鼻咽癌类器官疾病模型的建立与药敏初步试验

Establishment of an organoid model of nasopharyngeal carcinoma and its in vitro chemosensitivity profile

目的探索构建鼻咽癌类器官的体外培养方法,利用类器官模型进行个性化药物敏感性的初步检测。方法鼻咽镜活检或者鼻内镜手术获取鼻咽癌患者肿瘤组织,剪切、消化、过滤后获取鼻咽癌细胞,用基质胶重悬鼻咽癌细胞并接种于培养皿,3 D立体培养类器官;提取鼻咽癌类器官制成石蜡切片,进行形态学,Ki67、CD133免疫组化,CD44免疫荧光和EBER原位杂交鉴定,并与亲本肿瘤组织对比;鼻咽癌类器官培养5 d后,加入紫杉醇、顺铂、卡铂、吉西他滨和长春瑞滨处理,用中性红染色法测定细胞活力,分析药敏结果。结果鼻咽癌类器官核异型性明显,与亲本鼻咽癌组织一致。鼻咽癌类器官CD133免疫组化和CD44免疫荧光强阳性,提示鼻咽癌类器官是肿瘤干细胞的富集,Ki67细胞比例约占30%,类器官具备一定增殖能力。化疗药物作用后,鼻咽癌类器官对卡铂、顺铂、长春瑞滨高度敏感,吉西他滨中度敏感,替吉奥低度敏感。结论成功建立了鼻咽癌类器官体外培养体系,可初步用于体外药敏试验。

Objective To explore the method for in vitro culture of an organoid model of nasopharyngeal carcinoma(NPC)and examine its in vitro chemosensitivity profile.Methods Fresh specimens of NPC tissues were obtained from clinical patients during nasopharyngoscopic biopsy or nasal endoscopic surgeries.The tumor tissues were cut into pieces,digested with mixed digestive juice and filtered.The NPC cells were isolated by centrifugation,resuspended with Matrix gel and AMDM/F12,and inoculated in a Petri dish for three-dimensional culture of the organoids.Paraffin sections of the NPC organoids cultured for 5 to 7 d were prepared for morphological examination and immunohistochemistry for Ki67 and CD133,CD44 immunofluorescence assay,and EBER in situ hybridization.The NPC organoids cultured for 5 d were tested for chemosensitivity to paclitaxel,cisplatin,carboplatin,gemcitabine and vinorelbine by assessing the cell viability with neutral red staining following treatments with the drugs.Results The cultured NPC organoids showed obvious nuclear atypia,which was consistent with that of the source NPC tissues.The NPC organoids were strongly positive for CD133 and CD44,suggesting the enrichment of tumor stem cells in the organoids.Nearly 30%of the cells in the organoids were positive for Ki67,indicating the proliferative capacity of the organoids.Chemosensitivity tests showed that the NPC organoids were highly sensitive to carboplatin,cisplatin and vinorelbine,and moderately sensitive to gemcitabine,but with a low sensitivity to tigio.Conclusion We successfully established an in vitro culture system of NPC organoids,which can serve as a new NPC model for testing drug sensitivities.