Yang Ning, Abbaspour Ali, Considine James M, McGregor Stephanie M, Brooks Erin G, Naba Alexandra, Masters Kristyn S, Kreeger Pamela K
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA.
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
Acta Biomater. 2025 Sep 18. doi: 10.1016/j.actbio.2025.09.025.
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.
细胞外基质（ECM）在肿瘤进展中起着关键作用。在此，我们分析了高级别浆液性卵巢癌（HGSOC）正常大网膜和转移大网膜中的I型胶原蛋白和细胞纤连蛋白（cFN）。在HGSOC转移灶中，这两种蛋白质的水平均显著升高，且I型胶原纤维显著变粗。此外，ECM交联酶转谷氨酰胺酶2（TG2）在大网膜转移灶中增加，且在细胞外环境中具有酶活性。利用这些信息，单独或联合构建概括这些关键变化的ECM构建体，以研究它们对HGSOC细胞黏附的影响。据我们所知，这是首次使用TG2作为交联剂从多种ECM成分生成构建体的报告。在仅含胶原蛋白（coll）的凝胶上观察到HGSOC细胞黏附水平较低，而加入cFN或血浆纤连蛋白（pFN）可增加细胞黏附。TG2介导的coll/cFN水凝胶交联促进了HGSOC细胞黏附，而coll/pFN的交联则没有效果。细胞黏附取决于配体特性和纤维直径。当纤维厚度保持恒定时，由于β1整合素与cFN的EDA和RGD结构域相互作用，加入cFN相对于pFN或coll导致更高的HGSOC细胞黏附。同时，当凝胶组成保持恒定时，通过改变凝胶化温度增加纤维厚度，HGSOC细胞黏附增加。综合来看，我们的结果证明了与大网膜转移相关的ECM变化如何支持肿瘤进展，并为定制生物材料以支持细胞黏附的方法提供了见解。重要性声明：实体瘤中的ECM失调，蛋白质水平和物理组织发生改变。我们模拟了在晚期卵巢癌最常见转移部位大网膜中观察到的I型胶原蛋白、纤连蛋白和转谷氨酰胺酶2（TG2）的变化。虽然先前的研究通过聚乙二醇-肽凝胶或天然ECM混合物研究了ECM组合，但我们在此报告首次使用TG2交联多种ECM成分并生成纤维状材料。TG2交联增加了纤维厚度，这支持了肿瘤细胞黏附增加，对细胞纤连蛋白与血浆纤连蛋白观察到不同的效果。这些结果支持了ECM物理结构在指导细胞行为中的重要作用，并提供了一种实现这一目的的新仿生方法。
