结直肠癌腹膜转移患者来源的转移灶和腹水类器官模型的构建及鉴定OA

Background and purpose:Colorectal cancer peritoneal metastasis(CRC-PM)represents one of the lethal metastatic patterns of colorectal cancer and is frequently characterized by malignant ascites,diffuse peritoneal seeding,and poor responsiveness to systemic therapy.A major barrier to mechanistic and translational research in CRC-PM is the lack of reproducible,expandable in vitro models that faithfully capture patient's tumour biology.Patient-derived organoid(PDO)can be maintained long-term in three dimension(3D)culture while preserving tumour-associated phenotypes and genomic features;however,PDO establishment and rigorous characterization specifically for CRC-PM remain limited.Here,we aimed to establish stably expandable CRC-PM PDO derived from metastatic lesions and ascites,generate matched two dimension(2D)tumour cell lines,and evaluate their biological concordance with the corresponding tumour specimens across histomorphology,protein expression,and genomic profiles.Methods:Clinical specimens were obtained from patients with CRC-PM.Peritoneal metastatic lesions and ascites were processed by stepwise dissociation to establish 3D PDO cultures and matched PDO-derived 2D tumour cell lines.Organoid morphology was monitored longitudinally by bright-field microscopy.PDO was recovered and processed into cell blocks for H-E staining to assess epithelial/glandular architecture.Immunohistochemistry(IHC)was performed to compare pathological features between PDO and matched primary tumours;IHC signals were assessed using integrated optical density(IOD)for descriptive semi-quantification.Whole-exome sequencing(WES)was conducted on primary tumour tissues,PDO,and matched 2D cell lines to delineate somatic mutational landscapes and mutation-type compositions.Results:We successfully established CRC-PM PDO derived from both peritoneal metastatic lesions and ascites,which could be stably expanded and serially passaged,and generated the corresponding PDO-derived 2D tumour cell lines through repeated subculture.Both organoid models formed typical cystic and/or compact spheroid 3D structures.H-E staining of PDO cell blocks demonstrated epithelial,gland-like histological features.IHC analysis showed that PDO closely recapitulated the histomorphology and the expression patterns of key tumour markers(including CK20,E-cadherin,pan-cytokeratin and β-catenin)and Ki-67 proliferation index observed in the matched primary tumours,with no apparent differences in overall staining intensity across these markers.WES further confirmed a high degree of concordance in recurrently mutated genes(such as TTN and FAT2)and in mutation-type composition among PDO,matched 2D cell lines,and corresponding primary tumour tissues.Conclusion:We established and validated paired CRC-PM PDO derived from peritoneal metastatic lesions and ascites,together with matched PDO-derived tumour cell lines.These models largely preserve the key pathological and genomic features of the corresponding primary tumours and provide a reproducible experimental platform for mechanistic studies and personalised drug testing in CRC-PM.