Tumor Cell Arrest and Adhesion in the Microcirculation

描述（由申请人提供）：众所周知，循环肿瘤细胞在微血管系统中停滞，但这种停滞不是随机的。例如，乳腺癌细胞优先在肺、肝、脑和骨骼的小血管中停滞。负责这种优先阻止远处器官中乳腺癌细胞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与完整微血管中肿瘤转移之间的关系。本项目的目的是研究弯曲/拉伸微血管中局部剪切率和应力之间的关系，VEGF（血管内皮生长因子）诱导的微血管通透性过高，以及乳腺肿瘤细胞在完整微血管中的阻滞和粘附。在我们前期研究的基础上，我们将使用一种新开发的体内单血管灌注/弯曲方法，该方法可以沿着血管壁产生非均匀分布的剪切速率/应力沿着来验证两个假设：1）肿瘤细胞倾向于在微血管的毛细血管后小静脉中的较高剪切速率/应力和剪切速率/应力梯度的位置处停滞。更高的剪切速率/应力和剪切速率/应力梯度激活内皮细胞和肿瘤细胞（具体地，激活细胞粘附分子和内皮型一氧化氮合酶），以增加肿瘤细胞与血管壁的结合，并增加肿瘤细胞的积聚; 2）肿瘤细胞偏好于在具有增加的渗透性的微血管中停滞。增加的肿瘤细胞粘附到微血管壁并具有增加的渗透性，部分是由于径向压力梯度将细胞推向壁。这些想法将使用生理学，生物化学，数学和成像方法的组合进行探索。具体目标是：1）使用定量荧光视频和共聚焦显微镜来确定正常、非恶性（MCF-10 A）和恶性（Au-565）乳腺上皮细胞在大鼠肠系膜上的直的和弯曲/拉伸的微血管中的粘附率，所述粘附率在已知的整体流速下和a）在正常和VEGF渗透性增加的条件下，B）在用内皮细胞粘附分子的阻断抗体预处理后，c）在用肿瘤细胞粘附分子的阻断抗体预处理后和d）在用eNOS抑制剂预处理后，对微血管内皮细胞; 2）使用基于过滤器的粘附/迁移测定来确定上述细胞对/穿过在与目的1相同的条件下从肺、脑、肾和肌肉分离的微血管内皮细胞的培养细胞单层的粘附/迁移速率; 3）使用荧光视频和共聚焦显微镜来定量在不同总体流速下和在Aim 1中相同的条件a和d下直的和弯曲/拉伸的微血管中以及在Aim 1中相同的条件a和d下在肺和脑、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;和4）在已知的整体流速下和在VEGF正常和增加的渗透性条件下，通过数值模拟在直的和弯曲/拉伸的微血管中量化剪切速率、剪切应力、法向应力（压力）、速度和涡度分布。 公共卫生相关性：该项目将导致对流体动力学因子，细胞粘附分子和一氧化氮在肿瘤优先转移中的作用的定量理解，从而帮助定义一类新的癌症治疗药物设计靶点。我们希望，可以与传统疗法结合使用，以更有效地对抗这种恶性疾病的抑制剂，防止癌细胞停滞和粘附在微循环和试剂，提高微血管壁的完整性。

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. PUBLIC HEALTH RELEVANCE: This project will lead to a quantitative understanding of the role of hydrodynamic factors, cell adhesion molecules and nitric oxide in tumor preferential metastasis, and hence help define a new class of targets for therapeutic drug design for cancer. We hope that inhibitory reagents that prevent cancer cell arrest and adhesion in the microcirculation and reagents that enhance the microvessel wall integrity may be used in combination with traditional therapies to combat this malignant disease more effectively.