基于临床肿瘤标本的胃癌个体化转移模型的建立及评估

It is very important to establish an individual gastric tumor metastasis model to simulate clinical characteristics. The patient derived xenograft (PDX) model established by transplantation clinical fresh gastric cancer specimen into immunodeficiency mice well maintains the heterogeneity of the primary tumor, but there is little metastasis. It is reported that orthotopic transplantation can promote tumor metastasis due to provide a suitable microenvironment for tumor growth. Our study also found that six cases of gastric tumor tissue from PDX model after implanted into stomach of nude mice displayed distant organ metastasis. Moreover, 6 metastasis relative genes were highly expressed in these three PDX tumor tissues, including VEGF-A, VEGF-B, CD44, MMP-9, CXCR4 and NEDD9. So we speculate that gastric cancer tissues with high expression of these 6 genes have metastasis potential, can be further screened for individual metastasis model by orthotopic transplantation. This project is designed to detect the expression of these 6 genes, screening the high metastatic potential of gastric cancer PDX model. These tumor tissues will be implanted into stomach of severe combined immunodeficient NPG mice to obtain the individual gastric cancer metastatic model. By near infrared fluorescence (NIRF) animal imaging technique we evaluate the reliability of heptamethine carbocyanine dye for detection of metastatic tumors. By gene sequencing, IHC and FISH analysis the tumor related gene expression in metastasis model are detected. By PCR-Arrays analysis the metastasis relative genes of gastric cancer are screened. We will further standardized the establishment and evaluation methods of gastric cancer metastasis model, so as to provide an individual experimental model for the mechanism and treatment of gastric cancer metastasis.

建立模拟临床特征的个体化转移模型对于胃癌研究具有重要意义。通过肿瘤标本异种移植建立的PDX模型较好地保持了原发瘤的特征，但少有转移发生。由于原位移植可提供适宜的微环境促进肿瘤转移，我们将六例胃癌PDX模型的瘤组织移植裸鼠胃部后均发生转移，且6种转移相关基因VEGF-A，VEGF-B，CD44，MMP-9，CXCR4和NEDD9均高表达。推测这6种基因高表达的PDX模型瘤组织通过原位移植可获得胃癌个体化转移模型。本项目计划通过检测上述6种基因的表达，筛选出高转移潜能的胃癌PDX模型，将其瘤组织原位移植NPG小鼠获得转移模型；评估近红外荧光活体成像技术检测转移瘤的可靠性；通过基因测序、IHC和FISH分析转移模型中肿瘤相关基因与原发瘤的相似性和特异性；使用PCR-Array技术初步筛选出胃癌转移相关基因；规范模型的制备与评估方法，以期为胃癌转移发生机制和治疗研究提供良好的个体化实验模型。

本课题先后建立了25例人胃癌PDX模型，并筛选获得20例胃癌个体化转移模型。进一步通过PCR-Array技术比较了PDX模型肿瘤组织与癌旁组织中VEGF-A、VEGF-B、CD44、MMP-9、CXCR4和NEDD9基因表达水平，筛选出5例高转移潜能PDX模型进行研究，分别为C19751，C65175，C15715，B86917和B97493。通过基因测序和IHC分析了转移模型中肿瘤相关基因与原发瘤的相似性和特异性。将筛选的PDX模型注射肿瘤靶向性近红外荧光染料DZ-1，活体成像结果显示DZ-1荧光信号特异性集聚在肿瘤部位，同时在胃部、肝部和肺部出现荧光信号，提示高转移潜能胃癌PDX模型构建成功，此技术为胃癌转移模型的制备提供了可靠的检测方法。之后采用PCR-Array技术分析了三例配对标本（C19751原发瘤与转移瘤；C65175原发瘤和转移瘤；B97493原发瘤和转移瘤）转移相关基因的表达，成功筛选出转移相关基因MMP2、FXYD5和EPHB2。通过细胞划痕实验，Transwell实验和裸鼠体内移植实验证实MMP2、FXYD5可促进胃肿瘤细胞的侵袭和迁移，并且证实MMP2通过促进EMT及减少细胞凋亡促进胃癌转移，FXYD5主要通过EMT途径增强胃癌细胞的耐药性以及转移潜能。进一步研究了胃癌细胞中EPHB2的表达水平，筛选出与EPHB2表达相关的同源配体EFNA5，生信分析显示EPHB2在胃癌组织中高表达。以上研究结果显示，成功构建了胃癌转移性PDX模型，并筛选出转移相关基因MMP2、FXYD5和EPHB2，为胃癌转移发生机制和治疗研究提供了良好的个体化实验模型。