Understanding Mechanisms of Resistance to Anti-angiogenic Treatments

描述(申请人提供)：目前恶性胶质母细胞瘤(GBM，脑瘤)的存活期通常不到一年。GBM的特征是释放血管内皮生长因子(VEGF)，它是新血管形成(血管生成)和血管通透性的重要调节和促进因素。由于临床试验的初步阳性反应，人们认为以血管内皮生长因子或血管内皮生长因子受体(VEGFRs)为靶点的抗血管生成治疗将成为控制GBM的有效工具。然而，现在已经很好地认识到，阳性反应是暂时的，肿瘤会产生耐药性并最终进展，在某些情况下会出现更具侵袭性和侵袭性的表型。要克服这一失败，必须了解抗血管生成治疗耐药的基本机制。我们假设，使用VEGFR抑制剂(如维他那利)进行抗血管生成治疗将导致血管丢失和内皮细胞(EC)存活率下降，从而促进其他促血管生成因子的代偿性增加，如基质细胞衍生因子1(SDF-1)。SDF-1反过来将诱导内皮祖细胞在肿瘤周围或进入肿瘤内的动员、迁移和积聚。已知骨髓来源的前体细胞可以促进血管生成和促生长反应，并可能是耐药的机制之一。然而，目前尚不清楚抗血管内皮生长因子治疗期间内皮祖细胞是否在肿瘤中积聚，或者减少骨髓细胞或中和SDF-1是否有助于克服抗血管生成治疗的耐药性。使用以多个血管生成部位为靶点的新型化合物来克服抗血管生成耐药性将是有利的。HET0016是靶向多个血管生成部位的化合物之一。在本方案中，我们将建立裸鼠原位人脑胶质瘤模型。荷瘤动物将接受Vetanalib或HET0016单独或联合治疗。动态增强磁共振成像(DCE-MRI)将在基础和治疗条件下测定肿瘤的通透性转移常数(KTRANS)、分布(肿瘤血液)体积、肿瘤体积、增强方式和扩散参数。Western印迹和RT-PCR分析将确定不同血管生成因子/受体的表达水平，而免疫组织化学将评估血管密度和形态。我们预计，耐药肿瘤的大小、渗透性、肿瘤血流量和其他MRI参数将发生变化。这些数据可能提供代偿性/难治性血管生成的直接证据，解释某些肿瘤对抗VEGFR治疗变得无效的原因，并确定HET0016可能是克服肿瘤对抗血管生成治疗耐药性的辅助治疗。本课题组先前的研究表明，由于肿瘤释放趋化细胞因子(如HIF-1介导的SDF-1释放)，移植的EPC迁移并整合到肿瘤血管生成中。为了确定减少可用的BM细胞或阻断可获得的SDF-1是否会减少甚至克服对抗VEGFR或HET0016治疗的耐药性。我们将使用亚致死剂量辐射来降低骨髓释放促血管生成细胞的能力。在一系列实验中，我们还将使用特定的抗体或CXCR4拮抗剂来阻断SDF-1，从而降低内皮祖细胞迁移到肿瘤的能力。静脉注射内皮祖细胞的初始迁移和掺入将通过使用In-111标记细胞的SPECT进行检测，而给药细胞的长期掺入将通过使用磁性标记细胞的细胞磁共振进行检测。这些数据将通过免疫组织化学进行验证。我们希望这项实验方案的结果将有助于阐明抗血管生成治疗的耐药机制，并允许临床医生改变未来对GBM或其他实体瘤的治疗策略。

DESCRIPTION (provided by applicant): At present survival from malignant glioblastoma (GBM, brain tumor) is usually less than a year. GBM are characterized by release of vascular endothelial growth factor (VEGF), an important regulator and promoter of new vessel formation (angiogenesis) and vascular permeability. Because of initial positive responses noted in clinical trials, it was thought that anti-angiogenic therapy targeting VEGF or VEGF receptors (VEGFRs) would become an effective tool for controlling GBM. However, it is now well recognized that the positive responses are temporary and that the tumors develop resistance and eventually progress, and in some cases with a more aggressive and invasive phenotype. To overcome the failure, it is essential to understand the basic mechanism of resistance to anti-angiogenic therapy. We hypothesize that anti-angiogenic treatment using VEGFR inhibitors (such as vetanalib) will cause hypoxia due to vascular loss and decreased endothelial cell (EC) survival, which will enhance compensatory increases in other pro-angiogenic factors such as stromal-cell derived factor 1 (SDF-1). SDF-1 in turn will induce the mobilization, migration and accumulation of endothelial progenitor cells (EPCs) around or into the tumor. Bone marrow (BM) derived precursor cells are known to promote angiogenesis and pro-growth responses and may be a mechanism for resistance. However it is not known whether EPCs accumulate in the tumor during anti VEGF therapy, or whether decreasing BM cells or neutralizing SDF-1 helps overcome resistance to anti- angiogenic therapy. It would be advantageous to use novel compounds that target multiple sites of angiogenesis to overcome anti-angiogenic resistance. HET0016 is one of the compounds that target multiple sites of angiogenesis. In this proposal we'll make orthotopic human glioma tumor model in nude rat. Tumor bearing animals will be treated either with vetanalib, or HET0016 alone or in combination. Dynamic contrast enhanced MRI (DCE-MRI) will be performed to determine permeability transfer constant (Ktrans), distribution (tumor blood) volume, tumor volume, enhancement pattern, and diffusion parameters in the tumors under basal and treated conditions. Western blot and RT-PCR analysis will determine the expression level of different angiogenic factors/receptors while immunohistochemistry will assess the vascular density and morphology. We expect that treatment resistance tumor will show changes in size, permeability, tumor blood volume, and other MRI parameters. The data may give direct evidence of compensatory/refractory angiogenesis, explain why certain tumors become refractory to anti VEGFR therapy and identify HET0016 as a possible adjuvant therapy in surmounting the tumor resistance to anti-angiogenic therapy. Previous studies by our group showed that transplanted EPCs migrated and incorporated in the tumor angiogenesis due to chemotactic cytokines released from the tumors (such as HIF-1 mediated SDF-1 release). To determine whether decreasing available BM cells or blocking accessible SDF-1, will diminish or even overcome resistance to anti VEGFR or HET0016 therapy. We will use sub lethal irradiation to lower the ability of the BM to release pro-angiogenic cells. In a set of experiments we will also block SDF-1 using specific antibodies or CXCR4 antagonists and thus reduce the ability of EPCs to migrate to the tumor. The initial migration and incorporation of intravenously administered EPCs will be detected by SPECT using In-111 labeled cells and the long term incorporation of the administered cells will be detected by cellular MRI using magnetically labeled cells. These data will be validated by immunohistochemistry. We expect the results of this experimental proposal will shade lights on the mechanisms of resistance to anti- angiogenic treatments and allow clinician to change the strategy of future treatment for GBM or other solid tumors.