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 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.
Background: Cancer is becoming the top killer for people under age seventy-five in the United States. The cause of death is usually organ failure caused by metastatic tumors that are derived from the primary tumor. 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 lung, the liver, and the bones. The underlying mechanisms responsible for this preferential arrest of breast cancer cells in distant organs are not well understood. One difficulty is that there is no proper experimental method that can control all the hydrodynamic and microvessel factors and also represent the real physiological environment. Findings: Under NSF support, Professor Fu and her team have developed a new method, which enables in vivo cannulation and perfusion of a single microvessel under well-controlled conditions. Using this method, they have found that 1) Breast cancer cells like to adhere to the microvessels with higher permeability, which is induced by vascular endothelial growth factor (VEGF, a secretion from tumor and blood cells) (Fig. 1). The antibody targeting VEGF at tumor cell and that targeting VEGF receptor at the microvessel wall greatly decrease the tumor cell adhesion; 2) Adhesion of malignant tumor cells increases microvessel permeability via degradation of the surface glycocalyx layer (a very thin matrix-like sugar coating) of endothelial cells forming the microvessel wall (Fig. 2). Strengthening this layer by a plasma protein orosomucoid can prevent the increase in vessel permeability and reduce tumor cell adhesion; 3) Computational results show that the curvature of the microvessel and the interactions between tumor to tumor cells, tumor cells to endothelial cells forming the vessel wall determine the tumor cell arrest (slow down and rolling) and adhesion to the wall. The wall shear stress and its gradient do increase the tumor cell adhesion to the inner side wall of the curved microvessel. In addition to the shear stress, the higher vorticity (rotation of blood flow) at the inner turning point of branched microvasculature contributes to the arrest of tumor cells; 4) In vivo tumor cell arrest and adhesion experiments show that mechanical trapping due to microvessel size restriction is responsible for the majority arrest of rigid and partial arrest of flexible tumor cells. Different types of tumor cells (stiff or flexible) arrest at different sites of the microvasculature. Intellectual Merit: A new method was developed to enable the investigation of the tumor cell adhesion mechanisms under dynamic, quantitative and real physiological conditions. This bridges an important gap in our knowledge of molecular mechanisms acquired from cell culture study and our knowledge of physiological and pathological functions of hydrodynamic factors and microvessel endothelium obtained from animal studies during tumor metastasis. The Broader Impacts of this research include: - 1 - Benefits to society: A well-controlled in vivo single microvessel perfusion and immunostaining method has been developed. Tumor cell adhesion is one of the critical steps for tumor metastasis. This method can be used to investigate the tumor adhesion mechanisms under dynamic, quantitative and real physiological conditions. In addition to preserving the microvessel wall integrity as found by this project, targeting tumor cell adhesion may trigger new anti-metastatic therapies. This will benefit cancer patients. - 2 - Broadening participation of underrepresented groups: This project has trained fifteen students, two of them are African-American and five are women students. - 3 - Advancing discovery and understanding while promoting teaching, training, and learning: Ten original journal publications, two review articles and two book chapters have been produced under the partial support of this NSF grant, as well as many conference abstracts and presentations. This project has also provided engineering students an opportunity in conducting researches that are normally in medical schools. This definitely benefits them in their learning and training outside the traditional engineering fields. - 4 - Enhancing the infrastructure for research and education: This project brings new research direction at the City College of New York, and potential new collaborations and partnerships from the New York medical communities. - 5 - Results disseminated broadly to enhance scientific and technological understanding: In addition to publications in scientific journals and presentations in scientific conferences, ten invited seminars based on the findings from this project have been delivered at the City College, in the universities within the U.S. as well as in China, the United Kingdom and Europe.
