三维生物打印胆囊癌模型的构建与评价OA

Background and purpose:Gallbladder cancer is a highly aggressive gastrointestinal malignancy with a 5-year survival rate of less than 10%.Conventional two-dimensional(2D)cell cultures poorly mimic the tumor microenvironment,limiting translational drug screening.Three-dimensional(3D)bioprinting enables the construction of biomimetic tumor models with controllable structure and function.This study aimed to establish a 3D bioprinted gallbladder cancer model and compare its biological and pharmacological features with 2D cultures.Methods:The NOZ human gallbladder cancer cell line and GelMA hydrogel bioink were used to construct a 3D gallbladder cancer model via extrusion-based 3D bioprinting,aiming to simulate the tumor microenvironment.The morphology,viability,and proliferation of cells in the 3D constructs were assessed using microscopy,live/dead cell staining,and the cell counting kit-8(CCK-8)assay.Following RNA extraction,RNA sequencing was performed on the Illumina NovaSeqTM 6000 platform.Differential gene expression analysis was conducted using DESeq2,and the results for selected genes were validated by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR).Drug sensitivity was evaluated by determining the half-maximal inhibitory concentration(IC50)through dose-response experiments with gemcitabine(GEM),cisplatin(DDP),nab-paclitaxel and 5-fluorouracil(5-FU).Statistical analyses were performed using GraphPad Prism 9.0 and R software(version 4.4.0),and P<0.05 was considered statistically significant.Results:The 3D model showed stable lattice structures with high cell viability and spheroid formation,accompanied by significantly enhanced proliferation compared with 2D cultures(P<0.001).Transcriptome analysis revealed 617 differentially expressed genes(235 upregulated,382 downregulated),enriched in cell cycle regulation,cytokine signaling,and extracellular matrix remodeling.RTFQ-PCR confirmed consistency with RNA-seq results.Drug response assays demonstrated higher IC50 in 3D models versus 2D for all agents tested,indicating reduced chemosensitivity and stronger resemblance to clinical resistance.Conclusion:This study established a 3D bioprinted gallbladder cancer model.Compared with 2D culture,the 3D model showed lower sensitivity and higher IC50 to GEM,DDP,nab-paclitaxel,and 5-FU,consistent with drug-tolerant phenotypes of clinical solid tumors.Therefore,the 3D model is superior to the 2D model in recapitulating clinically relevant drug resistance and tolerance and can be utilized for investigating drug resistance mechanisms and for screening/validating candidate drugs or combination regimens.