Tumor-Associated Fibroblast-Derived Matrix Containing EDA-FN Alters Macrophage Behavior

Abstract Introduction Cancer progression is driven not only by tumor cells but also by interactions between the extracellular matrix (ECM), stromal cells, and immune cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major drivers of ECM remodeling, assembling ECM with aberrant organization. Extra domain A fibronectin (EDA-FN), a cellular FN containing an extra type III domain, is upregulated in the TME. EDA-FN regulates cellular behavior and has been associated with poor patient prognosis. Macrophages are among the most abundant immune cells within the TME, where they contribute to TME remodeling and inflammation to promote cancer cell invasion and metastasis. However, how the microarchitectural organization of tumor-associated matrices, enriched in EDA-FN, alters macrophage behavior remains largely understudied. Purpose Here, we developed a fibroblast-derived matrix platform that captures the structural imprint of tumor-associated EDA-enriched matrices and investigated how macrophages respond to these matrices in the absence of ongoing soluble factor cues. Method Human mammary fibroblasts (HMFs) preconditioned in incubated low-serum media and MDA-MB-231 metastatic breast cancer cell-conditioned media were cultured on polyacrylamide gels of 2 kPa and 20 kPa, respectively, followed by decellularization. Matrix organization, including fiber alignment, width, and intrafibrillar spacing, was quantified from confocal images. Decellularized HMF-derived matrices were subsequently reseeded with macrophages to assess macrophage morphology, phenotype, and matrix interactions. Results The combined effects of tumor-derived soluble factors and pathological stiffness induced a CAF-like phenotype in HMFs, accompanied by cytoskeletal reorganization and microarchitectural alterations of HMF-derived matrices. Tumor-associated matrices exhibited increased alignment, narrower fiber width, and enlarged intrafibrillar spacing compared to control matrices. These aberrant, tumor-associated matrix-derived features were associated with altered macrophage behavior, including heterogeneous morphology, enhanced localized EDA-FN matrix loss beneath the cell body, and a modest shift toward a CD206-dominant profile within a predominantly hybrid population. Conclusions These findings demonstrate the feasibility of obtaining HMF-derived matrices to investigate how matrix-derived cues in the absence of ongoing cellular and soluble factor signaling shape macrophage behavior. Furthermore, this platform may provide a framework for investigating matrix-targeting approaches to modulate macrophage function.