement of both SOCE and ER-depletion amplitude was observed at 4 mo of RUX, reaching HD values and corresponded to clinical improvement of MF. The ER depletion amplitude had a 2.5- to 3.1-fold increase from 4 to 10 mo of RUX (p<0.0001), whereas SOCE amplitude tended to fall (1.4- to 0.4-fold decrease; p<0.05). At the last time point, both SOCE and ER depletion amplitude returned to the untreated CALR-MF levels, consistent with disease progression. Conclusion We demonstrated significant dysregulation of SOCE in myelofibrosis. The SOCE was inhibited during RUX treatment and changed drastically at the end time point. The overexpression of Orai1 and STIM1 may be compensatory to SOCE inhibition by treatment.

Background. Research in oncology assumes establishment and usage of xenograft animal models, meeting the requirements of humanity and rationality. Although praised as promising field of research, preclinical studies of low-molecular-weight inhibitors, antitumor monoclonal antibodies, vaccines, cellular therapy products, CAR-T cells entail continuous control of tumor growth dynamics. Luciferase detection of bioluminescence requires injection of a reporter substrate. However, injections not only are laborious, time-consuming and expensive, but are also stressful for animals. Thus, a vast variety of new visualization methods is employed, including proteins of the far-red spectrum. Objective. The study aimed to compare detection efficiency of tumor growth dynamics in mice models of cervical cancer, applying a commercially available line of fluorescent red protein derivatives of Katushka clade and Renilla luciferase, that is commonly used in in vivo studies . Design and methods. Xenograft mice models were derived injecting modified HeLa cell line, that expresses fluorescent reporter proteins: Katushka, Katushka2S, TurboRFP, TurboFP650 and Renilla enzyme. Results. Spectral properties and emission wavelength of far-red fluorescent protein Katushka and Katushka2S outlines these markers within RFP derivatives lineage as outstanding instrument for in vivo tumor visualization. Conclusion. Detection of fluorescent far-red reporters Katushka and Katushka2S can be considered as a credible alternative to Renilla luciferase bioluminescence in experimental models in vivo on the part of immunotherapy research.

INTRODUCTION. The main risk to the clinical translatability of preclinical results for anticancer medicinal products is posed by the difficulty of simulating clinical conditions in an experimental model. With only 5% of product candidates proving clinically effective, the search for new approaches to the preclinical development of anticancer medicinal products is currently an active area of research in medicine.AIM. This study aimed to provide methodological support for planning experiments with modelling of neoplastic processes through analysis and classification of the methods used in preclinical studies of the efficacy of anticancer medicinal products in vivo.DISCUSSION. This article reviews the development of animal tumour models and the selection of cell lines and their testing for tumourigenicity and viability on a step-by-step basis. According to the study results, imaging systems, vital staining, and fluorescence- and luminescence-based methods can be used to assess the efficacy of anticancer medicinal products in both solid tumour models and haematological malignancy models. The article presents a schematic representation of the main types of mouse cancer models. However, no single animal species is universally suitable for in vivo cancer modelling. Researchers selecting models and considering their advantages and disadvantages should pay special attention to the similarity of disease mechanisms in animal models and humans at the tissue and molecular level, keeping in mind the aims of their research.CONCLUSIONS. The results of this comparative analysis of methods for preclinical efficacy evaluation of anticancer medicinal products are essential for designing experimental studies and ensuring the reliability of the results obtained. Choosing the correct research method will increase the chances of obtaining experimental data that can be successfully translated into clinical practice.

Цель. Создание анти-CD87 CAR T-лимфоцитов и оценка их функциональной активности in vitro. Материалы и методы. Выделенные из периферической крови здорового донора Т-лимфоциты трансдуцировали лентивирусным вектором, кодирующим гены анти-CD87-CAR, T2A и FusionRed. Эффективность трансдукции, оцениваемая по уровню сигнала репортерного белка FusionRed, субпопуляционный состав и функциональное состояние CAR T-лимфоцитов определены методом проточной цитометрии. Экспрессия интерферона-γ (IFN-γ) CAR T-лимфоцитами изучалась с помощью иммуноферментного анализа. Оценка цитотоксической активности CAR T-лимфоцитов выполнялась при сокультивировании с клетками-мишенями HeLa с помощью системы анализа клеток в реальном времени xCELLigence. Результаты. Эффективность трансдукции Т-лимфоцитов составила 8,4 %. Полученные CAR T-клетки содержали как маркеры активации CD27 и/или CD28 (92,91 % случаев), так и маркер истощения PD1 (20,66 %). В популяции CAR T-лимфоцитов фенотип Т-клеток центральной памяти составил 98,51 %, а соотношение CD4/CD8 — 1:7. Концентрация IFN-γ в среде после сокультивирования CAR T-лимфоцитов с клетками-мишенями оказалась значимо выше, чем в контрольных образцах. Показано, что полученные СAR T-лимфоциты проявляют специфическую цитотоксичность по отношению к клеткам-мишеням как немодифицированной, так и повышенной экспрессии антигена CD87 клеточных линий HeLa. Цитотоксичность оказалась более выраженной в отношении линии клеток с повышенной экспрессией антигена CD87. Заключение. Несмотря на повышенную экспрессию маркера истощения PD1, CAR Т-лимфоциты продемонстрировали специфическую секрецию IFN-γ и выраженную цитотоксическую активность при взаимодействии с антигеном CD87 на мембране клеток-мишеней. Следовательно, анти-CD87 CAR Т-лимфоциты могут использоваться для лечения как гематологических, так и солидных опухолей. Поскольку наблюдаемая разница в цитотоксичности не коррелирует линейно с плотностью антигена CD87 на поверхности атакуемых клеток, при применении CAR T-клеточного препарата in vivo необходимо исключить вероятность цитотоксического воздействия на здоровые клетки, экспрессирующие CD87.

Aim. To study antitumor cytotoxic effect of CAR-T NKG2D and CAR-T anti-CD19 in vitro and in vivo in order to compare antitumor activity of chimeric antigen receptors (CAR) with different structural and functional properties. Materials & Methods. CAR constructions were produced by molecular cloning. CAR-T cell populations were obtained by transduction of healthy donor T-lymphocytes with recombinant lentiviral particles coding CAR NKG2D or CD19 target antigen CAR sequences. CAR-T cell proportion was assessed by FusionRed fluorescence and EGFR membrane receptor imaging. Specific in vitro cytotoxic activity of CAR-T effector cells was analyzed by Real-Time Cytotoxicity Assay (RTCA) during co-cultivation with HeLa_CD19 target cell line using xCELLigence. Interferon-Y (IFN-y) synthesis in vitro and in vivo along with the degree of cytotoxic effect were analyzed by immunoassay of culture medium of co-cultivated effector cells and target cells as well as isolated auto-plasma from the peripheral blood of mice. To assess the in vivo functional activity, CAR-T cell populations were infused into immunodeficient NSG-SGM3 mice (10 000 000 cells/mouse) 12 days after HeLa_CD19 cell injection and confirmation of engraftment and tumor growth. Upon euthanasia, tumors were removed and fixed in paraffin to prepare histological sections. CAR-T cell tumor infiltration was assessed by CD3 antigen immunohistochemical staining. Results. The highest ligand (molecules MICA, ULBP1/2/3/4/5/6) expression levels were detected in HeLa cell line. The obtained NKG2D CAR-T cells showed a considerable cytotoxic activity against HeLa_CD19 target line (cell index [CI] = 1.27), which was, however, twice as low as that of CAR-T anti-CD19 (CI = 0.60) (p = 0.0038). IFN-y level during co-cultivation of CAR-T anti-CD19 with HeLa_CD19 at the ratio of Е/Т = 1:1 was 64,852 pcg/mL, which was 3.5 times higher than IFN-y level during co-cultivation of CAR-T NKG2D with HeLa_CD19 (18,635 pcg/mL) (p = 0.0360). The degree of tumor infiltration by CAR-T anti-CD19 cells was higher than that by CAR-T NKG2D. The absence of NKG2D proliferating CAR-T cells in mice peripheral blood confirms their low persistence. IFN-y concentration in mice auto-plasma was 11.89 pcg/mL after CAR-T anti-CD19 infusion and 0.57 pcg/mL after CAR-T NKG2D infusion (p = 0.0079). The mean weight of tumor xenografts in experimental groups 10 days after CAR-T anti-CD19 injection was 0.72 g (p = 0.0142), after Т-lymphocyte and NKG2D CAR-T cell infusions it was 2.12 g and 1.2 g, respectively. Conclusion. CAR-T anti-CD19 cells are characterized by more pronounced cytotoxic effect under both in vitro and in vivo experimental conditions compared with CAR-T NKG2D cells. The degree of CAR-T anti-CD19 proliferation and their infiltration in mice xenograft models is considerably higher than the levels reached with NKG2D CAR-T cell injections. A single CAR-T NKG2D injection results only in short-term tumor reduction.

Gilteritinib therapy is comparable in efficacy to salvage IC in patients with FLT3-mutated R/R AML but has been shown to be safer and have better survival rates. Gilteritinib is an optimal bridge therapy to allogeneic BMT.

Aim. To generate anti-CD87 CAR-T lymphocytes and to assess their in vitro functional activity. Materials & Methods. Т-lymphocytes isolated from healthy donor peripheral blood were transduced with the anti-CD87-CAR, T2A, and FusionRed gene coding lentiviral vector. Transduction efficacy assessed by reporter protein FusionRed signal, subpopulation structure, and functional status of CAR-T lymphocytes were determined by flow cytometry. Interferon-y (IFN-y) expression by CAR-T lymphocytes was analyzed using immunoassay. Cytotoxic activity of CAR-T lymphocytes was evaluated during their co-cultivation with HeLa target cells by means of xCELLigence real-time assay. Results. The efficacy of T-lymphocyte transduction was 8.4 %. The obtained CAR-T cells contained the markers of both CD27 and/or CD28 activation (92.91 % cases) and PD1 exhaustion (20.66 % cases). The population of CAR-T lymphocytes showed 98.51 % central memory T-cell phenotype and CD4/CD8 ratio of 1:7. IFN-y concentration in the medium after co-cultivation of CAR-T lymphocytes with target cells appeared to be significantly higher than in control samples. The study demonstrates that generated CAR-T lymphocytes manifest specific cytotoxicity towards target cells with both unmodified expression and overexpression of CD87 antigen in HeLa cell lines. Cytotoxicity proved to be more pronounced with respect to the cell line with CD87 antigen overexpression. Conclusion. Despite overexpression of PD1 exhaustion marker, CAR-T lymphocytes showed specific IFN-y secretion and pronounced cytotoxic activity in interaction with CD87 antigen on target cell membranes. Therefore, anti-CD87 CAR-T lymphocytes can be applied in the treatment of hematologic as well as solid tumors. Since the observed difference in cytotoxicity does not linearly correlate with CD87 antigen density on the surface of attacked cells, the in vivo administration of a CAR-T cell drug should be designed to prevent cytotoxic risk for CD87-expressing healthy cells.

Aim. To identify mutations in IDH1/IDH2, DNMT3A, and ASXL1 genes responsible for genome epigenetic regulation and their co-occurrence with FLT3, NPM1, and RUNX1 mutations in newly diagnosed adult acute myeloid leukemias (AML). Materials & Methods. The study included 56 patients with newly diagnosed AML treated at the VA Almazov National Medical Research Center. Among them there were 34 men and и 22 women aged 18-76 years (median 46 years). Mutation status of IDH1, IDH2, DNMT3A, and ASXL1 genes of epigenetic regulation was assessed by Sanger sequencing method. Molecular genetic analysis of FLT3, NPM1, and RUNX1-RUNX1T1 genes was performed using commercial kits. Results. Mutations in epigenetic regulation genes were detected in 14 (25 %) out of 56 patients. Mutation prevalence was not associated with risk groups (p = 0.072). IDH1/2 mutations were identified in 15.6 % of patients and were significantly oftener observed concurrent with NPM1 mutations (62.5 %; p = 0.01) compared to patients with wild-type IDH1/2. In most patients IDH1/2 mutations were associated with normal karyotype (p = 0.002). The DNMT3A (R882) mutation was identified in 4 (7.1 %) out of 56 patients within the analyzed group. In 6 patients (11.1 %) ASXL1 mutations were detected co-occurring with RUNX1-RUNX1T1 and FLT3-ITD mutations. Conclusion. Mutations in epigenetic regulation genes are often identified in AML patients and can be concurrent with abnormalities in NPM1, FLT3 и RUNX1 genes.

Цель. Выявление мутаций генов IDH1/IDH2, DNMT3A и ASXL1, ответственных за эпигенетическую регуляцию генома, при впервые диагностированных острых миелоидных лейкозах (ОМЛ) у взрослых и их сочетания с мутациями генов FLT3, NPM1, RUNX1. Материалы и методы. В исследование включено 56 пациентов с впервые выявленным ОМЛ, проходивших лечение в ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России. Среди них было 34 мужчины и 22 женщины в возрасте 18–76 лет (медиана 46 лет). Мутационный статус генов эпигенетической регуляции IDH1, IDH2, DNMT3A и ASXL1 определяли методом секвенирования по Сэнгеру. Молекулярно-генетический анализ генов FLT3, NPM1, RUNX1-RUNX1T1 выполняли с использованием коммерческих наборов. Результаты. Мутации генов эпигенетической регуляции обнаружены у 14 (25 %) из 56 пациентов. Распространенность мутаций не была связана с группами риска (p = 0,072). Мутации IDH1/2 выявлены у 15,6 % пациентов и статистически значимо чаще обнаруживались одновременно с мутациями NPM1 (62,5 %; p = 0,01) по сравнению с пациентами с диким типом IDH1/2. У большинства пациентов мутации IDH1/2 были связаны с нормальным кариотипом (p = 0,002). Мутация DNMT3A (R882) определена у 4 (7,1 %) из 56 пациентов анализируемой группы. У 6 (11,1 %) пациентов были идентифицированы мутации ASXL1, которые сочетались мутациями с RUNX1-RUNX1T1 и FLT3-ITD. Заключение. Мутации генов эпигенетической регуляции часто обнаруживаются у пациентов с ОМЛ и могут сочетаться с нарушениями в генах NPM1, FLT3 и RUNX1.

NK-cells as innate immunity elements manifest key reactions of antitumor immune response. NKG2D is an activating transmembrane receptor of NK-cells which is responsible for cytotoxicity initiation in response to the binding of specific ligands of genetically modified cells. Selective expression of NKG2D ligands provides a unique perspective on the therapy of wide variety of tumors. Acute myeloid leukemias (AML) are malignant hematological tumors with a high relapse risk. Due to the complexity of AML treatment strategy it is necessary to develop new approaches to tumor elimination using novel genetic constructs. Currently available CAR T-cell drugs with NKG2D receptor are successfully subjected to clinical studies in AML patients and prove their high therapeutic potential.

Актуальность. До настоящего времени показатели выживаемости пациентов с острыми миелоидными лейкозами (ОМЛ) остаются неудовлетворительными. Для успешного лечения ОМЛ необходимо создание персонализированных моделей этого заболевания. Наиболее перспективным направлением в данной области является разработка ксенографтных моделей от больных ОМЛ с использованием наиболее современной линии иммунодефицитных «гуманизированных» мышей NSG-SGM3. Цель. Создание ксенографтных моделей от больных ОМЛ с использованием иммунодефицитных мышей линии NSG-SGM3. Материалы и методы. Для создания PDX-моделей использовались образцы аспирата костного мозга 4 пациентов с впервые диагностированным ОМЛ, проходивших лечение в ФГБУ «НМИЦ им. В.А. Алмазова» МЗ РФ. Опухолевые клетки от пациентов трансплантировали мышам линии NSG-SGM3. Для проведения контрольного эксперимента мышам NSG-SGM3 вводили клетки линий ОМЛ: OCI-АМL2 и HL60. Эффективность приживления образцов опухоли оценивалась на основе физического состояния животных и лабораторных исследований (формула крови, мазок крови, ПЦР, проточная цитофлюориметрия). Результаты. Приживление введенных опухолевых клеток от 4 пациентов с ОМЛ достигнуто у половины (2 из 4) мышей. У 2 мышей с успешной трансплантацией имел место лейкоцитоз. Бластные клетки обнаруживались в периферической крови на 30-й день после трансплантации. У мышей с инъецированными клеточными линиями ОМЛ OCI-АМL2 и HL60 наблюдалось более агрессивное течение заболевания. Среди протестированных подходов для оценки приживления опухоли у мышей-реципиентов метод ПЦР отличался наибольшей чувствительностью. Заключение. Использование иммунодефицитных «гуманизированных» мышей линии NSG-SGM3 позволяет успешно получать ксенографтные модели от пациентов с ОМЛ.

Background. Up to the present the survival rates of acute myeloid leukemia (AML) patients have remained low. A successful AML management presupposes generating personalized models of the disease. The most promising research activity in this field is creation of AML patient-derived xenograft models using the advanced strain of immunodeficient humanized NSG-SGM3 mice. Aim. To generate AML patient-derived xenograft models using immunodeficient NSG-SGM3 mice. Materials & Methods. The creation of PDX models was based on bone marrow aspirates taken from 4 patients with newly diagnosed AML who were treated at the V.A. Almazov National Medical Research Center. Patient-derived tumor cells were transplanted to NSG-SGM3 mice. Test experiment consisted in injecting AML cells OCI-АМL2 and HL60 in NSG-SGM3 mice. The efficacy of tumor engraftment was evaluated in terms of physical condition of animals and laboratory tests (blood count, blood smear, PCR, and flow cytofluorometry). Results. The engraftment of applied tumor cells derived from AML patients was achieved in half (2 out of 4) of the transplanted tumor samples. In mice with successful transplantation leukocytosis was reported. Blast cells were identified in peripheral blood on Day 30 after transplantation. The mice with injected AML cells OCI-АМL2 and HL60 showed a more aggressive course of disease. Among tested approaches to evaluate tumor engraftment in mouse recipients, the PCR method was marked by highest sensitivity. Conclusion. The use of immunodeficient humanized NSG-SGM3 mice enables successful generation of AML patient-derived xenograft models.