Cancer progression depends on interactions between elements of the tumor microenvironment that include transformed cancer cells, normal stromal cells, and extracellular matrix (ECM). Non-malignant cells that play major roles in tumorigenesis include fibroblasts and macrophages. Fibroblasts are the main producers of ECM components. During cancer progression, they become activated and increase their production of ECM and proteases that degrade ECM proteins. Fibroblast Activation Protein-α (FAP) is a protease of the post-prolyl peptidase family that is primarily expressed by activated fibroblasts. It proteolytically cleaves type I collagen, which is a major component of ECM. Importantly, a regulatory immune role for tumor-associated fibroblasts (TAFs) was demonstrated by FAP-targeted deletion of TAFs in vivo which increased tumor killing by immune cells. Tumor-associated macrophages (TAMs) are key regulators of inflammatory and immune processes in the tumor microenvironment. Binding to ligands that include native and modified ECM components facilitates macrophage infiltration and accumulation in the tumor. Therefore, I hypothesize that FAP-mediated cleavage of type I collagen enhances macrophage adhesion via specific cell surface receptor(s).
To test this hypothesis, I first examined whether cleavage of type I collagen by FAP enhances macrophage adhesion. I found that FAP selectively cleaves type I collagen. When used as an adhesion substrate, FAP-cleaved type I collagen increased both the attachment and spread of primary macrophages in comparison to native collagen. Next, I demonstrated that chelating divalent cations did not inhibit macrophage attachment to FAP-cleaved collagen indicating that this effect is not dependent on integrins. However, treatment with polyinosine, a ligand for pattern recognition receptors, abolished macrophage adhesion on FAP-cleaved type I collagen. Further, FAP-mediated cleavage of collagen did not increase the adhesion of macrophages isolated from Class A Scavenger Receptor (SR-A) null mice. Together, these results demonstrate that macrophage adhesion to FAP-cleaved type I collagen is mediated by SR-A and identify a novel biological role for FAP-modified type I collagen.
Using immunohistochemistry (IHC), I showed that primary mammary tumors in the MMTV-PyMT mouse model of spontaneous breast cancer are characterized by a distinct and collagen-rich stromal compartment infiltrating epithelial cancer cell acini. Next, utilizing IHC and biochemical approaches I found that enzymatically active FAP is present on TAFs of MMTV-PyMT tumors and that SR-A-positive TAMs localize to stromal compartment of these tumors. These results demonstrate that FAP-positive fibroblasts and SR-A-positive macrophages localize to the same collagen-rich stromal compartment of MMTV-PyMT tumors and support the conclusion that FAP- and ECM-mediated interactions between fibroblast and macrophages could occur in vivo.
Overall, my findings demonstrate that FAP, a fibroblast-specific protease induced in tumor stroma, proteolytically cleaves type I collagen to create a substrate recognized by macrophage SR-A. This interaction results in enhanced macrophage adhesion characterized by increased attachment and a spread morphology. Extrapolating from previous studies, SR-A-mediated recognition of FAP-cleaved collagen would activate signaling cascades that differentially regulate the secretion of factors that regulate behavior of cells in the tumor microenvironment. Therefore, my studies describe a novel mode of communication between TAFs and TAMs and propose a stroma-based molecular mechanism that leads to tumor growth.
|Advisor:||Kelly, Thomas, Post, Steven R.|
|Commitee:||Nakagawa, Mayumi, Suva, Larry J., Ware, Jerry|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/01(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Pathology|
|Keywords:||Extracellular matrix, Fibroblast, Macrophage, Microenvironment, Protease, Stroma|
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