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<article article-type="research-article" dtd-version="1.3" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xml:lang="ru"><front><journal-meta><journal-id journal-id-type="publisher-id">oncotomsk</journal-id><journal-title-group><journal-title xml:lang="ru">Сибирский онкологический журнал</journal-title><trans-title-group xml:lang="en"><trans-title>Siberian journal of oncology</trans-title></trans-title-group></journal-title-group><issn pub-type="ppub">1814-4861</issn><issn pub-type="epub">2312-3168</issn><publisher><publisher-name>Tomsk National Research Medical Сепtеr of the Russian Academy of Sciences</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.21294/1814-4861-2022-21-6-59-67</article-id><article-id custom-type="elpub" pub-id-type="custom">oncotomsk-2375</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="section-heading" xml:lang="ru"><subject>ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ</subject></subj-group><subj-group subj-group-type="section-heading" xml:lang="en"><subject>LABORATORY AND EXPERIMENTAL STUDIES</subject></subj-group></article-categories><title-group><article-title>Оптимизация метода исследования маммосферообразования для оценки il-6 индуцированной стволовой пластичности дифференцированных клеток рака молочной железы</article-title><trans-title-group xml:lang="en"><trans-title>Optimization of mammosphere formation assay for quantification of il6-induced stemness in differentiated breast cancer cells</trans-title></trans-title-group></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0003-1659-8812</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Невская</surname><given-names>К. В.</given-names></name><name name-style="western" xml:lang="en"><surname>Nevskaya</surname><given-names>K. V.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Невская Ксения Владимировна, кандидат медицинских наук, научный сотрудник центра биологических исследований и биоинженерии центральной научно-исследовательской лаборатории, </p><p>634050, г. Томск, Московский тракт, 2</p></bio><bio xml:lang="en"><p>Ksenia V. Nevskaya, MD, PhD, Researcher at the Center for Biological Research and Bioengineering of the Central Research Laboratory, </p><p>2, Moskovsky tract, 634050, Tomsk</p></bio><email xlink:type="simple">nevskayaksenia@gmail.com</email><xref ref-type="aff" rid="aff-1"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0002-7168-1419</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Ефимова</surname><given-names>Л. В.</given-names></name><name name-style="western" xml:lang="en"><surname>Efimova</surname><given-names>L. V.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Ефимова Лина Викторовна, младший научный сотрудник центра биологических исследований и биоинженерии центральной научно-исследовательской лаборатории, </p><p>634050, г. Томск, Московский тракт, 2</p></bio><bio xml:lang="en"><p>Lina V. Efimova, Junior Researcher at the Center for Biological Research and Bioengineering of the Central Research Laboratory,  </p><p>2, Moskovsky tract, 634050, Tomsk</p></bio><xref ref-type="aff" rid="aff-1"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0001-8887-536X</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Корда</surname><given-names>П. В.</given-names></name><name name-style="western" xml:lang="en"><surname>Korda</surname><given-names>P. V.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Корда Полина Викторовна, студентка медико-биологического факультета, </p><p>634050, г. Томск, Московский тракт, 2</p></bio><bio xml:lang="en"><p>Polina V. Korda, Student,</p><p>2, Moskovsky tract, 634050, Tomsk</p></bio><xref ref-type="aff" rid="aff-1"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0002-6763-920X</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Долгашева</surname><given-names>Д. С.</given-names></name><name name-style="western" xml:lang="en"><surname>Dolgasheva</surname><given-names>D. S.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Долгашева Дарья Сергеевна, младший научный сотрудник лаборатории онковирусологии, </p><p>634009, г. Томск, пер. Кооперативный, 5</p></bio><bio xml:lang="en"><p>Daria S. Dolgasheva, Junior Researcher at the Laboratory of Viral Oncology, </p><p>5, Kooperativny St., 634009, Tomsk</p></bio><xref ref-type="aff" rid="aff-2"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0001-8815-2786</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Ибрагимова</surname><given-names>М. К.</given-names></name><name name-style="western" xml:lang="en"><surname>Ibragimova</surname><given-names>M. K.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Ибрагимова Марина Константиновна, кандидат биологических наук, старший научный сотрудник лаборатории онковирусологии, 634009, г. Томск, пер. Кооперативный, 5;</p><p>научный сотрудник лаборатории генетических технологий центральной научно-исследовательской лаборатории, 634050, г. Томск, Московский тракт, 2</p><p> </p></bio><bio xml:lang="en"><p>Marina K. Ibragimova, PhD, Senior Researcher at the Laboratory of Viral Oncology, 5, Kooperativny St., 634009, Tomsk;</p><p>Researcher of the Laboratory of Genetic Technologies of the Central ResearchLaboratory, 2, Moskovsky tract, 634050, Tomsk</p></bio><xref ref-type="aff" rid="aff-3"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0003-4630-4934</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Меняйло</surname><given-names>М. Е.</given-names></name><name name-style="western" xml:lang="en"><surname>Menyailo</surname><given-names>M. E.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Меняйло Максим Евгеньевич, кандидат медицинских наук, научный сотрудник лаборатории биологии опухолевой прогрессии,</p><p>634009, г. Томск, пер. Кооперативный, 5</p></bio><bio xml:lang="en"><p>Maksim E. Menyailo, MD, PhD, Researcher at the Laboratory of Biology of Tumor Progression,</p><p>5, Kooperativny St., 634009, Tomsk</p></bio><xref ref-type="aff" rid="aff-2"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0002-6104-4782</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Удут</surname><given-names>Е. В.</given-names></name><name name-style="western" xml:lang="en"><surname>Udut</surname><given-names>E. V.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Удут Елена Владимировна, доктор медицинских наук, профессор, заведующая центральной научно-исследовательской лабораторией, </p><p>634050, г. Томск, Московский тракт, 2</p></bio><bio xml:lang="en"><p>Elena V. Udut, MD, Professor, Head of the Central Research Laboratory,</p><p>2, Moskovsky tract, 634050, Tomsk</p></bio><xref ref-type="aff" rid="aff-1"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0002-0714-8927</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Литвяков</surname><given-names>Н. В.</given-names></name><name name-style="western" xml:lang="en"><surname>Litviakov</surname><given-names>N. V.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Литвяков Николай Васильевич, доктор биологических наук, профессор РАН,</p><p>научный сотрудник лаборатории генетических технологий центральной научно-исследовательской лаборатории, 634050, г. Томск, Московский тракт, 2;</p><p>заведующий лабораторией онковирусологии, 634009, г. Томск, пер. Кооперативный, 5</p></bio><bio xml:lang="en"><p>Nikolai V. Litvyakov, DSc, Professor of the Russian Academy of Sciences, Researcher of the Laboratory of Genetic Technologies of the Central Research Laboratory, 2, Moskovsky tract, 634050, Tomsk;</p><p>Head of the Laboratory of Viral Oncology, 5, Kooperativny St., 634009, Tomsk</p></bio><xref ref-type="aff" rid="aff-4"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">https://orcid.org/0000-0001-7604-9722</contrib-id><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Першина</surname><given-names>А. Г.</given-names></name><name name-style="western" xml:lang="en"><surname>Pershina</surname><given-names>A. G.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Першина Александра Геннадьевна, кандидат биологических наук, заведующая центром биологических исследований и биоинженерии центральной научно-исследовательской лаборатории, </p><p>634050, г. Томск, Московский тракт, 2</p></bio><bio xml:lang="en"><p>Alexandra G. Pershina, PhD, Head of the Center for Biological Research and Bioengineering of the Central Research Laboratory, </p><p>2, Moskovsky tract, 634050, Tomsk</p></bio><xref ref-type="aff" rid="aff-1"/></contrib></contrib-group><aff-alternatives id="aff-1"><aff xml:lang="ru"><institution>ФГБОУ ВО «Сибирский государственный медицинский университет» Минздрава России</institution><country>Россия</country></aff><aff xml:lang="en"><institution>Siberian State Medical University of the Ministry of Health of Russia</institution><country>Russian Federation</country></aff></aff-alternatives><aff-alternatives id="aff-2"><aff xml:lang="ru"><institution>Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук</institution><country>Россия</country></aff><aff xml:lang="en"><institution>Cancer Research Institute, Тomsk National Research Medical Center, Russian Academy of Sciences</institution><country>Russian Federation</country></aff></aff-alternatives><aff-alternatives id="aff-3"><aff xml:lang="ru"><institution>Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук;&#13;
ФГБОУ ВО «Сибирский государственный медицинский университет» Минздрава России</institution><country>Россия</country></aff><aff xml:lang="en"><institution>Cancer Research Institute, Тomsk National Research Medical Center, Russian Academy of Sciences;&#13;
Siberian State Medical University of the Ministry of Health of Russia</institution><country>Russian Federation</country></aff></aff-alternatives><aff-alternatives id="aff-4"><aff xml:lang="ru"><institution>ФГБОУ ВО «Сибирский государственный медицинский университет» Минздрава России;&#13;
Научно-исследовательский институт онкологии, Томский национальный исследовательский медицинский центр Российской академии наук</institution><country>Россия</country></aff><aff xml:lang="en"><institution>Siberian State Medical University of the Ministry of Health of Russia;&#13;
Cancer Research Institute, Тomsk National Research Medical Center, Russian Academy of Sciences</institution><country>Russian Federation</country></aff></aff-alternatives><pub-date pub-type="collection"><year>2022</year></pub-date><pub-date pub-type="epub"><day>30</day><month>12</month><year>2022</year></pub-date><volume>21</volume><issue>6</issue><fpage>59</fpage><lpage>67</lpage><permissions><copyright-statement>Copyright &amp;#x00A9; Невская К.В., Ефимова Л.В., Корда П.В., Долгашева Д.С., Ибрагимова М.К., Меняйло М.Е., Удут Е.В., Литвяков Н.В., Першина А.Г., 2023</copyright-statement><copyright-year>2023</copyright-year><copyright-holder xml:lang="ru">Невская К.В., Ефимова Л.В., Корда П.В., Долгашева Д.С., Ибрагимова М.К., Меняйло М.Е., Удут Е.В., Литвяков Н.В., Першина А.Г.</copyright-holder><copyright-holder xml:lang="en">Nevskaya K.V., Efimova L.V., Korda P.V., Dolgasheva D.S., Ibragimova M.K., Menyailo M.E., Udut E.V., Litviakov N.V., Pershina A.G.</copyright-holder><license xml:lang="ru" license-type="creative-commons-attribution" xlink:href="https://creativecommons.org/licenses/by/4.0/" xlink:type="simple"><license-p>Данная работа распространяется под лицензией Creative Commons Attribution 4.0.</license-p></license><license xml:lang="en" license-type="creative-commons-attribution" xlink:href="https://creativecommons.org/licenses/by/4.0/" xlink:type="simple"><license-p>This work is licensed under a Creative Commons Attribution 4.0 License.</license-p></license></permissions><self-uri xlink:href="https://www.siboncoj.ru/jour/article/view/2375">https://www.siboncoj.ru/jour/article/view/2375</self-uri><abstract><p>Цель исследования – оптимизация метода оценки образования маммосфер после индуцированной интерлейкином 6 (IL6) стволовой пластичности у дифференцированных (CD44– ) опухолевых клеток линии рака молочной железы человека T47D.</p><sec><title>Материал и методы</title><p>Материал и методы. Проведена оценка жизнеспособности, цитофлуориметрического профиля клеток и их способности к маммосферообразованию при различной исходной конфлюентности (40–50 % и 70–80 %) и методах снятия с поверхности пластика (использование TrypLE, аккутазы, скребка для клеток). Жизнеспособность клеток оценивали методом проточной цитометрии с использованием флуоресцентно-меченного АннексинV и йодистого пропидия. Динамику изменения экспрессии поверхностных маркеров клеток оценивали методом проточной цитометрии с флуоресцентно-меченными антителами к CD24 и CD44.</p></sec><sec><title>Результаты</title><p>Результаты. Применение скребка для снятия клеток приводило к существенному повышению количества клеток с цитофлуориметрическими признаками раннего и позднего апоптоза по сравнению с использованием TrypLE и аккутазы. Использование TrypLE приводило к снижению количества CD44+ клеток, тогда как аккутаза не влияла на представленность данного маркера. Количество маммосфер и их средний диаметр не отличались между исследуемыми группами. Стимуляция дифференцированных (CD44– CD24+) клеток линии T47D IL6 уже через 24 ч приводила к появлению популяций CD44+CD24+ прогениторных и CD44+CD24–/low опухолевых стволовых клеток. Дифференцированные клетки после стимуляции IL6 в течение 24 ч образовывали в 3 раза больше маммосфер по сравнению с контролем.</p></sec><sec><title>Заключение</title><p>Заключение. Для оценки маммосферообразования целесообразно использовать клетки с конфлюентностью до 80 % и применять аккутазу для снятия с поверхности пластика. Для стимуляции стволовой пластичности у дифференцированных клеток линии T47D достаточной является их инкубация с IL6 в течение 24 ч. </p></sec></abstract><trans-abstract xml:lang="en"><p>The aim of the study was mammosphere assay optimization for quantifcation of IL6-induced stemness in differentiated (СD44– ) T47D breast cancer cells.</p><sec><title>Material and Methods</title><p>Material and Methods. The effect of three commonly used cell-detaching methods (TrypLE, accutase, cell scrapper) at various confuence (40–50 % and 70–80 %) on cell viability, phenotypic profle and mammosphere formation was tested. The cell viability was examined using AnnexinV/propidium iodide assay. The phenotypic profle was analyzed by fow cytometry with fuorescent markers CD24 and CD44.</p></sec><sec><title>Results</title><p>Results. Detachment of the cells using scrapper led to substantial increase in early apoptotic and late apoptotic cells in comparison with TrypLE and accutase. Dissociation with TrypLE reduced the percentage of detected CD44+ positive cells, whereas accutase saved the surface marker. The number of mammosphere and their diameter did not differ between groups. Incubation of differentiated (CD44– CD24+) T47D cells with IL-6 for 24 hours resulted in an appearance of CD44+CD24+ and CD44+CD24–/low subpopulation. Furthermore, the differentiated cells after 24 hours of IL6 exposure formed 3 times more mammospheres compared to the control.</p></sec><sec><title>Conclusion</title><p>Conclusion. Usage of cells with confuence of no more than 80 % and accutase for detachment of cells is recommended for mammosphere assay. Incubation of CD44– CD24+ T47D cells with IL6 for 24 hours is suffcient for stimulation of stemness plasticity. </p></sec></trans-abstract><kwd-group xml:lang="ru"><kwd>рак молочной железы</kwd><kwd>маммосферообразование</kwd><kwd>интерлейкин 6</kwd><kwd>стволовая пластичность.</kwd></kwd-group><kwd-group xml:lang="en"><kwd>breast cancer</kwd><kwd>mammosphere formation assay</kwd><kwd>IL6</kwd><kwd>stemness</kwd></kwd-group><funding-group><funding-statement xml:lang="ru">Работа выполнена при финансовой поддержке Министерства науки и высшего образования Российской Федерации (соглашение от 29.09.2021 № 075-15-2021-1073).</funding-statement><funding-statement xml:lang="en">The study was funded by the Ministry of Science and Higher Education of the Russian Federation (agreement № 075-15-2021-1073, 29.09.2021).</funding-statement></funding-group></article-meta></front><back><ref-list><title>References</title><ref id="cit1"><label>1</label><citation-alternatives><mixed-citation xml:lang="ru">Fumagalli A., Oost K.C., Kester L., Morgner J., Bornes L., Bruens L., Spaargaren L., Azkanaz M., Schelfhorst T., Beerling E., Heinz M.C., Postrach D., Seinstra D., Sieuwerts A.M., Martens J.W.M., van der Elst S., van Baalen M., Bhowmick D., Vrisekoop N., Ellenbroek S.I.J., Suijkerbuijk S.J.E., Snippert H.J., van Rheenen J. 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