РАЗРАБОТКА И ХАРАКТЕРИСТИКА ТРЕХМЕРНЫХ КЛЕТОЧНЫХ МОДЕЛЕЙ СОЛИДНЫХ ОПУХОЛЕЙ ДЛЯ ИНДИВИДУАЛИЗАЦИИ ЛЕЧЕНИЯ ОНКОЛОГИЧЕСКИХ БОЛЬНЫХ

Background. To solve the problems of personalized medicine in oncology, preclinical studies based on the use of three-dimensional cellular models of tumors in vitro, including spheroids / tumoroids, are of great importance. They are an interesting tool for genetic, epigenetic, biomedical and pharmacological studies aiming to determine the most effective individual therapeutic approaches, since they allow modeling the dynamic evolution of a tumor disease from early stages to metastatic spread through interaction with the microenvironment.The purpose of the study was to compare characteristic features of formation and spatial organization of spheroids, obtained from solid malignant tumors cells with various histogenesis: melanomas, soft tissue sarcomas and osteosarcomas, epithelial tumors.Material and Methods. Solid tumor cell lines of patients who were treated from 2015 to 2021 were the basis for the creation of 3D-cell models. Fragments of tumor tissue were obtained intraoperatively: 15 samples of melanoma, 20 samples of soft tissue sarcomas and osteosarcomas, and 9 samples of epithelial tumors. All tumor cells were cultured for at least 10 passages. Methods of phase contrast, confocal microscopy, and histological techniques were used to study spheroids. Using ELISA methods and multiplex analysis, the supernatants of monolayer cell cultures and spheroids were studied for the presence of a wide range of biologically active substances that provide the processes of immunosuppression, invasion and metastasis.Results. The use of low adhesion surfaces was proven to be preferable to obtain spheroids of a given seed concentration and size of interest. The average cultivation time of spheroids was 4.7 days, and the optimal seeding concentration was 10,000 cells per well, while the spheroid diameter varied from 300 to 1000 μm depending on the type of malignant cells: the largest spheroids formed melanoma cultures. In general, the efficiency of spheroid formation was 88.6 % (39 out of 44). The introduction of fibroblasts into the 3D construct led to increasing in the invasive potential of tumor cells, which was associated with the production of IL8 (rho=0.636, p=0.035), HGF (rho=0.850, p=0.004), SCF (rho=0.857, p=0.014), FST (rho=0.685, p=0.029), Prolactin (rho=0.810, p=0.015), PECAM1 (rho=0.788, p=0.004).Conclusion. The technology of low-adhesive surfaces makes it possible to successfully create three-dimensional models of a tumor node from malignant tumors cells of various histogenesis. The colonization of a three-dimensional structure with fibroblasts enhances the biologically aggressive properties of tumor cells and demonstrates complex reciprocal interactions between the cellular elements of the tumor stroma and malignant cells, which brings the model closer to a real clinical situation. Введение. Для решения задач персонализированной медицины в онкологии важным становится этап предклинических исследований, основанный на использовании трехмерных клеточных моделей опухолей in vitro, в том числе сфероидов/тумороидов, которые представляют собой интересный инструмент для генетических, эпигенетических, биомедицинских и фармакологических исследований с целью определения наиболее эффективного индивидуального терапевтического подхода, так как позволяют моделировать динамическую эволюцию опухолевого заболевания от ранних стадий до метастатического распространения через взаимодействие с микроокружением.Цель исследования – провести сравнительные исследования особенностей формирования и пространственной организации сфероидов, полученных из клеток солидных злокачественных новообразований различного гистогенеза: меланом (МК), сарком мягких тканей и остеогенных сарком (СМТ/ОС), эпителиальных опухолей (ЭОп).Материал и методы. Основой для создания 3D-клеточных моделей служили культуры клеток солидных опухолей пациентов, которые проходили лечение в ФГБУ «НМИЦ онкологии им. Н.Н. Петрова» в 2015–21 гг. Фрагменты опухолевой ткани были получены интраоперационно: 15 образцов МК, 20 – СМТ/ОС и 9 – ЭОп. Все опухолевые клетки культивировали не менее 10 пассажей. Для изучения сфероидов были использованы методы фазовой контрастной, конфокальной микроскопии, гистологическая техника. С помощью методов ИФА и мультиплексного анализа были изучены супернатанты монослойных клеточных культур и сфероидов на предмет присутствия широкого спектра биологически активных веществ, обеспечивающих процессы иммуносупрессии, инвазии и метастазирования.Результаты. Использование низкоадгезивных поверхностей оказалось предпочтительным для получения сфероидов заданной посевной концентрации и интересующего размера. Среднее время культивирования сфероидов составило 4,7 сут, оптимальная посевная концентрация – 10 000 клеток на лунку, при этом диаметр сфероида варьировал от 300 до 1 000 мкм в зависимости от типа злокачественных клеток: самые крупные сфероиды формировали культуры МК. В целом эффективность образования сфероидов составила 88,6 % (39 из 44). Введение в 3D-конструкцию фибробластов приводило к усилению инвазивного потенциала опухолевых клеток, который был ассоциирован с продукцией IL8 (rho=0,636, p=0,035), HGF (rho=0,850, p=0,004), SCF (rho=0,857, p=0,014), FST (rho=0,685, p=0,029), Prolactin (rho=0,810, p=0,015), PECAM1 (rho=0,788, p=0,004). Заключение. Технология низкоадгезивных поверхностей позволяет успешно создавать трехмерные модели опухолевого узла из клеток злокачественных новообразований различного гистогенеза. Заселение трехмерной конструкции фибробластами усиливает биологически агрессивные свойства опухолевых клеток и демонстрирует сложные реципрокные взаимодействия между клеточными элементами стромы опухоли и малигнизированными клетками, что приближает модель к реальной клинической ситуации.

МЕТОДИЧЕСКИЕ АСПЕКТЫ ПРОВЕДЕНИЯ ФОТОДИНАМИЧЕСКОЙ ТЕРАПИИ СОЛИДНОЙ КАРЦИНОМЫ ЭРЛИХА У МЫШЕЙ ЛИНИИ BALB/C С РАЗЛИЧНОЙ ЛОКАЛИЗАЦИЕЙ ОПУХОЛИ

Background. Photodynamic Therapy is one of the treatment methods used in modern oncology. Evaluation of the efficacy in vivo of photosensitizers on tumor models is generally accepted, but the photodynamic  therapy technique in mice is not without drawbacks.The purpose of the study was evaluation of the efficacy of photodynamic therapy in mice with Ehrlich tumor model after subcutaneous and intracutaneous injection of tumor cells.Material and Methods. The study was conducted on BAL B/C mice of both sexes. Fotoditazin® and Radachlorin® were used as photosensitizers. For photoactivation, the Alod laser apparatus with a wavelength of 662 nm was used.Results. A comparison of photodynamic therapy with subcutaneous and intracutaneous localization of Ehrlich tumor was performed. It was shown that depending on the location and depth of inoculation of Ehrlich tumor, the pharmacokinetics (both the fluorescence intensity over time and the contrast ratio of the tumor/surrounding tissue) and pharmacodynamics (tumor growth inhibition, survival) of photosensitizers are significantly different. Higher contrast of the tumor/surrounding tissue is observed with intracutaneous localization of the tumor.Conclusion. A model with intracutaneous localization of Ehrlich tumor can be recommended for a primary assessment of efficacy; it allows the use of fewer animals in the experiment. When planning experiments to study photosensitizers and evaluating their results, the advantages and disadvantages of different methods for modeling tumors in mice should be taken into account.Введение. Фотодинамическая терапия – один из методов лечения, применяемых в  современной онкологии. Оценка эффективности фотосенсибилизаторов in vivo на  моделях опухолей является общепринятой, однако методика проведения  фотодинамической терапии у мышей не лишена недостатков. Цель исследования – оценить эффективность фотодинамической терапии у мышей на модели опухоли Эрлиха при подкожной и внутрикожной инъекции опухолевых клеток. Материал и методы. Исследование проведено на мышах BalB/c обоего пола. В качестве фотосенсибилизаторов использовали препараты Фотодитазин и Радахлорин.  Для фотоактивации применяли лазерный аппарат Алод с длиной волны 662 нм. Результаты. Проведено сравнение фотодинамической терапии при подкожной и внутрикожной локализации опухоли Эрлиха. Показано, что в зависимости от  локализации и глубины инъекции опухоли Эрлиха в значительной степени различаются фармакокинетика (как интенсивность флуоресценции во времени, так и контрастность по отношению опухоль/окружающие ткани) и фармакодинамика (торможение роста  опухоли, выживаемость животных) фотосенсибилизаторов. Большая контрастность опухоль/окружающие ткани наблюдается при внутрикожной локализации опухоли. Заключение. Модель с внутрикожной локализацией опухоли Эрлиха может быть рекомендована для первичной оценки эффективности и позволяет использовать меньшее число животных в эксперименте. При планировании экспериментов по изучению фотосенсибилизаторов и оценке их результатов должны учитываться преимущества и недостатки разных способов моделирования опухоли у мышей. 

Ascitic ovarian cancer is an adequate preclinical model of carcinomatosis to study intraperitoneal chemoperfusion treatment

To study the mechanisms underlying the effects of intraperitoneal chemoperfusion and to develop the optimal chemoperfusion regimen, an animal model of peritoneal carcinomatosis closely resembles a human model of peritoneal carcinomatosis is required. In our study, the model of peritoneal carcinomatosis in rats with ascitic ovarian cancer was used. material and methods. There were three groups of rats with ascitic ovarian cancer: 1 – the control group (n=15) having no treatment; 2 – rats receiving normothermic intraperitoneal chemoperfusion with cisplatin, 40 mg/kg (n=12); 3 – rats receiving hyperthermic intraperitoneal chemoperfusion with cisplatin, 20 mg/kg (n=14). All animals were euthanized with subsequent autopsy. results. Ascitic ovarian cancer developed in 100 % of the animals injected with the tumor cells. The median overall survival of rats in the control group was 9.5 days. At autopsy, all rats had ascites, and rats surviving 15‒17 days after the tumor cell injection had white tumor nodes measuring 1–3 mm in the greater omentum, intestinal mesentery, parietal and visceral peritoneum. The nodes were histologically verified as metastases of low-differentiated ovarian tumor. In 2 and 5 rats from groups 2 and 3 respectively, metastases in paratracheal lymph nodes and tumor hydrothorax were detected with no evidence of peritoneal carcinomatosis. The median survival of rats in groups 2 and 3 was significantly higher than that in the control group, being 37.5 and 25.5 months, respectively (р=0,256). conclusion. This in vivo study proved that localization of ascitic ovarian tumor, development of the tumor in all animals injected with tumor cells, fast ascites progression and peritoneal carcinomatosis make this ascitic ovarian cancer an adequate preclinical model of peritoneal carcinomatosis to study intraperitoneal chemoperfusion. Further studies are needed to understand the reasons and mechanisms of the tumor hydrothorax development in rats after intraperitoneal chemoperfusion

METHODOLOGICAL ASPECTS OF PHOTODYNAMIC THERAPY OF EHRLICH SOLID CARCINOMA IN BALB/C MOUSE STRAIN WITH VARIOUS TUMOR LOCALIZATION

Background. Photodynamic Therapy is one of the treatment methods used in modern oncology. Evaluation of the efficacy in vivo of photosensitizers on tumor models is generally accepted, but the photodynamic  therapy technique in mice is not without drawbacks.The purpose of the study was evaluation of the efficacy of photodynamic therapy in mice with Ehrlich tumor model after subcutaneous and intracutaneous injection of tumor cells.Material and Methods. The study was conducted on BAL B/C mice of both sexes. Fotoditazin® and Radachlorin® were used as photosensitizers. For photoactivation, the Alod laser apparatus with a wavelength of 662 nm was used.Results. A comparison of photodynamic therapy with subcutaneous and intracutaneous localization of Ehrlich tumor was performed. It was shown that depending on the location and depth of inoculation of Ehrlich tumor, the pharmacokinetics (both the fluorescence intensity over time and the contrast ratio of the tumor/surrounding tissue) and pharmacodynamics (tumor growth inhibition, survival) of photosensitizers are significantly different. Higher contrast of the tumor/surrounding tissue is observed with intracutaneous localization of the tumor.Conclusion. A model with intracutaneous localization of Ehrlich tumor can be recommended for a primary assessment of efficacy; it allows the use of fewer animals in the experiment. When planning experiments to study photosensitizers and evaluating their results, the advantages and disadvantages of different methods for modeling tumors in mice should be taken into account

STUDY OF THE EFFECT OF LOMUSTIN ON HER2-POSITIVE BREAST CANCER IN FVB/N HER-2 TRANSGENIC MICE

Because of the high risk of brain metastases from HER2-positive breast cancer, the study of the anticancer activity of drugs used to treat brain tumors, in particular lomustine, is of great importance. In the FVB/N Her-2 transgenic mice bearing HER2-positive breast cancer (BC HER2+), a single oral administration of lomustine at a dose of 50 mg/kg resulted in a significant tumor growth inhibition (up to 96 %, p<0.0001). The tumor growth index (TGI) expressed as a ratio between the areas under the kinetic curves of tumor growth in the study and control groups and amounted to 33 % (p<0.001) indicated the high activity of lomustine. However, the effect of lomustine on intramuscularly transplanted Ehrlich tumor was insignificant (tumor growth inhibition and tumor growth index were <39 % and 68 %, respectively). Lomustine administered orally at a single dose of 50 mg/kg 24 hours after intracranial transplantation of BC HER2+ increased the median survival time up to 30 days in FVB/N mice compared to 21 days in the control group mice (p<0.001). The high therapeutic effect of lomustine in HER2-positive breast cancer mice is likely can be explained by the biological characteristics of this tumor; therefore clinical trials of lomustine for HER2-positive tumors are needed