BRAF定义的犬尿路上皮癌类器官作为药物反应评估的比较肿瘤学平台

Canine urothelial carcinoma, also referred to as transitional cell carcinoma (TCC), is the most common malignancy of the canine urinary tract, with the BRAF V595E mutation reported in approximately 70-85% of cases. This mutation is the canine homolog of the human oncogenic BRAF mutation, which is frequently detected in melanoma and papillary thyroid carcinoma. However, whereas BRAF mutation is a potent therapeutic target in human cancers, clinical efficacy in canine TCCs remains unclear, highlighting the need for additional research. In this study, cancer organoids were established from surgically resected tissues of canine patients six BRAF V595E-mutated TCCs, two BRAF wildtype TCCs, and two cystitis samples. The organoids expressed urothelial markers (cytokeratin-7, uroplakin-3A) and retained BRAF status identical to the parental tumors. Organoids harboring BRAF mutation showed enhanced proliferation and motility, consistent with Ki-67 and vimentin expression. To validate these in vitro results, canine TCC organoids were injected into mice via the tail vein. After seven weeks, all injected mice developed multiple pulmonary nodules, confirming strong metastatic capacity. Tumors were re-cultured as organoids, which retained aggressive phenotypes ex vivo alongside sustained ERK phosphorylation and the BRAF V595E mutation. Organoid-based in vitro assays demonstrated that six targeted inhibitors commonly used in human cancers effectively reduced phosphorylated-ERK expression. Among these agents, sorafenib showed the strongest cytotoxic activity. In xenografted mice, six weeks of oral administration of sorafenib or vemurafenib, markedly reduced tumor formation. Mice treated with BRAF inhibitors also showed decreased p-ERK expression in metastatic lesions and lower BRAF mutant allele fractions in circulating tumor DNA, indicating suppression of metastatic progression through inhibition of the BRAF/MAPK pathway. Collectively, our findings demonstrate that canine TCC organoids function as a comparative model bridging human and canine cancers, enabling us to dissect BRAF-dependent oncogenic mechanisms, evaluate anti-metastatic drug efficacy, and monitor treatment response via ctDNA.