Effects of Hydrodynamic Factors on Tumor Cell Arrest and Adhesion in the Microcirculation

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、肝和骨的小血管中。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。虽然发现生化和机械因素都在微血管中的肿瘤细胞阻滞和粘附中起作用，但对它们的贡献的定量理解很差。我们研究的长期目标是阐明完整微血管中微循环诱导的机械因素、微血管通透性（血管完整性）、细胞粘附分子和肿瘤转移之间的关系。本项目旨在探讨弯曲/拉伸微血管中局部流体动力因子、血管内皮生长因子诱导的微血管高通透性与完整微血管中肿瘤细胞阻滞和粘附之间的关系。在初步研究的基础上，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，来验证两个假设：1)肿瘤细胞倾向于在微血管中剪切速率和剪切速率梯度较高的位置停止。较高的剪切速率/剪切速率梯度激活内皮细胞和肿瘤细胞，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通透性增加的微血管内。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。本研究将在大鼠肠系膜内单独灌注的微血管上进行一系列体内实验。定量荧光视频和共聚焦显微镜将用于测量荧光染色肿瘤细胞在不同流量和通透性条件下在直管和弯曲/拉伸微血管中的粘附率。数值模拟将用于量化每种实验条件下微血管内剪切速率和应力、压力、速度和涡度的分布。具体目的是：1)在已知体积流速下，测量正常、非恶性（MCF-10A）和恶性（MDA-MB-435）乳腺上皮细胞在直微血管中的粘附率；a)在正常和VEGF通透性增加的情况下；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后；2)在已知体积流速和实验1相同条件下，测量正常、MCF-10A和MDA-MB-435乳腺上皮细胞在弯曲/拉伸微血管中的粘附率；3)通过数值模拟，量化在已知容积流速和VEGF通透性正常和增加两种情况下，直管和弯曲/拉伸微血管的剪切速率、剪切应力、正应力（压力）、速度和涡量分布：a)微血管内无肿瘤细胞，b)有肿瘤细胞贴壁。本项目为定量评估正常和炎症条件下肿瘤和内皮细胞的流体动力学因子和粘附分子在肿瘤转移中的作用提供了直接工具，从而有助于确定癌症治疗药物设计的新靶点。希望在微循环中预防癌细胞阻滞和粘附的抑制试剂和增强微血管壁完整性的试剂可以与传统疗法相结合，更有效地对抗这种恶性疾病。与此同时，该项目将为研究生和本科生提供一个机会，在一个新的有前途的领域，即癌症治疗的工程方法，以及拓宽和多样化纽约城市学院这个少数民族服务机构的研究领域。

CBET-0754158FuIt 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 lung, liver, and bone. The underlying mechanisms responsible for this preferential arrest of breast cancer cells in distant organs are not well understood. Although both biochemical and mechanical factors are found to play a role in tumor cell arrest and adhesion in the microvasculature, the quantitative understanding of their contribution is poor. The long-term goal of our research is to elucidate the relationships between microcirculation-induced mechanical factors, microvascular permeability (vascular integrity), cell adhesion molecules, and tumor metastasis in intact microvessels. The objective of this project is to investigate the relationships between localized hydrodynamic factors in curved/stretched microvessels, VEGF (vascular endothelial growth factor)-induced microvascular hyperpermeability, and tumor cell arrest and adhesion in intact microvessels. On the basis of the preliminary studies, a newly developed in vivo single vessel perfusion/bending method, which can create non-uniformly distributed shear rates/stresses along the vessel wall, will be used to test two hypotheses: 1) Tumor cells prefer to arrest at the locations of the higher shear rates and shear rate gradients in the microvasculature. The higher shear rates/shear rate gradients activate the endothelial cells and the tumor cells 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 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.A series of in vivo experiments will be performed on individually perfused microvessels in rat mesentery. Quantitative fluorescence video and confocal microscopy will be used to measure the adhesion rates of fluorescently dyed tumor cells in straight and curved/stretched microvessels under various flow and permeability conditions. Numerical simulation will be employed to quantify the profiles of shear rates and stresses, pressures, velocities and vorticities in the microvessel for each experimental condition. Specific aims are: 1) to measure the adhesion rates of normal, non-malignant (MCF-10A), and malignant (MDA-MB-435) breast epithelial cells in the straight microvessels 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, and c) after pretreatment with the blocking antibodies to tumor cell adhesion molecules; 2) to measure the adhesion rates of normal, MCF-10A, and MDA-MB-435 breast epithelial cells in the curved/stretched microvessels under known bulk flow rates and under the same conditions as in Aim 1; 3) to 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 conditions of normal and increased permeability by VEGF for two cases: a) when there are no tumor cells in the microvessel, and b) when there are tumor cells attached to the wall. This project provides a direct tool in the quantitative assessment of the role of hydrodynamic factors and adhesion molecules of tumor and endothelial cells in tumor metastasis under normal and inflammatory conditions, and hence helps define a new class of targets for therapeutic drug design for cancer. It is hoped 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. Meanwhile, this project will provide an opportunity to train both graduate and undergraduate students in a new promising field, engineering approach to cancer therapy, as well as to broaden and diversify the research areas of the City College of New York, a minority serving institution.