Studies of the Tumor-Vasculature Interface : Role of TGF-beta 1-induced Epithelial to Mesenchymal Transition

Tumor metastasis is a complex multistep process. Among key steps that occur during metastatic spread are acquisition of tumor cell motility, intravasation of tumor cells into blood or lymphatic vessels and extravasation of tumor cells at distal sites. However, the precise mechanisms that govern these metastatic steps remain elusive. This thesis aimed to bridge the fields of tumor and vascular biology to provide new insights into the metastatic process. Results are presented indicating a role of the cytokine transforming growth factor beta (TGF-β) in activating breast cancer cells for dissemination through the lymphatic system through re-activation of a latent development process termed epithelial to mesenchymal transition (EMT). Furthermore, essential roles of the coxsackie-and adenovirus receptor (CAR) for lymph vessel development, and the sphingosine-1-phosphate receptor (S1PR1) for blood vessel stabilization are presented. We expect the findings to have impact on our understanding of the interface between tumor and vascular biology and to influence future strategies to target cancer metastasis. In paper I, we present data identifying an essential role of CAR for normal development of lymphatic vessels in the mouse. We show that genetic deletion of the CAR gene (Cxadr) from E12.5 during mouse development leads to subcutaneous edema, hemorrhage and embryonic death. The lymphatic vessels in CAR-deficient mice were dilated and structurally abnormal with the presence of gaps and holes at lymphatic endothelial cell-cell junctions. In addition, blood-filled lymphatics were observed in CAR-deficient mice suggesting an incomplete separation between the blood and lymphatic vascular systems. The data demonstrate that CAR plays a crucial role in the development of lymphatic vasculature in mice through formation of lymphatic endothelial cell-cell junctions. In paper II, we demonstrate that S1PR1 plays critical role in suppressing angiogenesis and promoting vascular stability during mouse development. S1PR1 signaling promotes cell-cell adhesion and prevents sprouting angiogenesis whereas S1PR1-deficiency leads to hypersprouting angiogenesis. These data suggest that S1PR1 signaling might protect developing blood vessels from abnormal angiogenic signals through promotion of vascular stability. In paper III, we show that TGF-β-induced EMT promotes chemotactic migration of tumor cells through the lymphatic system by mediating crosstalk between tumor cells and lymphatic endothelial cells through the chemokine receptor 7 (CCR7) and its chemokine ligand, CCL21. Reversal of EMT process through p38 MAPK inhibition inhibited tumor cell invasion in vitro and migration towards the lymphatics in vivo suggesting that p38 MAPK inhibition may be a useful therapeutic approach to inhibit tumor cell dissemination through the lymphatic system. In paper IV, we describe development of a novel co-culture system to study tumor cell migration and interaction with lymphatic endothelial cells within a 3-dimensional matrix component. This assay allows manipulation of tumor properties or matrix components and can be used as a platform to screen for pharmacological agents which inhibit tumor-endothelial interactions

Essential Role of the Coxsackie - and Adenovirus Receptor (CAR) in Development of the Lymphatic System in Mice.

Peer reviewe

Association of maternal Vitamin D status with glucose tolerance and caesarean section in a multi-ethnic Asian cohort: the growing up in Singapore towards healthy outcomes study

10.1371/journal.pone.0142239PLoS ONE10111-16GUSTO (Growing up towards Healthy Outcomes

Association between pre-biologic T2-biomaker combinations and response to biologics in patients with severe asthma

Funding This study was conducted by the Observational and Pragmatic Research Institute (OPRI) Pte Ltd and was partially funded by Optimum Patient Care Global (OPCG) and AstraZeneca Ltd. No funding was received by the OPRI for its contribution. The International Severe Asthma Registry (ISAR) is operated by OPCG and co-funded by OPCG and AstraZenecaPeer reviewe

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Validation of the Children's Eating Behavior Questionnaire in 3 year old children of a multi-ethnic Asian population: The GUSTO cohort study

APPETITE113100-10

Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking αVβ8-TGFβ signaling in the brain.

Vascular development of the central nervous system and blood-brain barrier (BBB) induction are closely linked processes. The role of factors that promote endothelial sprouting and vascular leak, such as vascular endothelial growth factor A, are well described, but the factors that suppress angiogenic sprouting and their impact on the BBB are poorly understood. Here, we show that integrin αVβ8 activates angiosuppressive TGFβ gradients in the brain, which inhibit endothelial cell sprouting. Loss of αVβ8 in the brain or downstream TGFβ1-TGFBR2-ALK5-Smad3 signaling in endothelial cells increases vascular sprouting, branching and proliferation, leading to vascular dysplasia and hemorrhage. Importantly, BBB function in Itgb8 mutants is intact during early stages of vascular dysgenesis before hemorrhage. By contrast, Pdgfb(ret/ret) mice, which exhibit severe BBB disruption and vascular leak due to pericyte deficiency, have comparatively normal vascular morphogenesis and do not exhibit brain hemorrhage. Our data therefore suggest that abnormal vascular sprouting and patterning, not BBB dysfunction, underlie developmental cerebral hemorrhage

The proportion of embryos with macroscopic phenotypic changes following tamoxifen administration at E12.5.

<p>Embryonic day (E) indicate day of analysis. The morning and afternoon of E16.5 is indicated as am and pm, respectively. Ctrl and cKO are tamoxifen-treated embryos with genotypes F/F and F/F;Cre, respectively. Animals with macroscopic phenotypic changes are indicated as number of animals (n) as well as % of animals. The total number of embryos analyzed is indicated within brackets. Phenotypic changes include edema, hemorrhage and reduced crown-rump length.</p