Dev Proj 3: Imaging Tumor HIF-1 activity in a Rat Orthotopic Brain Tumor Model...

试点项目3：用核磁共振成像技术对大鼠原位脑瘤模型中肿瘤HIF-1活性进行成像 派：胡小平博士。共同派：申贤锡博士和陈炳良博士。 背景和目的：多形性胶质母细胞瘤(Gbm)是一种致命的疾病，尽管具有侵袭性。 外科手术和辅助治疗。基底膜肿瘤的显著特征是存在 假性扁平坏死和血管生成--一种引导代谢需求的异常新生血管 为了肿瘤的生长。肿瘤细胞需要特殊的代谢产物才能发育成三维的 球体和实体瘤。异质性肿瘤微环境与 基底节异常血管化的发展，通常包括扩张的毛细血管和渗漏的壁和 与正常组织的规则、有序的血管系统相比，血流迟缓[1，2]。尽管不断地 肿瘤细胞努力招募新血管，缺氧发生在距离血液150^m的肿瘤肿块中 供应，并倾向于广泛存在于实体肿瘤。实体瘤中存活的低氧细胞与 放射治疗和某些化疗失败，对治疗结果产生负面影响。的发展。 新的治疗方法将受益于小动物肿瘤缺氧模型和可以可视化的成像技术 并监测与肿瘤生长或治疗相关的缺氧的发展或抑制。肿瘤 这些缺氧区的细胞表达低氧诱导因子(HIF)-1，一种促进生存的转录因子。在……下面 如此低的氧浓度，HIF-1a被稳定下来，并与HIF-1P二聚形成活性转录 因素。该二聚体与位于靶基因启动子上的DNA序列5‘-RCGTG-3’(HRE)结合。 这随后导致促进肿瘤生长和血管生成的因素上调，包括 糖酵解酶和血管内皮生长因子[3，4]。这些数据表明，HIF-1是一种 有希望成为治疗基底膜的新靶点。因此，非侵入性成像方法对于 一个用于确定HIF-1活性。用于实时监测即将到来的抗HIF-1治疗的治疗反应和 确定抗HIF-1治疗是否适合患者。此前，我们评估了2-甲氧基雌二醇 HIF-1抑制剂(2ME2)对9L大鼠原位胶质肉瘤模型具有治疗作用 MRI测量肿瘤体积，生物发光成像(BLI)检测HIF-1活性[5]。生成9L单元格 报告HIF-1活性，我们稳定地将9L大鼠胶质瘤细胞与荧光素酶表达载体(V6R)共转染 监测HIF-1活性和pCDNA3.1用于药物选择(荧光素酶载体：pCDNA 3.1的混合比例=5：1) 使用脂质体2000(Invitgen)。V6R HIF-1报告质粒含有荧光素酶基因，其 表达受来自血管内皮生长因子的缺氧反应元件(HRE)的六个串联拷贝的驱动 基因启动子[6]。单细胞G418抗性克隆在低氧条件下荧光素酶活性升高 条件进行了选择。将9L-V6R细胞立体激活注射到Fischer 344大鼠脑内，以 建立原位脑瘤模型。2ME2治疗始于肿瘤种植后第8天， 持续了9天。在2ME2治疗前后(第8天)监测肿瘤生长和药物反应 17)Gd后T1加权磁共振成像[5]。使用异种基因的BLI法监测肿瘤HIF-1活性 配备Living Image软件的小动物成像系统(IVIS成像系统)。在做完核磁共振和 BLI，随后进行组织学分析以阐明药物作用机制。通过以下方式治疗 2ME2(60~600 mg/kg/d)可剂量依赖性地缩小肿瘤体积[5]。这种影响也是 与皮蒙硝唑染色评估的肿瘤氧合改善有关，HIF-1α蛋白增加 在组织学上可见微管不稳定。尽管V6R驱动的荧光素酶的使用 成像能够显示体内HIF-1的活性(见图1)，BLI数据与 由于缺乏深层组织穿透和3D断层扫描信息，组织学数据不足。因此，a 该方法结合了像BLI这样的报告肿瘤活动的遗传过程的能力和高分辨率 三维解剖信息将满足肿瘤分子成像的迫切需求。尽管核磁共振提供了 在所有成像方式中，它具有优越的解剖细节和组织对比度，但其敏感性较低。 在分子成像中的应用。目前使用的大多数造影剂都是外源性的，不适合 直接监测体内的遗传事件。

Pilot project 3: Imaging tumor HIF-1 activity in a rat orthotopic brain tumor model by MRI PI: Xiaoping Hu, Ph.D. Co-Pis: Hyunsuk Shim, Ph.D. and Anthony Chan, Ph.D. Background and Specific Aims: Glioblastoma multiforme (GBM) is a fatal disease despite aggressive surgical and adjuvant therapies. The distinguishing hallmark of GBM tumors is the presence of pseudopalisading necrosis and angiogenesis, an abnormal neovasculature that channels the metabolic needs for tumor growth. Neoplastic cells have a special need for metabolites in order to develop into threedimensional spheroids and solid tumors. Heterogeneous tumor microenvironment is associated with the development of abnormal vascularization in GBM, often consisting of distended capillaries with leaky walls and sluggish flow as compared with the regular, ordered vasculature of normal tissues [1, 2]. Despite the constant effort of tumor cells to recruit new blood vessels, hypoxia occurs in tumor masses 150 ^m away from the blood supply and tends to be widespread in solid tumors. Viable hypoxic cells in solid tumors are associated with the failure of radiation and certain chemotherapies, negatively impacting treatment outcomes. The development of new therapies will benefit from small-animal tumor-hypoxia models and imaging techniques that can visualize and monitor the development or inhibition of hypoxia associated with the tumor growth or treatment. The tumor cells in these hypoxic areas express hypoxia-inducible factor (HIF)-1, a pro-survival transcription factor. Under such low oxygen concentrations, HIF-1a is stabilized and dimerizes with HIF-1P to form an active transcription factor. The dimer binds to the DNA sequence 5'-RCGTG-3' (HRE), located in the promoter of target genes. This subsequently leads to up-regulation of factors that promote tumor growth and angiogenesis, including glycolytic enzymes and vascular endothelial growth factor (VEGF) [3, 4]. These data suggest that HIF-1 is a promising new target for the treatment of GBM. Thus, non-invasive imaging methods are highly desirable for one to determine HIF-1 activity.to monitor therapeutic response in real-time upcoming anti-HIF-1 therapies and to determine if anti-HIF-1 therapy is suitable for patients. Previously, we evaluated whether 2-methoxyestradiol (2ME2), an HIF-1 inhibitor, has therapeutic potential for tumor in a 9L rat orthotopic gliosarcoma model using MRI, to measure tumor volume, and bioluminescence imaging (BLI) for HIF-1 activity [5]. To generate 9L cells reporting HIF-1 activity, we stably co-transfected 9L rat glioma cells with a luciferase expression vector (V6R) to monitor HIF-1 activity and pCDNA3.1 for drug selection (mixture ratio of luciferase vector: pCDNA 3.1 = 5:1) using Lipofectamine 2000 (Invitrogen). The V6R HIF-1 reporter plasmid contains a luciferase gene whose expression is driven by six tandem copies of the hypoxia-responsive element (HRE) derived from the VEGF gene promoter [6]. Single cell G418-resistant clones showing elevated luciferase activity under hypoxic conditions were selected. The 9L-V6R cells were stereotactivally injected into the brains of Fischer 344 rats to establish an orthotopic brain tumor model. 2ME2 treatment was initiated from 8th day of tumor implantation and lasted 9 days. Tumor growth and drug response were monitored before and after the 2ME2 treatment (days 8 and 17) by post-gadolinium T1-weighted MRI [5]. Tumor HIF-1 activity was monitored by BLI using a Xenogen Small Animal Imaging system (IVIS¿ Imaging System) equipped with Living Image software. After MRI and BLI, histological analysis was subsequently performed to elucidate the drug action mechanism. Treatment with 2ME2 (60 - 600 mg/kg/day) resulted in a dose-dependent reduction in tumor volume [5]. This effect was also associated with improved tumor oxygenation as assessed by Pimonidazole staining, increased HIF-1 a protein levels, and microtubule destabilization as seen with histology. Although the use of V6R-driven luciferase imaging was able to reveal the HIF-1 activity in vivo (see Fig. 1), the BLI data were not consistent with histological data due to a lack of deep tissue penetration and 3D tomographic information. Therefore, a method that combines the ability of reporting genetic processes of tumor activity like BLI and high resolution 3D anatomic information will fulfill the urgent need in molecular imaging of cancer. Although MRI provides superior anatomic details and tissue contrast among all imaging modalities, it suffers from low sensitivity in its application in molecular imaging. Most of contrast agents used currently are exogenous and inappropriate for directly monitoring the genetic events in vivo.