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

CBET-0754158Fu众所周知，循环中的肿瘤细胞停滞在微血管中，但这种停滞不是随机的。例如，乳腺癌细胞优先滞留在肺、肝和骨的小血管中。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。虽然生化和机械因素都被发现在肿瘤细胞在微血管系统中的阻止和黏附中发挥作用，但对它们的贡献的定量了解很少。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)、细胞黏附分子和完整微血管中的肿瘤转移之间的关系。本研究的目的是探讨弯曲/拉伸微血管局部流体动力学因素、血管内皮生长因子(VEGF)诱导的微血管高通透性与肿瘤细胞在完整微血管内的滞留和黏附之间的关系。在初步研究的基础上，一种新开发的体内单血管灌注/弯曲方法将用于检验两个假设：1)肿瘤细胞倾向于停滞在微血管中剪切率和剪切率梯度较高的位置。较高的剪切率/剪切率梯度激活血管内皮细胞和肿瘤细胞，增加肿瘤细胞与血管壁的结合，增加肿瘤细胞的聚集；2)肿瘤细胞倾向于滞留在通透性增加的微血管中。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。一系列体内实验将在大鼠肠系膜上单独灌流的微血管上进行。定量荧光视频和共聚焦显微镜将被用来测量不同流动和渗透条件下荧光染色的肿瘤细胞在直微血管和弯曲/拉伸微血管中的粘附率。数值模拟将被用来量化每个实验条件下微血管内的剪切率和应力、压力、速度和涡量的分布。具体目的是：1)测量正常、非恶性(MCF-10A)和恶性(MDA-MB-435)乳腺上皮细胞在已知体积流量和a)正常和血管通透性增加的条件下的乳腺上皮细胞在直微血管中的粘附率；b)用内皮细胞黏附分子阻断抗体预处理后，和c)肿瘤细胞黏附分子阻断抗体预处理后；2)测量正常、MCF-10A和MDA-MB-435乳腺上皮细胞在已知体积流量和在与目标1相同的条件下弯曲/拉伸微血管的黏附率；3)通过数值模拟在已知体积流量、正常通透性和血管通透性增强两种情况下(A)微血管内无肿瘤细胞和b)微血管壁上有肿瘤细胞附着时，通过数值模拟来量化血管内的剪切率、剪应力、法向应力(压力)、速度和涡量分布。该项目为定量评估正常和炎症条件下肿瘤和血管内皮细胞的流体动力学因素和黏附分子在肿瘤转移中的作用提供了直接的工具，从而有助于为癌症治疗药物设计确定一类新的靶点。人们希望，防止癌细胞在微循环中停滞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。与此同时，该项目将提供一个机会，在一个新的有前途的领域--癌症治疗的工程学方法--培训研究生和本科生，并拓宽和多样化纽约城市学院的研究领域，这是一个为少数族裔服务的机构。

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.