Kopyeva Irina, Bretherton Ross C, Ayers Jessica L, Yu Ming, Grady William M, DeForest Cole A
Department of Bioengineering, University of Washington, Seattle 98105, Washington, United States.
Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle 98105, Washington, United States.
ACS Biomater Sci Eng. 2025 May 12;11(5):2810-2823. doi: 10.1021/acsbiomaterials.4c01632. Epub 2025 Apr 30.
Colorectal cancer (CRC) studies in vitro have been conducted almost exclusively on 2D cell monolayers or suspension spheroid cultures. Though these platforms have shed light on many important aspects of CRC biology, they fail to recapitulate essential cell-matrix interactions that often define in vivo function. Toward filling this knowledge gap, synthetic hydrogel biomaterials with user-programmable matrix mechanics and biochemistry have gained popularity for culturing cells in a more physiologically relevant 3D context. Here, using a poly(ethylene glycol)-based hydrogel model, we systematically assess the role of matrix stiffness and fibronectin-derived RGDS adhesive peptide presentation on CRC colony morphology and proliferation. Highlighting platform generalizability, we demonstrate that these hydrogels can support the viability and promote spontaneous spheroid or multicellular aggregate formation of six CRC cell lines that are commonly utilized in biomedical research. These gels are engineered to be fully degradable via a "biologically invisible" sortase-mediated reaction, enabling the triggered recovery of single cells and spheroids for downstream analysis. Using these platforms, we establish that substrate mechanics play a significant role in colony growth: soft conditions (∼300 Pa) encourage robust colony formation, whereas stiffer (∼2 kPa) gels severely restrict growth. Tuning the RGDS concentration did not affect the colony morphology. Additionally, we observe that epidermal growth factor receptor (EGFR) signaling in Caco-2 cells is influenced by adhesion ligand identity─whether the adhesion peptide was derived from collagen type I (DGEA) or fibronectin (RGDS)─with DGEA yielding a marked decrease in the level of downstream protein kinase phosphorylation. Taken together, this study introduces a versatile method to culture and probe CRC cell-matrix interactions within engineered 3D biomaterials.
结直肠癌（CRC）的体外研究几乎完全是在二维细胞单层或悬浮球体培养物上进行的。尽管这些平台揭示了CRC生物学的许多重要方面，但它们无法重现通常定义体内功能的基本细胞-基质相互作用。为了填补这一知识空白，具有用户可编程基质力学和生物化学性质的合成水凝胶生物材料在更具生理相关性的三维环境中培养细胞方面受到了广泛关注。在这里，我们使用基于聚乙二醇的水凝胶模型，系统地评估了基质硬度和纤连蛋白衍生的RGDS黏附肽呈现对CRC集落形态和增殖的作用。突出平台的通用性，我们证明这些水凝胶可以支持六种常用于生物医学研究的CRC细胞系的活力，并促进自发球体或多细胞聚集体的形成。这些凝胶经过工程设计，可通过“生物不可见”的分选酶介导反应完全降解，从而能够触发回收单个细胞和球体用于下游分析。使用这些平台，我们确定底物力学在集落生长中起重要作用：柔软条件（约300 Pa）促进强大的集落形成，而较硬（约2 kPa）的凝胶则严重限制生长。调节RGDS浓度不影响集落形态。此外，我们观察到Caco-2细胞中的表皮生长因子受体（EGFR）信号受到黏附配体特性的影响——无论黏附肽是源自I型胶原（DGEA）还是纤连蛋白（RGDS）——DGEA会导致下游蛋白激酶磷酸化水平显著降低。综上所述，本研究介绍了一种在工程化三维生物材料中培养和探究CRC细胞-基质相互作用的通用方法。
