Research Grants Awarded
Synergistic Roles Of The Biophysical And Cellular Microenvironments In Breast Cancer Invasion And Metastasis
The tumor microenvironment is a complex milieu composed of multiple cell types, a constantly changing extracellular matrix, and myriad biochemical and biophysical signals. It is becoming increasingly apparent that the microenvironment surrounding a breast tumor plays critical roles in its growth and invasion; despite its importance, very little is known about how biophysical factors affect tumor invasion and metastasis. The significant contribution of the biophysical environment is often overlooked, where interstitial fluid flow can profoundly alter the gradients of signaling molecules, and impart forces to cancer cells and their stromal neighbors. We have previously demonstrated that cancer cells can sense slow interstitial fluid flow via autologously generated chemokine gradients and chemotact in the direction of flow, and also that fibroblasts differentiate into myofibroblasts (a hallmark of the stroma of aggressive breast tumors) in response to flow. These independent findings suggest that examining the interaction of three phenomena?breast cancer cell migration in response to flow, myofibroblast differentiation, and tumor-fibroblast interactions?may elucidate key mechanisms of breast cancer invasion and metastasis. Therefore, the objective of this proposal is to determine how the biophysical and cellular microenvironments, specifically interstitial flow and tumor-fibroblast crosstalk, interact to promote breast cancer invasion and metastasis via the lymphatic system. The proposed research will address two important questions: (1) do fibroblasts enhance or inhibit breast cancer cell invasion towards lymphatics (i.e., in the direction of interstitial flow); and (2) how does interstitial flow affect the tumor cell-fibroblast crosstalk? The specific aims will be to (1) develop an in vitro model breast tumor-stroma microenvironment; (2) determine whether fibroblasts enhance or hinder flow-induced breast cancer cell invasion; and (3) determine the effects of interstitial flow on breast cancer cell-fibroblast crosstalk. The central hypothesis of the study is that interstitial flow and tumor-fibroblast interactions will synergize to cause pro-invasive changes to the tumor microenvironment via matrix remodeling and growth factor and chemokine-mediated crosstalk. Human breast cancer cells will be cultured in a novel 3-D tissue engineered microenvironment that simulates the in vivo tumor stroma. This model will feature optimized co-culture of invasive breast tumor cells with fibroblasts, a 3-D collagen and Matrigel matrix, and interstitial fluid flow mimicking physiological and pathological conditions. Using this model, we will ascertain whether fibroblasts increase or decrease tumor invasion (including the effects of fibroblasts and flow on the migration of low metastatic and normal mammary epithelial cells), quantify changes in the physical microenvironment (e.g., matrix organization, composition, and permeability), and determine how these matrix changes promote invasion. We will also ask the question of how interstitial flow affects tumor-fibroblast crosstalk, focusing on a handful of candidate molecules. A range of strategies, including RNA silencing, neutralizing antibodies, pharmacological inhibitors, and engineered cell lines will be employed.
The mechanisms that drive tumor cells to invade and metastasize are still poorly understood, although there is growing evidence that stromal fibroblasts may play both supportive and inhibiting roles. Fluid flow is always directed from the tumor towards lymphatics, and since flow can both affect cell migration and fibroblast differentiation, it is likely that they critically affect metastasis by altering tumor-stroma crosstalk and directing signaling events that lead to invasion. This work will introduce a novel tissue engineered model of the human breast cancer microenvironment that will be invaluable in studying mechanisms of metastasis in vitro, and in the process establish the fundamental relationships between microenvironmental factors and invasion. Since no current therapies are effective at preventing metastasis, elucidating these relationships will provide information critical for rationally designing therapies to tackle the formidable problem cancer invasion.
The most deadly aspect of breast cancer is when it invades into the local tissue and spreads throughout the body via the lymphatic system. Despite its importance, very little is known about how tumor cells interact with their local environment (the tumor microenvironment) and how these interactions may lead to invasion. Since breast cancer usually spreads through lymphatic vessels to lymph nodes and beyond, we are particularly interested in factors related to the lymphatic system, which functions as a drainage system. This slow drainage of fluid through the tissue, called interstitial fluid flow, can change how bioactive molecules are distributed through and around the tumor, as well as apply physical stress to cancer cells and their neighbors, causing changes in cell behavior through gradients and forces. We believe that interstitial flow acts as a guide to breast cancer cells, pointing the way towards the lymphatic vessels and lymph nodes. Recent studies are revealing that interstitial flow can influence cancer cells and neighboring host cells, which we know to interact strongly with tumors, in ways that may be highly relevant to cancer metastasis.
Our goal is to answer two important questions: (1) how do surrounding host cells promote the movement of breast cancer cells towards the lymphatics (and ultimately the lymph node and beyond); and (2) how does interstitial flow affect the biochemical signals between tumor cells and host cells that lead to metastasis? These questions are critical because they will help us understand how a tumor invades into lymphatics, which eventually leads to metastasis. To address these questions, we need a model system that allows us to simultaneously study cancer cells interacting with host cells, in the presence of interstitial fluid. Unfortunately, no such model currently exists. Thus, our first specific aim is to develop this model so it can be used to study how breast cancer cells interact with their microenvironment. The model will consist of breast tumor cells, host tissue cells, a 3-D matrix, and, most significantly, interstitial fluid flow. In the second specific aim, we will determine whether the host tissue cells promote cancer cell invasion when exposed to interstitial flow. In the third specific aim, we will elucidate whether some of biochemical signals associated with lymph node metastasis are part of the signaling between breast tumors and surrounding host cells.
Our research will have several important outcomes. First, we will create a model system that empowers us to ask, and answer, questions about breast cancer in the context of the tumor?s microenvironment. This will provide a better understanding of the necessary steps in tumor cell migration, invasion, and metastasis. Since metastasis is the cause for the vast majority of cancer-related deaths, the second major outcome of this project is the development of tools that will allow us to identify the cellular pathways that are most critical to breast cancer invasion and metastasis. By learning about the key pieces in the invasion process, we can provide researchers and clinicians with new targets and strategies for therapy.