Selectins as mediators of lung metastasis

Lung metastasis remains the major cause of cancer related mortality in patients with breast, gastrointestinal, sarcoma, melanoma and kidney cancer. Here we characterize the expression of selectins during metastatic lung colonization and analyzed their function in the formation of pulmonary metastasis. E-selectin, together with VCAM-1, were detected 6h after the microvascular arrest of tumor cells indicating an inflammatory activation of the local endothelial cells. No E-selectin expression was detected in pre-metastatic lungs of mice carrying primary tumors. P- and L-selectin were present during initiating steps of lung colonization and correlated with the recruitment of platelets and leukocytes to metastatic tumor cells. Experimental metastasis was significantly reduced in the absence of P- or L-selectin while no attenuation of metastasis was observed in E-selectin-deficient mice. Collectively, selectins are upregulated within the metastatic microenvironment of tumor cells and the formation of a permissive metastatic microenvironment is facilitated by P- and L-selectin mediated interactions between tumor cells and blood components. E-selectin does not affect metastatic initiation in the lung tissue and its expression rather indicates a local activation of lung microvascular endothelial cell

P-selectin mediates metastatic progression through binding to sulfatides on tumor cells

Hematogenous carcinoma metastasis is associated with tumor cell emboli formation, which is now known to be facilitated by selectins. P-selectin-mediated interactions of platelets with cancer cells are based mostly on mucin- and glycosaminoglycan-type selectin ligands. We previously showed that mouse colon carcinoma cells (MC-38) carry P-selectin ligands of nonmucin origin, which were not identified. Here we show that P-selectin ligands recognized on MC-38 cells are sulfated glycolipids, thereby facilitating experimental metastasis in a syngeneic mouse model. Metabolic inhibition of sulfation by incubation of cells with sodium chlorate almost completely abrogated P-selectin binding. Metabolic labeling of MC-38 cells with (35)S sulfate revealed only a single band as detected by high-performance thin layer chromatography analysis of a total lipid extract. Matrix-assisted laser desorption/ionization tandem time-of-flight/time-of-flight analysis (MALDI-TOF-TOF) analysis of the purified sulfate-containing lipid fraction identified the selectin ligand to be a sulfated galactosylceramide SM4 (HSO(3)-3Galbeta-1Cer). Modulation of glycolipid biosynthesis in MC-38 cells altered P-selectin binding, thereby confirming sulfoglycolipids to be major P-selectin ligands. In addition, P-selectin was also found to recognize lactosylceramide sulfate SM3 (HSO(3)-3Galbeta-4Glcbeta-1Cer) and gangliotriaosylceramide sulfate SM2 [GalNAcbeta-4(HSO(3)-3)Galbeta-4Glcbeta-1Cer] in human hepatoma cells. Finally, the enzymatic removal of sulfation from the cell surface of MC-38 cells resulted in decreased P-selectin binding and led to attenuation of metastasis. Thus, SM4 sulfatide serves as a native ligand for P-selectin contributing to cell-cell interactions and to facilitation of metastasi

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

Inhibition of pulmonary metastasis in a human MT3 breast cancer xenograft model by dual liposomes preventing intravasal fibrin clot formation

International audienceThe process of metastasis formation in cancer is not completely understood and is the main reason cancer therapies fail. Previously, we showed that dual liposomes simultaneously containing the hemostatic inhibitor, dipyridamole and the anticancer drug, perifosine potently inhibited metastasis, causing a 90% reduction in the number of lung metastases in a murine experimental metastasis model. To gain deeper insight into the mechanisms leading to the inhibition of metastasis by these dual liposomes, in the present study, the development of metastases by MT3 breast cancer cells in a mouse xenograft model was analyzed in more detail with regard to tumor cell settlement and metastatic growth. We found that the development of lung metastases by MT3 tumor cells is essentially dependent on the formation of fibrin clots as a precondition for the pulmonary arrest of tumor cells and the subsequent intravascular expansion of micrometastases before their invasion into the surrounding tissue

In vivo tumor cell adhesion in the pulmonary microvasculature is exclusively mediated by tumor cell - endothelial cell interaction

<p>Abstract</p> <p>Background</p> <p>Metastasis formation is the leading cause of death among colon cancer patients. We established a new in-situ model of in vivo microscopy of the lung to analyse initiating events of metastatic tumor cell adhesion within this typical metastatic target of colon cancer.</p> <p>Methods</p> <p>Anaesthetized CD rats were mechanically ventilated and 10⁶human HT-29LMM and T84 colon cancer cells were injected intracardially as single cell suspensions. Quantitative in vivo microscopy of the lung was performed in 10 minute intervals for a total of 40 minutes beginning with the time of injection.</p> <p>Results</p> <p>After vehicle treatment of HT-29LMM controls 15.2 ± 5.3; 14.2 ± 7.5; 11.4 ± 5.5; and 15.4 ± 6.5 cells/20 microscopic fields were found adherent within the pulmonary microvasculature in each 10 minute interval. Similar numbers were found after injection of the lung metastasis derived T84 cell line and after treatment of HT-29LMM with unspecific mouse control-IgG. Subsequently, HT-29LMM cells were treated with function blocking antibodies against β1-, β4-, and αv-integrins wich also did not impair tumor cell adhesion in the lung. In contrast, after hydrolization of sialylated glycoproteins on the cells' surface by neuraminidase, we observed impairment of tumor cell adhesion by more than 50% (p < 0.05). The same degree of impairment was achieved by inhibition of P- and L-selectins via animal treatment with fucoidan (p < 0.05) and also by inhibition of the Thomson-Friedenreich (TF)-antigen (p < 0.05).</p> <p>Conclusions</p> <p>These results demonstrate that the initial colon cancer cell adhesion in the capillaries of the lung is predominantly mediated by tumor cell - endothelial cell interactions, possibly supported by platelets. In contrast to reports of earlier studies that metastatic tumor cell adhesion occurs through integrin mediated binding of extracellular matrix proteins in liver, in the lung, the continuously lined endothelium appears to be specifically targeted by circulating tumor cells.</p

A multi-targeted approach to suppress tumor-promoting inflammation

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes

Deletion of L-Selectin Increases Atherosclerosis Development in ApoE−/− Mice

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel−/−) animals were crossed with mice lacking Apolipoprotein E (ApoE−/−). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE−/− L-sel−/−) mice and the corresponding ApoE−/− controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE−/− L-sel−/− mice as compared to ApoE−/− controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE−/− L-sel−/− animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE−/− L-sel−/− and ApoE−/− mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE−/− L-sel−/− as compared to ApoE−/− mice. Leukocyte rolling on lesions in the aorta was similar in ApoE−/− L-sel−/− and ApoE−/− animals. ApoE−/− L-sel−/− mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE−/− controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis