Defined Extracellular Matrix Contexts Rewire 3D Breast Cancer Cell Models: Effects of Sulfated Hyaluronan in the Expression of Matrix Effector and Matrix-Based Cell Functional Properties

Abstract

Background: Breast cancer progression is strongly influenced by tumor cell interactions with the extracellular matrix (ECM). Three-dimensional (3D) cell culture models provide more relevant systems for studying aspects of the tumor microenvironment (TME), including tumor architecture, matrix remodeling and cell-ECM communication. Collagen type I (Col I), fibronectin (FN) and hyaluronan (HA) are key ECM components involved in breast cancer progression. At the same time, integrins act as major matrix-sensing receptors that translate ECM cues into cellular responses. In this study, we investigated how defined ECM contexts regulate functional cell properties in shERβ MDA-MB-231 and Hs578T advanced 3D breast cancer cell models. Methods: Cells were cultured in 2D and 3D systems under matrix-free conditions or in Col I, FN, and mixed Col I/FN environments, in the presence or absence of HA or sulfated HA (sHA). Spheroid growth, matrix-dependent spheroid organization and dissemination were evaluated. In parallel, selected collagen- and fibronectin-associated integrin subunits, together with HA-related molecules including HAS2, CD44, and RHAMM, were analyzed to assess receptor-mediated matrix- and HA-associated responses. Results: Matrix substrates strongly influenced breast cancer spheroid behavior. Col I promoted outward dissemination, whereas FN-containing environments favored compact organization and restricted spreading. HA supported spheroid-like organization in a matrix-dependent manner, whereas sHA disrupted organized spheroid assembly in ECM-rich conditions, resulting in looser structures or smaller cell colonies, depending on the matrix context. Gene expression analysis revealed model-dependent remodeling of matrix- and HA-associated programs, with broader adhesion-related responses in shERβ MDA-MB-231 cells and stronger dimensionality-driven regulation in Hs578T cells. Conclusions: Overall, these findings demonstrate that defined ECM contexts actively regulate breast cancer spheroid behavior and matrix-associated receptor programs. Col I, FN and mixed Col I/FN environments generated distinct 3D phenotypes, affecting spheroid growth, organization and dissemination, while HA/sHA further modified these matrix-dependent responses. This study supports sHA as a functional matrix molecule with anti-cancer relevance and highlights the value of 3D matrix-based models for investigating tumor–matrix communication in breast cancer.