Bibi, B. | bioRxiv Science

bioRxiv2025

3D Biomimetic Liver Cancer Model: Diethylnitrosamine-Induced Proteomic Dysregulations in Stromal-Epithelial Milieu

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.

cancer biology↗

bioRxiv2025

Differential Proteomic Analysis of DEN-Induced Hepatocellular Carcinoma in Male and Female Balb/c Mice Reveals Novel Gender Specific Markers

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of hepatic malignancy with a higher prevalence in males compared to females, however, the distinct underlying mechanisms contributing to this disparity remain poorly understood. MethodsIn this study, we aimed to investigate comparative proteome profiling of a diethylnitrosamine (DEN) induced HCC model in male and female Balb/c mice. We extracted proteins from liver tissue of DEN treated male and female mice and their corresponding controls and subjected them to mass spectrometry and subsequent bioinformatics analyses. ResultsWe identified 170 and 233 differentially expressed proteins (DEPs) in female and male mice, respectively. We identified chemical carcinogenesis and cortical cytoskeleton as the shared pathways between the two groups. In addition, we identified distinct signaling pathways in DEN-treated male and female mice. Female mice showed enrichment in oxidative phosphorylation, fatty acid biosynthesis, metabolism and degradation and cytochrome P450 clusters. In contrast, in male mice, these pathways were enriched in cholesterol metabolism, coagulation and mRNA processing. Further, we identified top ten genes ranked by highest maximal clique centrality, by protein-protein interaction analysis of differentially expressed proteins (DEPs) in both sexes. Of these hub genes, female mice showed upregulation of NDUFA8 and ATP5H and were associated with poor patient survival. On the other hand, In DEN-treated male mice upregulation of FGG, FGA, HPX and SERPINC1 were associated with poor survival. ConclusionIn conclusion, our research provides gender-specific proteomic signatures in DEN-induced HCC. The identification of proteins associated cholesterol metabolism and coagulation in males, and mitochondrial complex I proteins in females as prognostic markers suggests novel therapeutic targets that may inform gender-tailored treatment strategies for HCC. Simple SummaryHepatocellular carcinoma (HCC) is a common and deadly liver cancer that affects men more than women. To understand the biological reasons behind this difference, we developed a liver cancer model in male and female mice using a chemical called diethylnitrosamine (DEN). We then studied the proteins involved in tumor development using advanced techniques like mass spectrometry and bioinformatics. We found that different sets of proteins and biological pathways were active in males and females. In males, ribosomal and RNA-binding proteins were linked to worse survival, while in females, mitochondrial proteins were more important. These findings suggest that men and women may need different strategies for diagnosing and treating HCC, and they offer new gender-specific targets for future therapies.

cancer biology↗