Tumor Cell Arrest and Adhesion in the Microcirculation

描述(申请人提供)：众所周知，循环中的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，乳腺癌细胞优先滞留在肺、肝、脑和骨骼的小血管中。导致乳腺癌细胞优先滞留在远处器官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)、细胞黏附分子、一氧化氮和细胞因子与完整微血管内肿瘤转移的关系。本项目的目的是研究弯曲/拉伸微血管局部剪切率和应力、血管内皮生长因子(VEGF)诱导的微血管高通透性与乳腺肿瘤细胞在完整微血管中的滞留和黏附的关系。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流/弯曲方法来检验两个假设：1)肿瘤细胞更喜欢停滞在微血管的毛细血管后小静脉中剪切率/应力和剪切率/应力梯度较高的位置。较高的剪切率/应力和剪切率/应力梯度激活内皮细胞和肿瘤细胞(特别是激活细胞黏附分子和内皮型一氧化氮合酶)，增加肿瘤细胞与血管壁的结合，增加肿瘤细胞的积聚；2)随着通透性的增加，肿瘤细胞更喜欢滞留在微血管中。随着通透性的增加，肿瘤细胞与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朝向管壁。这些想法将使用生理、生化、数学和成像方法的组合来探索。其具体目的是：1)利用荧光定量视频和共聚焦显微镜，在已知的整体流量下和a)在正常和血管通透性增加的情况下，用荧光定量视频和共聚焦显微镜测定正常、非恶性(MCF-10A)和恶性(AU-565)大鼠肠系膜上皮细胞在直微血管和弯曲微血管(AU-565)上的粘附率；2)在与目标1相同的条件下，采用基于滤膜的黏附/迁移实验测定上述细胞对肺、脑、肾和肌肉微血管内皮细胞培养单层的黏附/迁移率；3)在目标1的不同体积流量和a、d相同条件下，利用荧光摄像和共聚焦显微镜定量测定不同体积流量和a、d相同条件下，目标1的肺、脑、肾小球和骨骼肌微血管内皮细胞培养单层细胞中一氧化氮的产生；4)利用血管内皮细胞生长因子对已知体积流量、渗透性正常和渗透性增强的微血管内切变率、切应力、法向应力(压力)、速度和涡量分布进行了数值模拟。 公共卫生相关性：该项目将有助于定量了解流体动力因素、细胞黏附分子和一氧化氮在肿瘤优先转移中的作用，从而有助于为癌症治疗药物设计确定一类新的靶点。我们希望阻止癌细胞在微循环中停止和黏附的抑制性试剂和增强微血管壁完整性的试剂可以与传统疗法结合使用，以更有效地对抗这种恶性疾病。

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.