Tumor models for the study of inflammation and oncogenesis

小鼠肿瘤模型是最有用的，当它们复制尽可能多的已知分子损伤，以便最准确地描述身体对肿瘤发生的生理反应。有了体细胞工程(睡美人/SB)，我们可以轻松地同时重现多个病变，而且规模是其他方法无法比拟的。肝细胞癌是世界上第三大致癌原因，由于许多流行病学和生物学原因，如肥胖，其发病率不断上升。此外，肝脏也是源于其他器官实体瘤的转移的经常储存库。我们发展了由癌基因引发的肝肿瘤，它概括了肝细胞癌和肝细胞腺瘤的发展和进展。我们的第一个目标是利用报告基因来优化和量化肝脏肿瘤的发展。到目前为止，我们已经通过SB-AKT和SB-β-连环蛋白(CAT)的共同传递复制了肝细胞腺瘤(HCA)，并通过SB-MET和SB-CAT的共同传递复制了肝细胞(HCC)。相比之下，单独使用这些癌基因并不会导致肿瘤的形成。我们正在使用共同传递的赤子荧光素酶转座子来跟踪肿瘤的生长，从癌基因启动的肝细胞到公开的肿瘤形成。Gaussia荧光素酶被分泌到血液中，从而允许对基因传递和肿瘤发展的非侵入性、定量测量。此外，表达红色或绿色荧光蛋白的共传递转座子将用于标记癌基因启动的肝细胞，使用肿瘤成像和流式细胞仪分选。我们的第二个目标是确定体内AKT+CAT和MET+CAT致癌途径的免疫学贡献。使用RAG1-/-免疫缺陷小鼠，我们发现这些小鼠对AKT+CAT(HCA)肝肿瘤的发展具有显著的保护作用。这一发现表明，宿主免疫系统在AKT+CAT启动的HCA的发展过程中相互协作，并提供了必要的贡献因素。相反，当使用MET+CAT时，RAG1-/-小鼠更快地死于肿瘤发展，这表明在MET+CAT启动的肝细胞癌的免疫介导监测中发挥了重要作用。我们正在进行的使用T细胞和B细胞缺陷小鼠的研究表明，AKT+CAT诱导的肝骨病的发展和整体肿瘤的生长依赖于B细胞，而不是CD4+或CD8+T细胞。B细胞缺陷小鼠的肝脏脂肪变性也显著减少，这是非酒精性脂肪性肝病的一个特征，也是肝脏肿瘤发展的易感性。B细胞的促癌作用是有争议的，但与某些慢性炎症模型中这些细胞的促癌作用是一致的。我们假设，当启动突变与促进肿瘤的炎性微环境相结合时，肿瘤就会发生。因此，在本项目的第三个目标中，我们将确定那些能够调节AKT+CAT或MET+CAT启动的肿瘤的细胞因子和炎症网络。我们使用免疫缺陷小鼠的研究结果发现，在AKT+CAT模型中，TNFR1和LTbetaR信号转导具有关键的肿瘤促进作用，因为这些受体或其配体缺陷的小鼠减少了肿瘤生长并提高了存活率。尤其是B细胞相关的LTβ，在AKT+CAT诱导的肝纤维化和肝脂肪变性中可能起着不可或缺的作用。我们正在监测野生型和B细胞缺陷小鼠的肿瘤进展，这些小鼠已经接受了LTbetaR激动剂和拮抗性LTbetaR结合试剂的治疗。其他B细胞衍生因子的参与也将被调查。MET+CAT的其他研究将完成，以调查肿瘤进展为癌症的癌基因特异性炎症介质。总而言之，本项目中描述的方法将为我们提供准确的肝细胞癌和肝细胞癌的临床前模型，并促进更有效地针对肿瘤发生的治疗方法的发展。

Mouse tumor models are most useful when they reproduce as many known molecular lesions as possible, so as to most accurately portray the bodys physiological response to tumorigenesis. With somatic cell engineering (Sleeping Beauty/SB), we can recapitulate multiple lesions simultaneously with ease and scale not possible by other means. Hepatocellular carcinoma is the third leading cause of cancer worldwide, with increasing incidence due to a number of epidemiologic and biologic reasons, such as obesity. Moreover, the liver is a frequent depot for metastases originating from solid tumors arising in other organs as well. We have developed oncogene-initiated liver tumors which recapitulate the development and progression of hepatocellular carcinoma (HCC) and hepatocellular adenoma (HCA). Our first Aim is to optimize and quantitate hepatic tumor development, using reporter genes. To date, we have reproducibly generated hepatocellular adenomas (HCA) by the co-delivery of SB-AKT and SB-beta-catenin (CAT), and hepatocellular (HCC) by the co-delivery of SB-MET and SB-CAT. In contrast, individual delivery these oncogenes does not induce tumor formation. We are using a co-delivered Gaussia luciferase transposon to track tumor growth from oncogene-initiated hepatocytes to overt tumor formation. Gaussia luciferase is secreted into the blood thereby allowing for a non-invasive, quantitative measure of gene delivery and tumor development. Additionally co-delivered transposons expressing red or green fluorescent protein will be used to tag oncogene-initiated hepatocytes using tumor imaging and flow cytometric sorting. Our second Aim is to define the immunological contributions to AKT+CAT and MET+CAT oncogenic pathways in vivo. Using RAG1-/- immunodeficient mice, we found that these mice were significantly protected from the development of AKT+CAT (HCA) liver tumors. This finding suggests that the host immune system collaborates and provides essential contributing factors towards the development of AKT+CAT initiated HCA. In contrast, RAG1-/- mice succumbed more rapidly to tumor development when MET+CAT was used, suggesting an important role for immune-mediated surveillance of MET+CAT initiated HCC. Our ongoing studies using T cell- and B cell-deficient mice suggest that AKT+CAT induced hepatosteatosis development and overall tumor growth is dependent on B cells, but not CD4+ or CD8+ T cells. B cell deficient mice also have a dramatic reduction in hepatic steatosis, a characteristic feature underlying non-alcoholic fatty liver disease and predisposition to liver tumor development. The tumor-promoting role of B cells is controversial, but consistent with a tumor-promoting role that has been proposed for these cells in some chronic inflammatory models.We hypothesize that tumors can develop when initiating mutations are coupled with inflammatory microenvironments that enhance tumor promotion. Therefore, in the third Aim of this project, we will identify those cytokines and inflammatory networks capable of modulating AKT+CAT or MET+CAT initiated tumors. Our results from studies using immunodeficient mice have identified a critical tumor promoting role for TNFR1 and LTbetaR signaling in the AKT+CAT model, since mice deficient in these receptors or their ligands have reduced tumor growth and improved survival. B cell associated LT beta, in particular, may therefore play an indispensable role in AKT+CAT induced HCA and hepatic steatosis. We are monitoring tumor progression in wildtype and B cell deficient mice that have been treated with LTbetaR agonistic and antagonistic LTbetaR-binding reagents. The involvement of additional B cell-derived factors will also be investigated. Additional studies with MET+CAT will be completed to investigate oncogene specific inflammatory-mediators of tumor progression to carcinoma. Collectively, the approaches described in this project will provide us with accurate pre-clinical models for HCA and HCC and facilitate the development of therapeutics that more effectively target tumorigenesis.