Tumor Cell Arrest and Adhesion in the Microcirculation

微循环中的肿瘤细胞阻滞和粘附

• 批准号: 8471667
• 负责人: BINGMEI M. FU
• 金额: $ 14.33万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471667
• 关键词: Adhesions; Area; Award; Binding; Biochemical; Biological Assay; Biomedical Engineering; Blocking Antibodies; Blood Vessels; Blood capillaries; Brain; Breast; Breast Cancer Cell; CD34 gene; Cancer Biology; Cardiovascular system; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cells; Cellular biology; Collaborations; Communities; Confocal Microscopy; Cultured Cells; Cyclic AMP; Cytoskeleton; Data; Development; Disease; Distant; Drug Design; Educational process of instructing; Endothelial Cells; Engineering; Epithelial Cells; Extracellular Matrix; Fluorescence; Funding Agency; Goals; Grant; Human; Image; In Vitro; Inflammatory; Integrins; Intercellular adhesion molecule 1; Journals; Kidney; Kidney Glomerulus; Knowledge; L-Selectin; Laboratories; Language; Lead; Liver; Location; Lung; Malignant - descriptor; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Measures; Mechanics; Memorial Sloan-Kettering Cancer Center; Mesentery; Methods; Microcirculation; Microvascular Permeability; Molecular; Molecular Biology; Molecular and Cellular Biology; Mucin-1 Staining Method; Muscle; NG-Nitroarginine Methyl Ester; Neoplasm Circulating Cells; Neoplasm Metastasis; Nitric Oxide; Non-Malignant; Oncogenes; Organ; P-Selectin; P-selectin ligand protein; Paper; Pathology; Perfusion; Permeability; Physiological; Physiology; Play; Production; Proteins; Publications; Publishing; Radial; Rattus; Reagent; Research; Role; Shapes; Signal Pathway; Skeleton; Stress; Stretching; Students; Testing; TimeLine; Training; United States National Institutes of Health; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factors; Video Microscopy; Work; anticancer research; base; bone; cancer cell; capillary; career; cell motility; chemokine; combat; cytokine; experience; graduate student; human NOS3 protein; improved; in vivo; inhibitor/antagonist; laminin-5; monolayer; neoplastic cell; omega-N-Methylarginine; pressure; prevent; professor; public health relevance; receptor; research study; shear stress; simulation; skills; therapeutic target; traditional therapy; tumor; undergraduate student; venule

DESCRIPTION (provided by applicant): It is widely known that circulating tumor cells arrest in the microvasculature, but this arrest is not random. For example, breast cancer cells preferentially arrest in the small blood vessels of the lungs, liver, brain and bones. The underlying mechanisms responsible for this preferential arrest of breast cancer cells in distant organs are not well understood. The long-term goal of our research is to elucidate the relationships between microcirculation-induced mechanical factors, microvascular permeability (vascular integrity), cell adhesion molecules, nitric oxide and cytokines, and tumor metastasis in intact microvessels. The objective of this project is to investigate the relationships between localized shear rates and stresses in curved/stretched microvessels, VEGF (vascular endothelial growth factor)-induced microvascular hyperpermeability, and mammary tumor cell arrest and adhesion in intact microvessels. On the basis of our preliminary studies, we shall use a newly developed in vivo single vessel perfusion/bending method that can create non-uniformly distributed shear rates/stresses along the vessel wall to test two hypotheses: 1) Tumor cells prefer to arrest at the locations of higher shear rates/stresses and shear rate/stress gradients in the post-capillary venules of microvasculature. The higher shear rates/stresses and shear rate/stress gradients activate the endothelial cells and the tumor cells (specifically, activate cell adhesion molecules and endothelial nitric oxide synthase) to increase the binding of tumor cells to the vessel wall and to increase the accumulation of tumor cells; 2) Tumor cells prefer to arrest in the microvessel with the increased permeability. The increased tumor cell adhesion to the microvessel wall with increased permeability is partially due to the radial pressure gradient that drives the cells towards the wall. These ideas will be explored using a combination of physiological, biochemical, mathematical and imaging approaches. Specific aims are: 1) use quantitative fluorescence video and confocal microscopy to determine the adhesion rates of normal, non-malignant (MCF-10A), and malignant (AU-565) breast epithelial cells in straight and curved/stretched microvessels on rat mesentery under known bulk flow rates and a) under conditions of normal and increased permeability by VEGF, b) after pretreatment with the blocking antibodies to endothelial cell adhesion molecules, c) after pretreatment with the blocking antibodies to tumor cell adhesion molecules and d) after pretreatment with eNOS inhibitors to microvessel endothelial cells; 2) use filter-based adhesion/transmigration assays to determine the adhesion/transmigration rates of above cells to/across cultured cell monolayers of microvascular endothelial cells isolated from the lung, brain, kidney and muscle under the same conditions as in Aim 1; 3) use fluorescence video and confocal microscopy to quantify the nitric oxide production in straight and curved/stretched microvessels under various bulk flow rates and under the same conditions a and d in Aim 1, and in cultured cell monolayer of lung and brain, kidney glomerulus and skeleton muscle microvascular endothelial cells under the same conditions a and d in Aim 1; and 4) quantify the shear rate, shear stress, normal stress (pressure), velocity and vorticity profiles by numerical simulation in the straight and curved/stretched microvessels under known bulk flow rates and under the conditions of normal and increased permeability by VEGF.

描述（由申请人提供）：众所周知，循环肿瘤细胞在微脉管系统中停滞，但这种停滞不是随机的。例如，乳腺癌细胞优先停留在肺、肝、脑和骨骼的小血管中。乳腺癌细胞在远处器官中优先停滞的潜在机制尚不清楚。我们研究的长期目标是阐明微循环诱导的机械因素、微血管通透性（血管完整性）、细胞粘附分子、一氧化氮和细胞因子以及完整微血管中肿瘤转移之间的关系。该项目的目的是研究弯曲/拉伸微血管中局部剪切速率和应力、VEGF（血管内皮生长因子）诱导的微血管通透性过高以及完整微血管中乳腺肿瘤细胞停滞和粘附之间的关系。在我们初步研究的基础上，我们将使用一种新开发的体内单血管灌注/弯曲方法，该方法可以沿血管壁产生非均匀分布的剪切速率/应力来测试两个假设：1）肿瘤细胞更喜欢停留在微脉管系统毛细血管后微静脉中较高剪切速率/应力和剪切速率/应力梯度的位置。较高的剪切速率/应力和剪切速率/应力梯度激活内皮细胞和肿瘤细胞（具体来说，激活细胞粘附分子和内皮一氧化氮合酶），增加肿瘤细胞与血管壁的结合，增加肿瘤细胞的积累； 2）肿瘤细胞更喜欢停留在通透性增加的微血管中。肿瘤细胞对微血管壁的粘附增加且渗透性增加，部分原因是驱动细胞朝向血管壁的径向压力梯度。这些想法将结合生理、生化、数学和成像方法进行探索。具体目标是：1) 使用定量荧光视频和共聚焦显微镜来确定大鼠肠系膜上直和弯曲/拉伸微血管中正常、非恶性 (MCF-10A) 和恶性 (AU-565) 乳腺上皮细胞的粘附率，在已知的总体流速下和 a) 在正常和 VEGF 通透性增加的条件下，b) 在用阻断预处理后 抗内皮细胞粘附分子的抗体，c)用抗肿瘤细胞粘附分子的封闭抗体预处理后和d)用eNOS抑制剂预处理微血管内皮细胞后； 2)使用基于过滤器的粘附/迁移测定来确定上述细胞在与目标1相同的条件下与从肺、脑、肾和肌肉分离的微血管内皮细胞的培养细胞单层之间的粘附/迁移率； 3)使用荧光视频和共聚焦显微镜来量化在不同总体流速和目标1中相同条件a和d下直线和弯曲/拉伸微血管中的一氧化氮产生，以及在目标1中相同条件a和d下肺和脑、肾小球和骨骼肌微血管内皮细胞的培养细胞单层中的一氧化氮产生； 4) 在已知体积流量以及 VEGF 正常渗透率和增加渗透率的条件下，通过数值模拟对直和弯曲/拉伸微容器中的剪切速率、剪切应力、法向应力（压力）、速度和涡度剖面进行量化。