Connolly Nina P, Stokum Jesse A, Schneider Craig S, Ozawa Tatsuya, Xu Su, Galisteo Rebeca, Castellani Rudolph J, Kim Anthony J, Simard J Marc, Winkles Jeffrey A, Holland Eric C, Woodworth Graeme F
Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
PLoS One. 2017 Mar 30;12(3):e0174557. doi: 10.1371/journal.pone.0174557. eCollection 2017.
Previously rodent preclinical research in gliomas frequently involved implantation of cell lines such as C6 and 9L into the rat brain. More recently, mouse models have taken over, the genetic manipulability of the mouse allowing the creation of genetically accurate models outweighed the disadvantage of its smaller brain size that limited time allowed for tumor progression. Here we illustrate a method that allows glioma formation in the rat using the replication competent avian-like sarcoma (RCAS) virus / tumor virus receptor-A (tv-a) transgenic system of post-natal cell type-specific gene transfer. The RCAS/tv-a model has emerged as a particularly versatile and accurate modeling technology by enabling spatial, temporal, and cell type-specific control of individual gene transformations and providing de novo formed glial tumors with distinct molecular subtypes mirroring human GBM. Nestin promoter-driven tv-a (Ntv-a) transgenic Sprague-Dawley rat founder lines were created and RCAS PDGFA and p53 shRNA constructs were used to initiate intracranial brain tumor formation. Tumor formation and progression were confirmed and visualized by magnetic resonance imaging (MRI) and spectroscopy. The tumors were analyzed using histopathological and immunofluorescent techniques. All experimental animals developed large, heterogeneous brain tumors that closely resembled human GBM. Median survival was 92 days from tumor initiation and 62 days from the first point of tumor visualization on MRI. Each tumor-bearing animal showed time dependent evidence of malignant progression to high-grade glioma by MRI and neurological examination. Post-mortem tumor analysis demonstrated the presence of several key characteristics of human GBM, including high levels of tumor cell proliferation, pseudopalisading necrosis, microvascular proliferation, invasion of tumor cells into surrounding tissues, peri-tumoral reactive astrogliosis, lymphocyte infiltration, presence of numerous tumor-associated microglia- and bone marrow-derived macrophages, and the formation of stem-like cell niches within the tumor. This transgenic rat model may enable detailed interspecies comparisons of fundamental cancer pathways and clinically relevant experimental imaging procedures and interventions that are limited by the smaller size of the mouse brain.
以前,啮齿动物在神经胶质瘤方面的临床前研究经常涉及将C6和9L等细胞系植入大鼠脑内。最近,小鼠模型占据了主导地位,小鼠的基因可操作性使得创建基因精确模型的优势超过了其脑体积较小限制肿瘤进展时间的劣势。在此,我们阐述一种利用具有复制能力的禽肉瘤病毒(RCAS)/肿瘤病毒受体-A(tv-a)转基因系统在大鼠中形成神经胶质瘤的方法,该系统可实现出生后细胞类型特异性基因转移。RCAS/tv-a模型已成为一种特别通用且精确的建模技术,它能够对单个基因转化进行空间、时间和细胞类型特异性控制,并为新生的胶质肿瘤提供具有不同分子亚型的肿瘤,这些亚型反映了人类胶质母细胞瘤(GBM)的特征。构建了巢蛋白启动子驱动的tv-a(Ntv-a)转基因斯普拉格-道利大鼠始祖品系,并使用RCAS PDGFA和p53 shRNA构建体启动颅内脑肿瘤形成。通过磁共振成像(MRI)和光谱学确认并观察到肿瘤的形成和进展。使用组织病理学和免疫荧光技术对肿瘤进行分析。所有实验动物都形成了大的、异质性脑肿瘤,与人类GBM极为相似。从肿瘤起始算起,中位生存期为92天,从MRI上首次观察到肿瘤算起为62天。通过MRI和神经学检查,每只荷瘤动物都显示出恶性进展为高级别神经胶质瘤的时间依赖性证据。死后肿瘤分析表明存在人类GBM的几个关键特征,包括高水平的肿瘤细胞增殖、假栅栏状坏死、微血管增殖、肿瘤细胞侵入周围组织、肿瘤周围反应性星形胶质细胞增生、淋巴细胞浸润、存在大量肿瘤相关小胶质细胞和骨髓来源的巨噬细胞,以及肿瘤内形成干细胞样微环境。这种转基因大鼠模型可能有助于对基本癌症途径以及受小鼠脑体积较小限制的临床相关实验成像程序和干预措施进行详细的种间比较。