During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm 2 ), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.Cancer metastasis, a multistep process in which cancer cells migrate or flow from the primary tumor site to a distal location, causes over 90% of cancer-related deaths 1,2 . Over 50% patients with colorectal cancer, for example, develop distant metastasis, making the colorectal cancer the second leading cause of cancer deaths in the United States 2 . During metastasis, circulating tumor cells (CTCs) are transported through the blood circulatory system and are subjected to hemodynamic forces 3 . Although it is known that fluid shear-forces resulted from the bloodstream cause destructions of CTCs and only a small fraction of CTCs can survive and generate metastasis 1,4,5 , the effect of circulatory shear flow on the viability and proliferation of CTCs remains elusive.Progress has been made to understand the mechanism of shear stress in the regulation of cancer cells. However, the majority of studies investigate the effects of shear on cells that are immobilized in micro-wells or adhered to microchannels [6][7][8] . The effect of shear on circulating cancer cells in suspension, however, remains less understood. Approaches, such as cone-and-plate viscometer and stirring bath, have been developed to study the effect of shear on cell suspensions [9][10][11] . However, the shear conditions are less physiologically relevant, and thus are marginally effective to evaluate the effect of circulatory shear stress on CTCs. Most importantly, previous studies have mainly focused on cell viability after shear stimulation 6,9,12 , the proliferation of cells that are survived from shear, which plays an important role in the development of secondary tumors, remains unknown.Here, we developed a microfluidic circulatory system to study the effect of shear stress on the viability an...

Wnt11 plays an essential role in gastrointestinal epithelial proliferation, and previous investigations have focused on development and immune responses. However, the roles of how enteric bacteria regulate Wnt11 and how Wnt11 modulates the host response to pathogenic bacteria remain unexplored. This study investigated the effects of Salmonella infection on Wnt activation in intestinal epithelial cells. We found that Wnt11 mRNA and protein expression were elevated after Salmonella colonization. Wnt11 protein secretion in epithelial cells was also elevated after bacterial infection. Furthermore, we demonstrated that pathogenic Salmonella regulated Wnt11 expression and localization in vivo. We found a decrease in Salmonella invasion in cells with Wnt11 overexpression compared with cells with normal Wnt11 level. IL-8 mRNA in Wnt11-transfected cells was low; however, it was enhanced in cells with a low level of Wnt11 expression. Functionally, Wnt11 overexpression inhibited Salmonella-induced apoptosis. AvrA is a known bacterial effector protein that stabilizes β-catenin, the downstream regulator of Wnt signaling, and inhibits bacterially induced intestinal inflammation. We observed that Wnt11 expression, secretion, and transcriptional activity were regulated by Salmonella AvrA. Overall, Wnt11 is involved in the protection of the host intestinal cells by blocking the invasion of pathogenic bacteria, suppressing inflammation, and inhibiting apoptosis. Wnt11 is a novel and important contributor to intestinal homeostasis and host defense.

The tissue inhibitor of metalloproteinase (TIMP) family, including TIMP-2, regulates the activity of multifunctional metalloproteinases in pathogenesis of melanoma. The Wnt/β-catenin pathway is constitutively activated and plays a critical role in melanoma progression. However, the relationship between TIMP-2 expression and β-catenin activity is still unclear. We hypothesize that TIMP-2 over expression inhibits the activation of the Wnt/β-catenin pathway in melanoma cells. Protein expression, distribution, and transcriptional activity of β-catenin were assayed in established stable melanoma cell lines: parental A2058 expressing, A2058 T2-1 over-expressing (T2-1), and A2058 T2R-7 under-expressing (T2R-7) TIMP-2. Compared to T2-1 cells at the basal level, T2R-7 showed significantly lower amount protein and weaker immunofluorescence staining of β-catenin. This regulation is through posttranslational level via ubiquitination. Functionally, proliferation and cell growth were lower in T2R-7 compared to A2058 and T2-1. Lithium treatment was used to mimics activation of the Wnt/β-catenin pathway. In T2R-7 cells under-expressing TIMP2, lithium significantly increased total β-catenin, nuclear β-catenin, and its downstream protein phosphor-c-Myc (S62). Nuclear β-catenin staining was enhanced in T2R-7. Beta-catenin transcriptional activity and cell proliferation were also increased significantly. Axins inhibit β-catenin pathway via GSK-3 β. We further found the ratio of p-GSK-3 β (S9) to β-catenin and protein levels of Axins were significantly lower, whereas downstream Wnt 11 was high in T2R-7 treated with lithium. Collectively, the high level of TIMP-2 protein inhibits the activation of the Wnt/β-catenin pathway, thus suppressing proliferation. Insights in the molecular mechanisms of TIMP-2 may provide promising opportunities for anti-proliferative therapeutic intervention.