Bezdenezhnykh N, Semesiuk N, Lykhova O, Zhylchuk V, Kudryavets Y
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine.
Rivne Region Oncology Hospital, Rivne 33001, Ukraine.
Exp Oncol. 2014 Jun;36(2):72-8.
Cell and tissue homeostasis results from the dynamic balance of cell - cell and cell - extracellular component crosstalk that regulates proliferation, differentiation, and apoptosis of cells as well as secretion and activation of soluble factors and/or deposition of extracellular matrix (ECM) components.
The aim of the work was to study the crosstalk between tumor cells and stromal cell components using noncontact co-cultivation in vitro system.
Human and rat breast cancer (BC) cell lines, normal human fibroblasts (NHF) and endothelial cells, and aspirates of bone marrow (BM) of BC patients with different clinical course of the disease (groups "Remission" (BM-R) and "Progression" (BM-P)) were used in noncontact co-cultivation system in vitro. The cell growth, expression of epithelial-mesenchymal transition (EMT) and tumor stem cell markers (E-cadherin, vimentin, CD44), Ki-67, p21 and Slug were investigated using immunocytochemical analysis.
Analysis of expression of E- and N-cadherin, vimentin and Slug in BC cells has shown that T-47D and MRS-T5 cells possess mesenchymal phenotype, while MCF-7 and MRS cells possess mostly epithelial phenotype with a part of cells with mesenchymal patterns. Upon noncontact co-cultivation of fibroblasts with Т-47D or MRS-Т5 cells, BC cells acquired higher proliferative activity compared to the control cells (р < 0.05) or MCF-7 and MRS cells co-cultivated with fibroblasts. Upon noncontact co-cultivation of Т-47D cells with normal fibroblasts and BM cells from BC patients from group "Progression" there were observed increased quantity of CD44(+) Т-47D cells (by 26%), decreased quantity of Е-cadherin(+) Т-47D cells, and appearance of vimentin-positive cells. In co-cultivation variant Т-47D + NHF + BM-R ("Remission") the quantity of CD44(+) Т-47D cells significantly decreased (р < 0.005) and E-cadherin expression remained unaltered compared to control cells. At the same time, in NHF cell population (co-cultivation variant Т-47D + NHF + BM-P) there was detected significant increase of quantity of р21+-cells (р < 0.005), cytoplasmic localization of p21, and nuclear localization of Slug. Expression of vimentin did not alter in any variant of co-cultivation.
The new integration cell system for investigation of the mechanisms of interaction between tumor cells and the tumor microenvironment in vitro was developed. The significant changes in proliferative activity of TC dependently on its -ЕМT-status were detected after their interaction with fibroblasts and endothelial cells in noncontact co-cultivation system. BM cells of BC patients had different modifying influence on TC dependent on clinical BC course. The activation of ЕМT program was revealed in TC upon noncontact co-cultivation with BM cells of BC patients with progression of the disease.
细胞和组织的稳态源于细胞 - 细胞及细胞 - 细胞外成分相互作用的动态平衡，这种平衡调节细胞的增殖、分化和凋亡，以及可溶性因子的分泌与激活和/或细胞外基质（ECM）成分的沉积。
本研究旨在利用体外非接触共培养系统，研究肿瘤细胞与基质细胞成分之间的相互作用。
将人及大鼠乳腺癌（BC）细胞系、正常人成纤维细胞（NHF）和内皮细胞，以及患有不同临床病程的BC患者的骨髓（BM）抽吸物（“缓解期”（BM - R）组和“进展期”（BM - P）组）用于体外非接触共培养系统。采用免疫细胞化学分析法研究细胞生长、上皮 - 间质转化（EMT）和肿瘤干细胞标志物（E - 钙黏蛋白、波形蛋白、CD44）、Ki - 67、p21和Slug的表达。
对BC细胞中E - 钙黏蛋白、N - 钙黏蛋白、波形蛋白和Slug表达的分析表明，T - 47D和MRS - T5细胞具有间充质表型，而MCF - 7和MRS细胞主要具有上皮表型，部分细胞具有间充质模式。在成纤维细胞与T - 47D或MRS - T5细胞非接触共培养时，与对照细胞（p < 0.05）或与成纤维细胞共培养的MCF - 7和MRS细胞相比，BC细胞获得了更高的增殖活性。在T - 47D细胞与正常成纤维细胞以及“进展期”BC患者的BM细胞非接触共培养时，观察到CD44(+) T - 47D细胞数量增加（增加26%），E - 钙黏蛋白(+) T - 47D细胞数量减少，并且出现波形蛋白阳性细胞。在共培养变体T - 47D + NHF + BM - R（“缓解期”）中，与对照细胞相比，CD44(+) T - 47D细胞数量显著减少（p < 0.005），E - 钙黏蛋白表达未改变。同时，在NHF细胞群体中（共培养变体T - 47D + NHF + BM - P），检测到p21+细胞数量显著增加（p < 0.005），p21的细胞质定位以及Slug的核定位。在任何共培养变体中波形蛋白的表达均未改变。
开发了一种用于体外研究肿瘤细胞与肿瘤微环境相互作用机制的新型整合细胞系统。在非接触共培养系统中，肿瘤细胞（TC）与成纤维细胞和内皮细胞相互作用后，其增殖活性根据其EMT状态发生了显著变化。BC患者的BM细胞根据BC临床病程对TC具有不同的调节作用。在与疾病进展期BC患者的BM细胞非接触共培养时，TC中EMT程序被激活。
