Understanding Mechanisms of Resistance to Anti-angiogenic Treatments

描述（由申请人提供）：目前恶性胶质母细胞瘤（GBM，脑肿瘤）的生存期通常不到一年。GBM的特征在于血管内皮生长因子（VEGF）的释放，VEGF是新血管形成（血管生成）和血管渗透性的重要调节剂和促进剂。由于在临床试验中观察到的最初的阳性反应，认为靶向VEGF或VEGF受体（VEGFR）的抗血管生成疗法将成为控制GBM的有效工具。然而，现在已经充分认识到，阳性反应是暂时的，并且肿瘤产生耐药性并最终进展，并且在某些情况下具有更具侵袭性和侵袭性的表型。为了克服这一失败，有必要了解抗血管生成治疗抵抗的基本机制。我们假设，使用VEGFR抑制剂（如vetanalib）进行抗血管生成治疗将导致血管损失和内皮细胞（EC）存活率降低导致缺氧，这将增强其他促血管生成因子（如基质细胞衍生因子1（SDF-1））的代偿性增加。SDF-1又会诱导内皮祖细胞（EPC）在肿瘤周围或肿瘤内的动员、迁移和积聚。已知骨髓（BM）来源的前体细胞促进血管生成和促生长反应，并且可能是抗性的机制。然而，尚不清楚抗VEGF治疗期间EPCs是否在肿瘤中积累，或者减少BM细胞或中和SDF-1是否有助于克服抗血管生成治疗的抗性。使用靶向多个血管生成位点的新型化合物来克服抗血管生成抗性将是有利的。HET 0016是靶向多个血管生成位点的化合物之一。本研究拟建立裸鼠原位人脑胶质瘤模型。荷瘤动物将用vetanalib或HET 0016单独或联合治疗。将进行动态对比增强MRI（DCE-MRI），以确定基础和治疗条件下肿瘤的渗透性传递常数（Ktranss）、分布（肿瘤血液）体积、肿瘤体积、增强模式和扩散参数。Western blot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。我们预期治疗抗性肿瘤将显示大小、渗透性、肿瘤血容量和其他MRI参数的变化。这些数据可能提供代偿性/难治性血管生成的直接证据，解释为什么某些肿瘤对抗VEGFR治疗变得难治，并确定HET 0016作为克服肿瘤对抗血管生成治疗耐药性的可能辅助治疗。本课题组前期的研究表明，移植的EPCs通过肿瘤释放的趋化因子（如HIF-1介导的SDF-1释放）迁移并整合到肿瘤血管生成中。为了确定减少可用的BM细胞或阻断可接近的SDF-1是否会减少甚至克服对抗VEGFR或HET 0016治疗的抗性。我们将使用亚致死辐射来降低BM释放促血管生成细胞的能力。在一组实验中，我们还将使用特异性抗体或CXCR 4拮抗剂阻断SDF-1，从而降低EPCs迁移到肿瘤的能力。将使用In-111标记的细胞通过SPECT检测静脉内给药的EPC的初始迁移和掺入，并将使用磁性标记的细胞通过细胞MRI检测给药细胞的长期掺入。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揭示抗血管生成治疗的耐药机制，并允许临床医生改变GBM或其他实体瘤的未来治疗策略。

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.