Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells

Background: 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. Aim: The aim of the work was to study the crosstalk between tumor cells and stromal cell components using noncontact co-cultivation in vitro system. Materials and Methods: 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. Results: 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. Conclusion: 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. Key Words: breast cancer, epithelial-mesenchymal transition, microenvironment, bone marrow, co-cultivation

Nonchromosomal cytogenetic analysis of mammal somatic cells

The mutational events that take place in mammalian somatic cells influenced with different endogenous and exogenous factors are presented in this review. The nonchromosomal method of research allows taking into account the complex cell characteristics without time-consuming analysis of the chromosomes as such. As a result, the information can be obtained about the mitotic (phases of mitosis, the number of nuclei per cell, micronuclei, pathology of mitosis) and vital (mitotic index, apoptosis) cell statuses, as well as about the state of chromosomal integrity (the presence of nucleoplasmic bridges, nucleus protrusions, chromosome fragmentation, micronuclei). Depending on the material studied (erythrocytes and lymphocytes of peripheral blood, buccal cells, permanent cell lines etc.), a complex of cytogenetic characteristics can be selected for each case which is the most informative for determination of the mutational spectra in mammalian somatic cells

Disseminated tumor cells and enhanced level of some cytokines in bone marrow and peripheral blood of breast cancer patients as predictive factors of tumor progression

Background: In recent years, the presence of disseminated tumor cells (DTC) in bone marrow (BM) of patients with breast cancer (BC) is considered as an important clinical feature of the distribution process. However, relapse often occurs in spite of the negative results of the bone marrow cytology. This suggests the need to find additional signs of the possibility for predict the recurrence. Aim: to detect DTC in BM and determinate cytokine status of peripheral blood (PB) and BM of primary BC patients for prognosis of tumor recurrence. Patients and methods: 72 BC patients with histologically proven diagnosis were enrolled into study. 31 patients with progression of disease and 41 patients with clinical stabilization (conditional remission) were included to “progression” and “remission” group respectively. This division of BC patients was conditional and was made during the 3 years study. The presence of DTC in BM was detected by immunocytochemical analysis. Plasma levels of TNF-α, M-CSF, IFN-α were defined by bioassay tests. Plasma levels of IL-6, TGF-β1 and VEGF were determined by ELISA. BM and PB BC patients were obtained before treatment. Results: In our study DTC in samples of BM were detected in 50% BC patients of progression group. It was found that most significant addition markers of tumor progression with presence of DTC in BM are the levels of cytokines such as TNF in BM and PB, CSF-1 and IL-6 in PB and endogenous IFN in BM and PB of BC patients. In patients of disease “progression” group the levels of TNF in BM were increased by 45.8% (p < 0.01), the levels of CSF-1 and IL-6 in PB were increased more than by 70–80% (p < 0.05). Conclusion: Comprehensive detection DTC in BM and identification the level of TNF, IFN, CSF-1, IL-6 in PB and BM of BC patients could be one of the ways for prognosis the metastatic process and correction antitumor individualized therapy

Broad-spectrum Anti-tumor and Anti-metastatic DNA Vaccine Based on p62- encoding Vector

Abstract Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti- cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in three models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), and S37 sarcoma. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti- metastatic vaccine feasible for further development and clinical trials