恶性腹膜间皮瘤裸小鼠原位模型的建立及其生物学性状鉴定

Establishment and characterization of patient derived xenograft model of malignant peritoneal mesothelioma in nude mice

目的建立恶性腹膜间皮瘤(MPM)原位人源动物模型(PDX模型),鉴定其生物学特征,为研究MPM病理机制及探索诊治新技术提供实验平台。方法将术中MPM标本接种于BALB/c-nu/nu裸小鼠皮下,进行3次传代,稳定后取第4代皮下瘤制成瘤细胞匀浆,以100、200μL剂量接种于裸小鼠腹腔,建立PDX模型,鉴定其生物学性状。结果成功建立MPM裸小鼠皮下瘤及腹腔瘤模型,皮下瘤模型接种第20天成瘤,第20~29天为缓慢生长期,第30~57天为快速生长期。根据接种瘤细胞剂量(100、200μL)及解剖时机(接种第14、69天)不同,腹腔瘤模型成功模拟了MPM临床早、晚期。MPM模型肿瘤组织HE染色为上皮样间皮瘤,肿瘤侵袭肝、脾、胰腺、肠系膜等脏器。免疫组织化学示Calretinin、细胞角蛋白(CK)5/6、WT1、Ki-67阳性。全外显子测序PDX模型及患者肿瘤组织分别发现26、36个较高频率基因突变,模型和患者共有基因突变21个。结论建立MPM的PDX模型,具备恶性程度高、生长速度快、侵袭性强的生物学特征,可为后续的MPM研究提供技术平台。

Objective To establish patient derived xenograft(PDX)model of malignant peritoneal mesothelioma(MPM),and to identify the key characteristics of tumor biology of the model,so as to provide an experiment platform for studying the pathologic mechanisms and new therapeutic strategies for MPM.Methods Surgically excised MPM tumor tissues were inoculated subcutaneously in BALB/c-nu/nu mice for 3 stable passages.In the 4th passage,the subcutaneous tumors were harvested under aseptic conditions,cleaned and made into MPM tumor cell homogenate.Four nude mice(two males and two females)were selected and one male and one female nude mouse were inoculated in the abdominal cavity at the dose of 100μL,others were inoculated at a dose of 200μL.The PDX model of MPM was established.The changes of body mass in nude mice were measured regularly,the extent of abdominal and pelvic tumors was judged by experimental peritoneal cancer index(ePCI)score,and the pathologic characteristics of tumors were analyzed.Results The subcutaneous and abdominal animal models of MPM were successfully established.The subcutaneous tumor model grew into tumor on the 20th day,followed by a slow growth stage between the 20th and 29th day,then a rapid growth stage between the 30th and 57th day.According to the dose of tumor cells(100,200μL)and timing(14th and 69th days after grafting),the abdominal tumor model successfully simulated the early and late clinical stages of MPM.The HE staining results of the MPM nude mice model showed that the tumor was epithelial mesothelioma and invaded most of the organs,including liver,spleen,pancreas,mesentery.Immunohistochemical staining for calretinin,cytokeratin 5/6,WT1 and Ki-67 were positive.Whole-genome exon sequencing identified 26 and 36 high frequency gene mutations in tumors derived from the PDX model and clinical sample from patients,including 21 common gene mutations.Conclusions The PDX model of MPM is established.The model is characterized by highly malignant tumor with rapid growth and high invasiveness.