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中阻滞，但这种阻滞不是随机的。例如，乳腺癌细胞优先停留在肺、肝、脑和骨骼的小血管中。导致乳腺癌细胞在远处器官中优先被抑制的潜在机制尚不清楚。我们的长期研究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肿瘤转移之间的关系。本项目旨在探讨弯曲/拉伸微血管的局部剪切速率和应力、血管内皮生长因子（VEGF）诱导的微血管高通透性以及完整微血管中乳腺肿瘤细胞阻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，以验证两个假设：1)肿瘤细胞倾向于在微血管毛细血管后小静脉中较高剪切速率/应力和剪切速率/应力梯度的位置停止。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（具体而言，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)肿瘤细胞倾向于停留在微血管内，微血管通透性增加。随着渗透性的增加，肿瘤细胞对微血管壁的粘附性增加，部分原因是径向压力梯度驱使细胞向微血管壁移动。这些想法将使用生理，生化，数学和成像方法的组合进行探索。具体目标是：1)利用定量荧光视频和共聚焦显微镜测定在已知体积流速下，正常、非恶性（MCF-10A）和恶性（au565）乳腺上皮细胞在大鼠肠系膜直、弯/拉伸微血管中的粘附率；a)在VEGF通透性正常和增加的情况下；b)内皮细胞粘附分子阻断抗体预处理后；c)肿瘤细胞粘附分子阻断抗体预处理和d)微血管内皮细胞eNOS抑制剂预处理；2)使用基于过滤器的粘附/迁移试验，在与第1项相同的条件下，确定上述细胞对从肺、脑、肾和肌肉分离的微血管内皮细胞培养单层细胞的粘附/迁移率；3)利用荧光视频和共聚焦显微镜定量测定不同容积流量下，Aim 1中相同条件a和d下直、弯/拉伸微血管，以及Aim 1中相同条件a和d下肺、脑、肾小球和骨骼肌微血管内皮细胞单层培养细胞的一氧化氮产量；4)通过数值模拟，量化了在已知容积流量和VEGF正常和增加渗透性条件下，直管和弯曲/拉伸微血管的剪切速率、剪切应力、法向应力（压力）、速度和涡量分布。

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.