6 research outputs found
Новые подходы в 3D-моделировании роста in vitro первичных культур злокачественных глиом
Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors.Введение. Заболеваемость глиомами головного мозга прочно занимает лидирующую позицию среди всех опухолей центральной нервной системы – 40–50 % выявленных случаев, более половины из них представлены глиобластомой. Существующие клеточные линии и методы культивирования не отражают всех особенностей трехмерной (3D) организации нативной глиобластомы. Применение темозоломида приводит к развитию лекарственной устойчивости и острого рецидива с последующим плохим клиническим исходом. Развитие резистентности в значительной степени связано с наличием в популяции опухолевых стволовых клеток и внутриопухолевой гетерогенностью. Получение 3D-культур из первичного материала позволит сохранить пул стволовых клеток и специфические для конкретной опухоли особенности.Цель исследования – получить 3D-модель на основе первичных клеточных культур, позволяющую сохранить гетерогенную популяцию и исходный фенотип опухолевых клеток.Материалы и методы. Использованы клетки линии человеческой глиомы U-87MG и первичная клеточная культура GBM002, полученная из операционного материала, с подтвержденным диагнозом глиобластомы. Из клеточных линий были получены нейросферы, рост которых контролировали с помощью автоматической системы для клеточного анализа InCell Analyzer 6000. Для определения содержания клеток CD133+ использовали метод проточной цитометрии. Оценку экспрессии в нейросферах рецепторных тирозинкиназ VEGFR1, VEGFR2 (рецепторы эндотелиального фактора роста 1‑го и 2‑го типов), FGFR2 (рецептор фактора роста фибробластов 2‑го типа) и маркера гипоксии HIF-1α (фактор, индуцируемый гипоксией, 1α) проводили с помощью конфокальной микроскопии.Результаты. Клетки глиобластомы GBM002, выделенные из операционного материала, образовывали нейросферы, при этом повышалось количество клеток CD133+ с 1–2 до 16–19 % по сравнению с двухмерными культурами. При длительном культивировании клеток с нецитотоксическими дозами темозоломида установлено, что такие клетки образуют нейросферы меньшего размера по сравнению с контрольными клетками. Показано, что экспрессия рецепторных тирозинкиназ при культивировании клеток глиобластомы GBM002 в нейросферах отличается от таковой в двухмерных культурах. Установлено, что в нейросферах в значительной степени увеличивается экспрессия FGFR2 и VEGFR1.Заключение. 3D-культивирование первичных культур позволяет получать более гетерогенную популяцию опухолевых клеток, отражающую пространственную неоднородность клеток, повышать пул стволовых клеток и воссоздавать условия гипоксии внутри микроопухолей головного мозга
Экспрессия ростовых факторов и рецепторных тирозинкиназ в клетках первичной опухоли опухолевого тромба у больных почечно-клеточным раком
Objective: to assess the expression and prognostic value of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2) and their receptors VEGFR-1, -2; FGFR-1, -2, as well as platelet-derived growth factor receptors (PDGFR-α, PDGFR-β) in paired samples of primary tumors and tumor thrombi in renal cell carcinoma (RCC).Materials and methods. Expression of VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β was studied in paired surgical samples of primary tumors and tumor thrombi in 25 patients with clear cell RCC pT3a–T4N0–1M0–1 and tumor venous thrombosis by immunohistochemical assay using the appropriate Abcam/Santa Cruz Biotech antibodies from the immunohistochemical staining kit Invitrogen. Expression levels were evaluated by a semi-quantitative method (H-score). The analysis of the correlation between expression levels of VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β and RCC characteristics, as well as evaluation of their influence on the outcome of RCC were performed.Results. VEGF-A, FGF-2, as well as VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β were expressed in the cytoplasm and on the membrane of the primary tumor and tumor thrombus cells in RCC patients. Tumor thrombus cells were characterized by lower expression of VEGFR-1, VEGFR-2, PDGFR-α (p <0.05 for all) and tendency to lower expression of VEGF-A (p = 0.060), FGF-2 (p = 0.046), FGFR-1 (p = 0.077) and FGFR-2 (p = 0.090) compared with primary tumor cells. RCC Furman grade correlated with the expression levels of VEGFR-1 (p = 0.035) and FGFR-1 (p = 0.022) in the primary tumor cells, tumor invasion into venous wall correlated with the expression levels of VEGFR-1 (p = 0.023) and FGFR-2 (p = 0.005) on the thrombus cells. VEGFR-2 overexpression in the primary tumor cells was associated with significant decrease of overall survival (OS) rate (p = 0.011). There was a tendency to OS deterioration in cases with overexpression of VEGFR-2 (p = 0.093) and VEGF-A (p = 0.095) in the tumor thrombus cells. One-year OS in patients with ³2 identified risk factors was 27.3 %, <2 risk factors – 87.5 % (p = 0.004).Conclusion. Tumor thrombus cells in RCC patients expressed VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β less active than the cells of the primary tumor. Overexpression of growth factors and tyrosine kinases correlated with RCC Furman grade and tumor venous wall invasion. Overexpression of VEGFR-2 in both primary tumor and thrombus cells in combination with hypoexpression of VEGF-A in the thrombus negatively influenced on OS.Цель исследования – провести изучение экспрессии и прогностической значимости фактора роста эндотелия сосудов (VEGF-A), фактора роста фибробластов 2 (FGF-2) и их рецепторов VEGFR-1, -2; FGFR-1, -2, а также рецепторов фактора роста тромбоцитарного происхождения (PDGFR-α, PDGFR-β) в клетках парных образцов первичной опухоли и опухолевого тромба у больных почечно-клеточным раком (ПКР).Материалы и методы. Экспрессию VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β изучали в парных операционных образцах опухоли почки и опухолевого тромба 25 больных светлоклеточным ПКР рТ3а–Т4N0–1M0–1, осложненным опухолевым венозным тромбозом, при помощи иммуногистохимического окрашивания с полуколичественной оценкой. Провели анализ корреляции выявленных уровней экспрессии ростовых факторов и рецепторных тирозинкиназ с характеристиками опухолевого процесса и оценку их влияния на исход ПКР.Результаты. В цитоплазме и на мембране клеток первичной опухоли и опухолевого тромба у больных ПКР экспрессировались VEGF-A, FGF-2, а также VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β. Клетки опухолевого тромба характеризовались более низкой экспрессией VEGFR-1, -2, PDGFR-α (р <0,05 для всех) и тенденцией к более низкой экспрессии VEGF-A (p = 0,060), FGF-2 (р = 0,046), FGFR-1 (p = 0,077) и FGFR-2 (p = 0,090) по сравнению с клетками первичной опухоли почки. Была выявлена прямая корреляция между степенью дифференцировки G и уровнями экспрессии VEGFR-1 (р = 0,035) и FGFR-1 (р = 0,022) в клетках первичной опухоли, а также между инвазией опухолевого тромба в венозную стенку и уровнями экспрессии VEGFR-1 (р = 0,023) и FGFR-2 (р = 0,005) на клетках тромба. Было отмечено неблагоприятное влияние на общую выживаемость (ОВ) больных ПКР гиперэкспрессии VEGFR-2 в клетках первичной опухоли (р = 0,011), а также тенденция к снижению ОВ при гиперэкспрессии VEGFR-2 (р = 0,093) и гипоэкспрессии VEGF-A (р = 0,095) в клетках опухолевого тромба. Однолетняя ОВ пациентов с ³2 выделенными факторами риска – 27,3 %, <2 факторами риска – 87,5 % (р = 0,004).Заключение. Клетки опухолевого тромба у больных ПКР экспрессируют VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β менее активно, чем клетки первичной опухоли. Гиперэкспрессия ростовых факторов и тирозинкиназ коррелирует со степенью дифференцировки G и инвазией венозной стенки. Гиперэкспрессия VEGFR-2 в первичной опухоли и тромбе в сочетании с гипоэкспрессией VEGF-A в тромбе ассоциирована со снижением ОВ
New approaches in 3D modeling of in vitro growth of primary cultures of malignant gliomas
Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors
Expression of growth factors and tyrosine kinase receptors in the primary tumor and tumor thrombus cells in patients with renal cell carcinoma
Objective: to assess the expression and prognostic value of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2) and their receptors VEGFR-1, -2; FGFR-1, -2, as well as platelet-derived growth factor receptors (PDGFR-α, PDGFR-β) in paired samples of primary tumors and tumor thrombi in renal cell carcinoma (RCC).Materials and methods. Expression of VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β was studied in paired surgical samples of primary tumors and tumor thrombi in 25 patients with clear cell RCC pT3a–T4N0–1M0–1 and tumor venous thrombosis by immunohistochemical assay using the appropriate Abcam/Santa Cruz Biotech antibodies from the immunohistochemical staining kit Invitrogen. Expression levels were evaluated by a semi-quantitative method (H-score). The analysis of the correlation between expression levels of VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β and RCC characteristics, as well as evaluation of their influence on the outcome of RCC were performed.Results. VEGF-A, FGF-2, as well as VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β were expressed in the cytoplasm and on the membrane of the primary tumor and tumor thrombus cells in RCC patients. Tumor thrombus cells were characterized by lower expression of VEGFR-1, VEGFR-2, PDGFR-α (p <0.05 for all) and tendency to lower expression of VEGF-A (p = 0.060), FGF-2 (p = 0.046), FGFR-1 (p = 0.077) and FGFR-2 (p = 0.090) compared with primary tumor cells. RCC Furman grade correlated with the expression levels of VEGFR-1 (p = 0.035) and FGFR-1 (p = 0.022) in the primary tumor cells, tumor invasion into venous wall correlated with the expression levels of VEGFR-1 (p = 0.023) and FGFR-2 (p = 0.005) on the thrombus cells. VEGFR-2 overexpression in the primary tumor cells was associated with significant decrease of overall survival (OS) rate (p = 0.011). There was a tendency to OS deterioration in cases with overexpression of VEGFR-2 (p = 0.093) and VEGF-A (p = 0.095) in the tumor thrombus cells. One-year OS in patients with ³2 identified risk factors was 27.3 %, <2 risk factors – 87.5 % (p = 0.004).Conclusion. Tumor thrombus cells in RCC patients expressed VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β less active than the cells of the primary tumor. Overexpression of growth factors and tyrosine kinases correlated with RCC Furman grade and tumor venous wall invasion. Overexpression of VEGFR-2 in both primary tumor and thrombus cells in combination with hypoexpression of VEGF-A in the thrombus negatively influenced on OS