Overexpression of adaptor protein Ruk/CIN85 in mouse breast adenocarcinoma 4T1 cells induces an increased migration rate and invasion potential

Aim. To study the effect of adaptor protein Ruk/CIN85 overexpression on the dynamics of migration and Matrigel invasion as well as transendothelial migration of murine 4T1 breast adenocarcinoma cells. Methods. Dynamics of 4T1 cells migration/invasion was monitored in real time using the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16. Transendothelial migration (TEM) of 4T1 cells was performed through the layer of primary mouse lung endothelial cells seeded on gelatin-coated 24-well transwell inserts (8-μm pores).The two-tailed Student’s t-test for unequal variances was used for statistical analysis. Results. Ruk/CIN85-overexpression in 4T1 cells are indices a significantly increased motility, Matrigel invasiveness and migration through endothelial cells layer. Conclusions. The Ruk/CIN85 adaptor protein may play a potential role in the control of metastasis in vivo.Мета. Дослідити вплив надекспресії адаптерного протеїну Ruk/CIN85 на динаміку міграції й інвазії через Матригель, а також на ефективність трансендотеліальної міграції клітин аденокарциноми молочної залози миші лінії 4Т1. Методи. Динаміку міграції/інвазії клітин 4Т1 аналізували в режимі реального часу за допомогою приладу XCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument, оснащеного імпедансним планшетом CIM-plate 16. Трансендотеліальну міграцію (ТЕМ) клітин 4Т1 здійснювали через шар первинних ендотеліоцитів легені миші, висіяних на мембрану (розмір пор 8 μм) камери Бойдена. Для статистичного аналізу використовували двовибірковий t-тест Ст’юдента для незалежних вибірок з нерівними дисперсіями. Результати. Встановлено, що надекспресія Ruk/CIN85 у клітинах лінії 4Т1 супроводжується значним зростанням рухливості, здатності до інвазії через Матригель та шар ендотеліальних клітин. Висновки. Отримані результати вказують на потенційну роль адаптерного протеїну Ruk/CIN85 у контролі метастазування in vivo.Цель. Исследовать влияние сверхэкспрессии адаптерного протеина Ruk/CIN85 на динамику миграции и инвазии через Матригель, а также на эффективность трансэндотелиальной миграции клеток аденокарциномы молочной железы мыши линии 4Т1. Методы. Динамику миграции/инвазии клеток 4Т1 анализировали в режиме реального времени с помощью прибора XCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument, оснащенного импедансным планшетом CIM-plate 16. Трансэндотелиальную миграцию (ТЭМ) клеток 4Т1 осуществляли через слой первичных эндотелиоцитов легкого мыши, высеянных на мембрану (размер пор 8 μм) камеры Бойдена. Для статистического анализа использовали двухвыборочный t-тест Стьюдента для независимых выборок с неравными дисперсиями. Результаты. Установлено, что сверхэкспрессия адаптерного протеина Ruk/CIN85 в клетках линии 4Т1 сопровождается значительным ростом подвижности, способности к инвазии через Матригель и слой эндотелиальных клеток. Выводы. Полученные результаты указывают на потенциальную роль адаптерного протеина Ruk/CIN85 в контроле метастазирования in vivo

Expression dynamics of metalloproteinases during mandibular bone formation: association with Myb transcription factor

As the dentition forms and becomes functional, the alveolar bone is remodelled. Metalloproteinases are known to contribute to this process, but new regulators are emerging and their contextualization is challenging. This applies to Myb, a transcription factor recently reported to be involved in bone development and regeneration. The regulatory effect of Myb on Mmps expression has mostly been investigated in tumorigenesis, where Myb impacted the expression of Mmp1, Mmp2, Mmp7, and Mmp9. The aim of this investigation was to evaluate the regulatory influence of the Myb on Mmps gene expression, impacting osteogenesis and mandibular bone formation. For that purpose, knock-out mouse model was used. Gene expression of bone-related Mmps and the key osteoblastic transcription factors Runx2 and Sp7 was analysed in Myb knock-out mice mandibles at the survival limit. Out of the metalloproteinases under study, Mmp13 was significantly downregulated. The impact of Myb on the expression of Mmp13 was confirmed by the overexpression of Myb in calvarial-derived cells causing upregulation of Mmp13. Expression of Mmp13 in the context of other Mmps during mandibular/alveolar bone development was followed in vivo along with Myb, Sp7 and Runx2. The most significant changes were observed in the expression of Mmp9 and Mmp13. These MMPs and MYB were further localized in situ by immunohistochemistry and were identified in pre/osteoblastic cells as well as in pre/osteocytes. In conclusion, these results provide a comprehensive insight into the expression dynamics of bone related Mmps during mandibular/alveolar bone formation and point to Myb as another potential regulator of Mmp13

Monocyte induction of e-selectin-mediated endothelial activation releases VE-Cadherin junctions to promote tumor cell extravasation in the metastasis cascade

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell-activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin-deficient mice. Tumor cell-derived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin-dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Cancer Res; 76(18); 5302-12. ©2016 AACR

CCL2 is a vascular permeability factor inducing CCR2-dependent endothelial retraction during lung metastasis.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2 ec KO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2 ec KO and Ccr2 fl / fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2 ec KO and Ccr2 fl / fl littermates the absence of vascular permeability induction was observed only in Ccr2 ec KO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. Implications: The findings provide mechanistic insight into how CCL2–CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis

Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist – an initial in vitro study

Josef Skopalik,1 Katerina Polakova,2 Marketa Havrdova,2 Ivan Justan,1 Massimiliano Magro,3 David Milde,2 Lucia Knopfova,4 Jan Smarda,4 Helena Polakova,1 Eva Gabrielova,5 Fabio Vianello,2,3 Jaroslav Michalek,1 Radek Zboril21Department of Pharmacology, Masaryk University, Brno, Czech Republic; 2Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry and Analytical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic; 3Department of Comparative Biomedicine and Food Science, University of Padua, Padova, Italy; 4Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; 5Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky University, Olomouc, Czech RepublicObjective: Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1–100×106 cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles – SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system.Methods: MSCs from rat and human donors were isolated, and then incubated at different concentrations (10–200 µg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied.Results: SAMNs do not affect MSC viability if the concentration does not exceed 100 µg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days.Conclusion: Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.Keywords: mesenchymal stromal cells, stem cell tracking, magnetic resonance imaging, superparamagnetic iron oxide nanoparticles, stem cell labellin

Transcription factor c-Myb inhibits breast cancer lung metastasis by suppression of tumor cell seeding

Metastasis accounts for most of cancer-related deaths. Paracrine signaling between tumor cells and the stroma induces changes in the tumor microenvironment required for metastasis. Transcription factor c-Myb was associated with breast cancer (BC) progression but its role in metastasis remains unclear. Here we show that increased c-Myb expression in BC cells inhibits spontaneous lung metastasis through impaired tumor cell extravasation. On contrary, BC cells with increased lung metastatic capacity exhibited low c-Myb levels. We identified a specific inflammatory signature, including Ccl2 chemokine, that was expressed in lung metastatic cells but was suppressed in tumor cells with higher c-Myb levels. Tumor cell-derived Ccl2 expression facilitated lung metastasis and rescued trans-endothelial migration of c-Myb overexpressing cells. Clinical data show that the identified inflammatory signature, together with a MYB expression, predicts lung metastasis relapse in BC patients. These results demonstrate that the c-Myb-regulated transcriptional program in BCs results in a blunted inflammatory response and consequently suppresses lung metastasis.Oncogene advance online publication, 30 October 2017; doi:10.1038/onc.2017.392