Introduction: The extracellular matrix (ECM) is a fibrillar structure that provides biochemical and mechanical cues to cells. Fibronectin (Fn), a fundamental EMC protein, is conformationally flexible and is implicated as a critical mechanoregulator of the ECM. The early ECM assembled by breast tumor conditioned stromal cells, comprising only Fn, is structurally and mechanically altered, and mediates an integrin-switch in newly attached cells[1]. Since collagen (Col I) deposition depends on underlying Fn matrices[2] and Col I modifications facilitate breast tumorigenesis[3], we sought to understand how the materials properties of tumor conditioned Fn would affect downstream Col I deposition and contribute to changes in the breast stroma during tumorigenesis. We assessed: (i) ECM composition and structure, (ii) Fn conformation dynamics via Förster resonance energy transfer (FRET), and (iii) matrix metalloproteinase (MMP) contributions to altered ECM assembly.
Materials & Methods: Pre-adipocytes were maintained in tumor or control conditioning media up to 24 hours before a timepoint (1, 5, 9d), when exogenous Fn (10% FRET-labeled for FRET experiments) was provided. Samples were either immunostained for Fn and Col I, or directly confocal imaged for FRET. Fn and Col I fiber diameter and linearity (short length/full length) were analyzed from immunostained images. Fluorescent intensity ratios (FRET = IA/ID) discriminated unfolded (low FRET/blue pixels), from relaxed Fn fibers (high FRET/yellow pixels). Parallel experiments were run with a broad spectrum MMP inhibitor, Batimastat to assess any changes in tumor-conditioned ECM evolution.
Results and Discussion: Our results revealed that tumor-conditioned cells initially deposited thin, linearized, and unfolded Fn fibers (Fig 1a-c), likely contributing to altered cell-ECM and Fn-Col interactions by exposing/disrupting cryptic binding sites[4]. This early altered Fn ECM was replaced by thick Fn and Col I fibers (Fig 1a-c). At all times, tumor-conditioned Fn fibers were more unfolded (Fig 1d-e). And both tumor-conditioned and control Fn relaxed when Col I was present (9d) (Fig 1d-e), likely due to Col I bearing most of the tension in the ECM[5]. Furthermore, tumor-conditioned cells with inhibited MMP activity resulted in an ECM comprising mostly thick Fn with altered linearity dynamics (Fig 1f-h). Tumor conditioned cells, unable to proteolytically degrade and remodel the previous Fn network, may have been unable to expose Col I binding sites for downstream Col I deposition, and thus continued to deposit Fn on previously assembled Fn.
Conclusions: Our findings suggest that the initial physical and conformational state of tumor-conditioned Fn fibers and active MMPs regulate Col I deposition and alterations in breast tumor stroma. Once tumor-conditioned Col I is deposited, its bulk tissue topology may be the driving force of altered tumor stroma mechanotransduction.
References:
[1] Wang K, Andresen Eguiluz RC, Wu F, Seo BR, Fischbach C, Gourdon D. Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells. Biomaterials 2015;54:63–71. doi:10.1016/j.biomaterials.2015.03.019.
[2] Sottile J, Hocking DC. Fibronectin Polymerization Regulates the Composition and Stability of Extracellular Matrix Fibrils and Cell-Matrix Adhesions. Molecular Biology of the Cell 2002;13:3546–59. doi:10.1091/mbc.E02.
[3] Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, et al. Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling. Cell 2009;139:891–906. doi:10.1016/j.cell.2009.10.027.
[4] Bradshaw MJ, Smith ML. Multiscale relationships between fibronectin structure and functional properties. Acta Biomaterialia 2014;10:1524–31.
[5] Kubow, KE, Vukmirovic R, Zhe L, Klotzsch E, Smith ML, Gourdon D, Luna S, Vogel V. Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix. Nature Communications 2015;6, 8026.