Genetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

基因工程小鼠（GEM）是癌症建模的金标准。然而，利用目前使用GEM的现有技术，从成年基因完整细胞中的单个肿瘤起始上皮细胞中严格地逐步概括癌变是不可行的。在以前的研究中，我们通过从成年动物体内分离器官，然后在类器官中进行基因工程和在裸鼠中进行皮下接种，部分地克服了这一挑战。尽管建立了适用于各种组织的合适的体外致癌模型，但肿瘤的发展仍然是异位的，并且是在免疫缺陷条件下发生的。因此，有必要进一步完善以建立理想的模型。由于缺乏胆囊（GB）特异的Cre，因此预后较差，模型很少我们假设开发出真实的模型将极大地有益于GB癌症研究。在这里，我们建立了使用慢病毒Cre体外产生的具有突变型Kras和Trp53缺失的GB类器官的新型模型转导和CRISPR / Cas9基因编辑。在同基因小鼠中，将类器官源性皮下肿瘤片段缝合到GB的外表面，该小鼠发育出原位肿瘤，其组织学和转录特征类似于人GB癌症。该模型揭示了皮下和原位肿瘤中免疫细胞类似亚群的浸润，证实了在致癌过程中适当的免疫环境。此外，我们在大型队列中准确验证了吉西他滨（一种用于GB癌症的常见治疗剂）的体内疗效。综上所述，该模型可作为药物发现和精密医学领域中GB癌症患者的有希望的化身。