The tumor microenvironment (TME) governs all aspects of cancer progression and in vitro 3D cell culture platforms are increasingly developed to emulate the interactions between components of the stromal tissues and cancer cells. However, conventional cell culture platforms are inadequate in recapitulating the TME, which has complex compositions and dynamically changing matrix mechanics. In this study, we developed a dynamic gelatin-hyaluronic acid hybrid hydrogel system through integrating modular thiol-norbornene photopolymerization and enzyme-triggered on-demand matrix stiffening. In particular, gelatin was dually modified with norbornene and 4-hydroxyphenylacetic acid to render this bioactive protein photo-crosslinkable (through thiol-norbornene gelation) and responsive to tyrosinase-triggered on-demand stiffening (through HPA dimerization). In addition to the modified gelatin that provides basic cell adhesive motifs and protease cleavable sequences, hyaluronic acid (HA), an essential tumor matrix, was modularly and covalently incorporated into the cell-laden gel network. We systematically characterized macromer modification, gel crosslinking, as well as enzyme-triggered stiffening and degradation. We also evaluated the influence of matrix composition and dynamic stiffening on pancreatic ductal adenocarcinoma (PDAC) cell fate in 3D. We found that either HA-containing matrix or a dynamically stiffened microenvironment inhibited PDAC cell growth. Interestingly, these two factors synergistically induced cell phenotypic changes that resembled cell migration and/or invasion in 3D. Additional mRNA expression array analyses revealed changes unique to the presence of HA, to a stiffened microenvironment, or to the combination of both. Finally, we presented immunostaining and mRNA expression data to demonstrate that these irregular PDAC cell phenotypes were a result of matrix-induced epithelial-mesenchymal transition (EMT).

肿瘤微环境（TME）控制着癌症进展和体外的所有方面越来越多地开发3D细胞培养平台来模拟基质组织成分与癌细胞之间的相互作用。然而，常规的细胞培养平台不足以概括TME，TME具有复杂的成分和动态变化的基质力学。在这项研究中，我们通过集成模块硫醇-降冰片烯光聚合和酶触发的按需基质硬化，开发了动态明胶-透明质酸杂化水凝胶系统。特别是，用降冰片烯和4-羟苯基乙酸对明胶进行了双重修饰，以使该生物活性蛋白可光交联（通过硫醇-降冰片烯凝胶化）并响应酪氨酸酶触发的按需变硬（通过HPA二聚化）。除了提供基本细胞粘附基序和蛋白酶可裂解序列的修饰明胶外，透明质酸（HA）（一种必需的肿瘤基质）也被模块化并共价地掺入了充满细胞的凝胶网络中。我们系统地表征了大分子单体的修饰，凝胶交联以及酶触发的硬化和降解。我们还评估了基质成分和动态硬化对3D胰腺导管腺癌（PDAC）细胞命运的影响。我们发现，含HA的基质或动态硬化的微环境均会抑制PDAC细胞的生长。有趣的是，这两个因素协同诱导了细胞表型变化，类似于3D中的细胞迁移和/或入侵。额外的mRNA表达阵列分析揭示了HA所特有的变化，硬化的微环境，或两者兼而有之。最后，我们提供了免疫染色和mRNA表达数据，以证明这些不规则的PDAC细胞表型是基质诱导的上皮-间质转化（EMT）的结果。