Establishment and characterization of new breast and ovarian cancer cell lines as a model for studying cellular plasticity in vitro

Aim: To compare biological properties of primary tumor cells isolated from malignant effusion of cancer patients with the same cells of permanent lines established during their long-term cultivation in vitro and to assess the impact of phenotypic conversion that was caused by changes in their microenvironment on their behavioral characteristics. Materials and Methods: The study was performed on primary cell cultures from pleural effusion or ascites of breast and ovarian cancer and permanent cell lines derived from them, namely permanent ovarian cancer cell line I, permanent ovarian cancer cell line II and permanent breast cancer cell line I. Biological characteristics were studied using standard cell culture methods and immunocytochemical assays. Results: Three new cell lines were established from breast and ovarian cancer and cell morphology, migration activity, the kinetics of growth, colony forming activity in semisolid agar and sensitivity to anticancer drug were examined. These characteristics were compared with those of the primary tumor cells. It has been shown that among the primary tumor cells from malignant effusion, cells with mesenchymal characteristics were the most prevalent. Cultivation of primary cancer cells in vitro leads to a phenotypic change of their population: it becomes more homogeneous in morphology with predominantly epithelial-like cells. Also, later after a number of cell doublings in vitro, the cell population changes to include cells primarily with immunophenotypic properties characteristic of epithelial cells. These changes include increase in number of E-cadherin-positive cells and a decrease in number of vimentin and α- smooth muscle actin-positive cells. It was found that significant changes in expression of epithelial-mesenchymal transition associated proteins in cells during their cultivation in vitro in new microenvironment are accompanied by a rapid change in their sensitivity to anticancer drugs. Conclusions: The new breast and ovarian cancer cell lines were established and characterized. The induction of phenotypic transdifferentiation in malignant cells from pleural effusion and ascites can be an important approach for suppressing the progression of neoplastic process

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

Suppression of tumorigenicity and metastatic potential of melanoma cells by transduction of interferon gene

The aim of this study was to investigate an inhibitory effect of baculovirus-mediated transduction of the murine interferon-beta gene on mouse melanoma in vitro and in vivo. Methods. Studies were performed on B16 mouse melanoma (MM-4 cell line). Transduction, immunocytochemical and tumor cell biology approaches have been used in this study. Results. Transduction of MM-4 cells by the recombinant baculovirus with IFN-beta gene is accompanied by morphological changes of tumor cells, suppression of cell proliferation, significant inhibition of platting efficiency of cells and their colonies formation in semisolid agar. Moreover, transduction of melanoma MM-4 cells by the baculovirus IFN-transgene leads to inhibition of tumorigenicity and metastatic ability of the cells in vivo. The intravenous administration of recombinant baculovirus vector with IFN gene inhibits growth of metastases induced in the lungs of mice by intravenously injected tumor cells. Conclusions. Transduction of mouse melanoma cells by the recombinant baculovirus with murine IFN-beta gene inhibits their proliferative potential, tumorigenicity and metastatic activity.Мета. Вивчення інгібувального ефекту трансдукції клітин меланоми миші рекомбінантним бакуловірусом з геном інтерферону-бета миші in vitro та in vivo. Методи. Дослідження проведено на моделі меланоми миші B16 (клітинна лінія MM-4) з використаннням трансдукції клітин рекомбінантним бакуловірусом, імуноцитохімічного аналізу та методів дослідження біології пухлинної клітини. Результати. Трансдукція клітин ММ-4 рекомбінантним бакуловірусом з геном ІФН-бета миші призводить до змін морфології пухлинних клітин, пригнічення клітинної проліферації, значного інгібування посадкової ефективності та колонієутворювальної активності у напіврідкому агарі. Крім того, трансдукція клітин меланоми ММ-4 бакуловірусом з геном ІФН-бета пригнічує онкогенність і метастатичний потенціал клітин in vivo. Внутрішньовенне введення рекомбінантного бакуловірусного вектора з геном ІФН-бета інгібує ріст метастазів меланоми в легенях мишей, яким внутрішньовенно введено пухлинні клітини. Висновки. Трансдукція клітин меланоми миші рекомбінантним бакуловірусом з геном ІФНбета миші пригнічує їхній проліферативний потенціал, а також онкогенну і метастатичну активність.Цель. Изучение ингибирующего эффекта трансдукции клеток меланомы мыши рекомбинантным бакуловирусом с геном интерферона-бета мыши in vitro и in vivo. Методы. Исследование проведено на модели меланомы мыши B16 (клеточная линия MM-4) с использованием трансдукции клеток рекомбинантным бакуловирусом, иммуноцитохимического анализа и методов исследования биологии опухолевой клетки. Результаты. Трансдукция клеток ММ-4 рекомбинантным бакуловирусом с геном ИФН-бета мыши приводит к изменениям морфологии опухолевых клеток, подавлению клеточной пролиферации, значительному ингибированию посадочной эффективности и колониеобразующей активности в полужидком агаре. Кроме того, трансдукция клеток меланомы ММ-4 бакуловирусом с геном ИФН-бета подавляет онкогенность и метастатический потенциал клеток in vivo. Внутривенное введение рекомбинантного бакуловирусного вектора с геном ИФНбета ингибирует рост метастазов меланомы в легких мышей, которым внутривенно введены опухолевые клетки. Выводы. Трансдукция клеток меланомы мыши рекомбинантным бакуловирусом с геном ИФН-бета мыши подавляет их пролиферативный потенциал, онкогенность и метастатическую активность

Endogenous production of IL-1B by breast cancer cells drives metastasis and colonisation of the bone microenvironment

Background: Breast cancer bone metastases are incurable highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL-1B by tumor cells drives metastasis and growth in bone. Methods: Tumor/stromal IL-B and IL-1R1 expression was assessed in patient samples and effects of the IL-1R antagonist, Anakinra or the IL-1B antibody Canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell-derived IL-1B on bone colonisation and parameters associated with metastasis were measured in MDA-MB-231, MCF7 and T47D cells transfected with IL-1B/control. Results: In tissue samples from >1300 patients with stage II/III breast cancer, IL-1B in tumor cells correlated with relapse in bone (hazard ratio 1.85; 95% CI 1.05-3.26; P=0.02) and other sites (hazard ratio 2.09; 95% CI 1.26-3.48; P=0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or Canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL-1B by tumor cells promoted EMT (altered E-Cadherin, N-Cadherin and G-Catenin), invasion, migration and bone colonisation. Contact between tumor and osteoblasts or bone marrow cells increased IL-1B secretion from all three cell types. IL-1B alone did not stimulate tumor cell proliferation. Instead, IL-1B caused expansion of the bone metastatic niche leading to tumor proliferation. Conclusion: Pharmacological inhibition of IL-1B has potential as a novel treatment for breast cancer metastasis

Risk of hospitalization and death due to bone fractures after breast cancer: a registry-based cohort study

BACKGROUND: Bone fractures may have an impact on prognosis of breast cancer. The long-term risks of bone fracture in breast cancer patients have not been thoroughly studied. METHODS: Poisson regression was used to investigate the incidence of hospitalisation due to bone fracture comparing women with and without breast cancer based on Swedish National registers. Cox regression was used to investigate the risk of being hospitalised with bone fracture, and subsequent risk of death, in a regional cohort of breast cancer patients. RESULTS: For breast cancer patients, the 5-year risk of bone fracture hospitalisation was 4.8% and the 30-day risk of death following a bone fracture hospitalisation was 2.0%. Compared with the general population, breast cancer patients had incidence rate ratios of 1.25 (95% CI: 1.23-1.28) and 1.18 (95% CI: 1.14-1.22) for hospitalisation due to any bone fracture and hip fracture, respectively. These ratios remained significantly increased for 10 years. Comorbidities (Charlson Comorbidity Index 1) were associated with the risk of being hospitalised with bone fracture. Women taking aromatase inhibitors were at an increased risk as compared with women taking tamoxifen (HR=1.48; 95% CI: 0.98-2.22). Breast cancer patients hospitalised for a bone fracture showed a higher risk of death (HR=1.83; 95% CI: 1.50-2.22) compared with those without bone fracture. CONCLUSIONS: Women with a previous breast cancer diagnosis are at an increased risk of hospitalisation due to a bone fracture, particularly if they have other comorbidities.Swedish Research CouncilSwedish Cancer SocietyFORTEAccepte

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

High levels of proinflammatory cytokines tumor necrosis factor alpha, interleukin 1β and interleukin 6 in bone marrow and peripheral blood of breast cancer patients as predictors of relapse

Breast cancer provides a typical example of an inflammation-linked malignant disease. The inflammatory components present in the tumor microenvironment contribute to the further progression of the disease. The present study was conducted to evaluate the correlation between levels of proinflammation cytokines tumor necrosis factor α, interleukin 1β, interleukin 6, C-reactive protein and tumor recurrence in breast cancer patients. Seventy two breast cancer patients with histologically proven diagnosis and 15 healthy donors were enrolled into study. Thirty one patients with progression of the disease and 41 patients with clinical stabilization (conditional remission) were included to “progression” and “remission” groups respectively. This division of breast cancer patients was made during the 3 years study. The levels of proinflammation cytokines – tumor necrosis factor α, interleukin 1β, interleukin 6 and C-reactive protein were revealed. This cytokines in bone marrow and peripheral blood act as a specific microenvironment and was found that the elevated levels of this cytokines were strongly associated with progression of breast cancer. The data showed that most prominent predictive markers of tumor recurrence are high levels of indicated cytokines in combination with other markers (C-reactive protein; disseminated tumor cells in bone marrow). Determination of the high levels of tumor necrosis factor, interleukin 1β, interleukin 6 in bone marrow and peripheral blood of breast cancer patients are important to establish the features of bone marrow microenvironment for prediction of metastasis and correction antitumor therapy