Fibronectin composition and transglutaminase 2 cross-linking cooperatively regulate ovarian cancer cell adhesion in ECM-mimetic constructs

The extracellular matrix (ECM) plays a crucial role in tumor progression. Here, we analyzed collagen I and cellular fibronectin (cFN) in normal omentum and metastatic omentum from high-grade serous ovarian cancer (HGSOC). The levels of both proteins were significantly elevated and collagen I fibers were significantly thicker in HGSOC metastases. Moreover, the ECM cross-linking enzyme transglutaminase 2 (TG2) was increased in omental metastases, where it is enzymatically active in the extracellular environment. This information was used to develop ECM constructs recapitulating these key changes, alone and in combination, to investigate their impact on HGSOC cell adhesion. To our knowledge, this is the first report using TG2 as a cross-linking agent to generate constructs from multiple ECM components. Low levels of HGSOC cell adhesion were observed on colIagen-only (coll) gels, while inclusion of cFN or plasma fibronection (pFN) increased cell adhesion. TG2-mediated cross-linking of colI/cFN hydrogels promoted HGSOC cell adhesion, while cross-linking of coll/pFN had no effect. Cell adhesion was dependent on ligand identity and fiber diameter. When fiber thickness was held constant, the inclusion of cFN led to greater HGSOC cell adhesion relative to pFN or coll, due to interactions of β1 integrins with the EDA and RGD domains of cFN. Meanwhile, when gel composition was held constant, HGSOC cell adhesion increased as fiber thickness was increased through modifications to gelation temperature. Combined, our results demonstrate how ECM changes associated with omental metastasis can support tumor progression and provide insights into methods to tailor biomaterials to support cell adhesion. STATEMENT OF SIGNIFICANCE: The ECM is dysregulated in solid tumors, with altered protein levels and physical organization. We modeled changes in collagen I, fibronectin, and transglutaminase 2 (TG2) observed in the omentum, the most common metastatic site of advanced ovarian cancer. While prior studies have examined ECM combinations through PEG-peptide gels or blends of native ECM, we report here the first use of TG2 to cross-link multiple ECM components and generate a fibrillar material. TG2 cross-linking increased fiber thickness, which supported increased tumor cell adhesion, with differential effects observed for cellular vs. plasma fibronectin. These results support the important role of the physical structure of the ECM in directing cellular behaviors and provide a new method of biomimicry to achieve this end.