Sulfated hyaluronan–mediated regulation of extracellular matrix effectors, cellular function and small extracellular vesicle communication in 2D and 3D breast cancer models
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Date
2026-02-10
Authors
Σπανόπουλος, Κωνσταντίνος
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Abstract
The extracellular matrix (ECM) is a central regulator of tumor progression, influencing cancer cell proliferation, invasion, therapy resistance, and intercellular communication. Hyaluronan (HA), a major ECM glycosaminoglycan synthesized predominantly by HA synthase 2 (HAS2), plays a key role in these processes through modulation of cell adhesion, motility, growth factor signaling, and receptor-mediated pathways. HA has been implicated in the regulation of extracellular vesicle (EV) biogenesis and cargo composition, thereby contributing to intercellular communication and the establishment of pre-metastatic niches. Chemically modified derivatives such as sulfated hyaluronan (sHA) have been proposed to interfere with HA–receptor interactions and downstream signaling. Notably, sHA exhibits reduced recognition and degradation by hyaluronidases (HYALs), potentially enhancing its stability and prolonging its modulatory effects within the tumor microenvironment. While two-dimensional (2D) cell culture systems remain widely used, three-dimensional (3D) spheroid models more accurately recapitulate tumor architecture, ECM organization, and diffusion gradients, providing a physiologically relevant platform for studying ECM-driven tumor behavior.
In this study, we investigated the effects of low-molecular-weight HA (50 kDa) and HA sulfated by chemical synthesis (sHA) on the breast cancer cell lines MDA-MB-231 and MCF-7 using both 2D and 3D cell models. Gene and protein expression of ECM components, HA receptors, epithelial-to-mesenchymal transition (EMT) markers, and matrix metalloproteinases (MMPs) were performed. EVs were isolated and characterized to assess sHA-dependent changes potentially linked to pre-metastatic niche formation. The contribution of endogenous HA synthesis was further examined through HAS2 silencing, followed by functional assays and ultrastructural analysis using scanning electron microscopy (SEM). 3D spheroids derived were also evaluated for growth kinetics and gene expression patterns associated with ECM remodeling, EMT, and invasiveness.
The results demonstrate that sHA exerts striking anticancer effects compared to HA, particularly in the highly aggressive MDA-MB-231 breast cancer cells. Moreover, sHA treatment led to the marked modulation of EMT-related biomarkers and MMPs expression, suggesting a reduction in invasive and metastatic potential. In parallel, alterations in EV-associated parameters indicate a possible impairment of mechanisms involved in pre-metastatic niche formation. In advanced 3D models, sHA significantly affected spheroid growth in both breast cancer cell lines, with a stronger inhibitory effect observed in MDA-MB-231 spheroids, highlighting cell line-specific responses linked to tumor aggressiveness. Differences between 2D and 3D outcomes further emphasize the importance of culture dimensionality in accurately capturing ECM-mediated effects. Collectively, these findings identify sHA as a promising modulator of breast cancer progression, capable of attenuating EMT-associated and pro-metastatic features, particularly in aggressive tumor phenotypes. Moreover, this work underscores the regulatory roles of HAS2 and extracellular vesicles in ECM-driven tumor communication and highlights the value of 3D spheroid models for translational cancer research.
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Keywords
Hyaluronic acid, Sulfated hyaluronan, HAS2, Breast cancer, 3D cancer cell culture