Salihah Salmma S, Bibi Bareerah, Khan Sehrish, Tahir Muhammad, Mahmood Sana, Vosough Massoud, Razzoli Agnese, Sidoli Simone, Gul Asma
Department of Biological Sciences, International Islamic University Islamabad, Pakistan.
School of Interdisciplinary Engineering & Science (SINES), NUST, Islamabad, Pakistan.
bioRxiv. 2025 May 22:2025.05.17.654447. doi: 10.1101/2025.05.17.654447.
Hydrogel-based three-dimensional (3D) co-culture systems are emerging as biomimetic platforms that more accurately recapitulate tissue architecture and microenvironmental interactions compared to conventional two-dimensional (2D) cultures. This study introduces an engineered 3D liver-like model to investigate compartment-specific responses to the potent hepatocarcinogen Diethylnitrosamine (DEN), with a focus on early events in carcinogenesis and tumor-stroma interactions. AML12 and 3T3 cell lines were treated with DEN or vehicle either in 2D culture or in 3D hydrogels in four experimental groups: (1) DEN-treated AML12 with vehicle-treated 3T3, (2) DEN-treated 3T3 with vehicle-treated AML12, (3) both cell types DEN-treated, and (4) both vehicle-treated. The cultured recombinants were subjected to proteomic profiling via mass spectrometry, followed by bioinformatics analysis and the results were validated through immunocytochemical staining (ICC). Gene ontology analysis revealed that cytoskeletal, RNA metabolism, and scaffold/adaptor proteins were among the most significantly enriched in 3D versus 2D models. Structural proteins emerged exclusively in mixed 3D co-cultures, reinforcing the organotypic nature of the system. Enriched pathways in 3D included intermediate filament organization, actin dynamics, and focal adhesion-pathways closely associated with liver carcinogenesis. Protein-protein interaction analysis demonstrated maximal network complexity in 3D cultures where both compartments were DEN-exposed. Survival analysis further identified poor-prognosis biomarkers (KRT20, KRT15, KRT14) uniquely enriched in this condition. ICC staining supported the proteomic findings. This organoid-like 3D co-culture model provides a physiologically relevant platform for investigating early-stage liver carcinogenesis and highlights the critical role of stromal-epithelial interactions. Its ability to replicate organ-level complexity and generate clinically relevant proteomic signatures supports its utility in translational cancer research and future drug discovery applications.
基于水凝胶的三维（3D）共培养系统正在成为一种仿生平台，与传统的二维（2D）培养相比，它能更准确地重现组织结构和微环境相互作用。本研究引入了一种工程化的3D肝样模型，以研究对强效肝癌致癌物二乙基亚硝胺（DEN）的特定隔室反应，重点关注致癌作用和肿瘤-基质相互作用中的早期事件。AML12和3T3细胞系在四个实验组中分别在2D培养或3D水凝胶中用DEN或溶剂处理：（1）用溶剂处理的3T3与用DEN处理的AML12共培养，（2）用溶剂处理的AML12与用DEN处理的3T3共培养，（3）两种细胞类型均用DEN处理，（4）两种细胞类型均用溶剂处理。对培养的重组体进行质谱蛋白质组分析，随后进行生物信息学分析，并通过免疫细胞化学染色（ICC）验证结果。基因本体分析表明，与2D模型相比，细胞骨架、RNA代谢和支架/衔接蛋白在3D模型中富集最为显著。结构蛋白仅出现在混合3D共培养中，强化了该系统的类器官性质。3D中富集的通路包括中间丝组织、肌动蛋白动力学和与肝癌发生密切相关的粘着斑通路。蛋白质-蛋白质相互作用分析表明，在两个隔室都暴露于DEN的3D培养中，网络复杂性最大。生存分析进一步确定了在这种情况下独特富集的预后不良生物标志物（KRT20、KRT15、KRT14）。ICC染色支持了蛋白质组学研究结果。这种类器官样的3D共培养模型为研究早期肝癌发生提供了一个生理相关平台，并突出了基质-上皮相互作用的关键作用。其复制器官水平复杂性并生成临床相关蛋白质组特征的能力支持了其在转化癌症研究和未来药物发现应用中的效用。
