Tumor-Mimetic Fibrillar Fibronectin Constructs Decorated with Hyaluronan Govern the Metastatic Potential of Breast Tumor Cells

The extracellular matrix (ECM) is a complex web of interwoven proteins and glycans that influence cell fate through biochemical and biophysical cues, where desmoplastic ECM signatures demarcate the progression of metastatic breast cancer. Accumulation of ECM glycan hyaluronan (HA) in breast tumor stroma has been correlated with poor patient prognosis. While this clinical correlation is well established, the functional role stromal HA serves in metastatic disease progression remains unclear but is suspected to be underpinned by differential HA molecular weight (Mw).<br/><br/>Fibronectin (Fn) is the bedrock of many native tissues, existing as a fibrous protein network that sequesters cell signaling factors and other ECM macromolecules and has been implicated in diseased tissue remodeling [1]. Given the complexity of native tissue-genesis, controlling the presentation of multivariant ECM components such as HA, Fn and collagen (COL) remains incredibly challenging using cell-based approaches. Engineered materials can be leveraged to resolve these outstanding ECM-related biological questions, but current platforms fail to utilize native proteins and glycans. Hence, it remains an outstanding challenge to recapitulate fibrillar ECM morphology in a scalable, three-dimensional (3D) platform with defined multi-component heterogeneity utilizing native materials.<br/><br/>Our lab recently pioneered an <i>in vitro</i> technique enabling the creation of 3D fibrillar Fn networks suspended across hyper-porous polymer scaffolds on the millimeter length scale by mimicking Fn fibrillogenesis that can be controlled to define fibril architecture [2,3]. This is achieved by shearing a native Fn solution across a polymeric scaffold at the solution/air interface, which promotes the formation of robust 3D fibrillar protein networks, herein referred to as engineered ECMs (EECMs). We showed Fn EECMs enrich for tumorigenic stem-like tumor cells [2] making them an ideal candidate for the creation of defined tumor niches to model the complexity of stromal ECM dynamics during metastatic disease progression.<br/><br/>In this work, we demonstrate the creation of defined multi-component fibrillar engineered ECMs comprised of native Fn, COL-I and HA. This was achieved by taking advantage of Fn’s binding activity, assembly modalities and developing novel bio-conjugation strategies. We show that Fn-based EECMs embody characteristics of fibrillar cell secreted tissue including conformationally active assembled, insoluble fibrillar structures with ECM component co-assembly. We further demonstrate remarkable control over the co-presentation of high and low Mw HA in porous, 3D fibrillar constructs for the first time in an engineered system using native materials. We confirmed Fn-HA EECMs retain critical biological characteristics compared to tumor associated cell secreted matrices. Finally, rationally designed Fn-HA EECMs enabled the study of HA’s role in influencing metastatic breast tumors. Conformationally active Fn increased metastatic tumor cell potential which may be enhanced or perturbed by HA in a Mw dependent manner.<br/><br/>Tumor-mimetic EECM marks a substantial advancement in the field of naturally derived material engineering where the findings presented here underscore the broader utility of native material systems. With further work, EECMs can be leveraged to dissect ECM-derived epigenetic regulation of the cellular milieu in the tumor microenvironment with potential application in personalized cancer treatment.<br/><br/>[1] Zollinger, AJ & Smith, ML DOI: 10.1016/j.matbio.2016.07.011<br/>[2] Jordahl, S, Neale DB, Lahann, J et al. DOI: 10.1002/adma.201904580<br/>[3] Neale, DB, Lahann, J et al. DOI: 10.1002/sstr.202000137