SCF as a Novel CNS and Glioma-Derived Angiogenic Factor and SC Chemotaxic Factor

在正常情况下，正常大脑中很少或没有SCF表达；然而，我们发现它在胶质瘤细胞系和胶质瘤中都有高水平的表达与非肿瘤大脑相比。此外，与低级别胶质瘤相比，高级别胶质瘤中SCF的表达水平有统计学意义。由于与低级别胶质瘤相比，高级别胶质瘤的特点是肿瘤相关血管生成的数量要大得多，因此SCF表达与胶质瘤级别的增加呈正相关，这与SCF在胶质瘤相关血管生成中的潜在作用是一致的。我们已经证明，SCF受体c-Kit在所有内皮细胞（ECs）表面表达，并且在基础培养基中BMVEC-b、HUVEC和HMVEC-d暴露于SCF导致胸苷结合和细胞增殖，即使在缺乏其他细胞因子（如VEGF）的低浓度下，也以剂量依赖的方式出现在所有3种EC系中。在体外伤口愈合实验和毛细管形成实验中，SCF还能诱导EC迁移和分化。这些数据证明了SCF在体外诱导BMVEC-b增殖、迁移和分化的能力。接下来，我们将单独浸渍SCF、b-FGF（阳性对照）或载药的Matrigel皮下植入成年SCID小鼠。实验结果表明，SCF能够促进体内血管生成。通过类似的技术，我们也证明了抑制胶质瘤细胞中的SCF可以显著抑制胶质瘤诱导的血管生成。我们接下来评估抑制SCF是否会影响颅内胶质瘤动物的存活。将U373/as-SCF或U373/载体细胞立体定向植入成年胸腺nu/nu小鼠的大脑皮层下。Kaplan-Meier生存曲线的Log-rank分析显示，携带U373/as-SCF的小鼠与携带U373/病媒对照的小鼠相比具有显著的生存优势（P<0.05），尽管两种细胞类型在体外的生长速度相同。为了在实际肿瘤样本中证实这些结果，对胶质母细胞瘤患者的多个手术标本进行免疫组织化学分析显示，在继发于肿瘤和神经元相关的SCF表达的胶质瘤细胞浸润的大脑皮层中，SCF的深度表达。总之，SCF表达似乎在浸润性胶质瘤的侵袭性前部最为显著，表明其在肿瘤进展中的作用。鉴于我们的数据表明SCF在肿瘤和宿主细胞诱导的血管生成中的重要性，我们假设格列卫以前未被认识到的主要抗肿瘤机制可能是通过其有效抑制c-kit信号传导的能力作为抗血管生成剂。因此，我们开始了一系列的体内实验，以观察格列卫对胶质瘤介导的血管生成的影响。因此，我们将开展一系列临床前研究，评估格列卫与特异性VEGF抑制剂（LY317615、阿瓦斯汀等）的联合应用。然而，格列卫通过完整血脑屏障的渗透性差，也将迫使我们筛选其他对c-kit有活性但可能具有更有利药代动力学的酪氨酸激酶抑制剂。

Under normal conditions, little or no SCF expression is detectable in normal cerebrum; however we found it to be expressed at high levels both in glioma cells lines and in gliomas when compared to non-tumor brain. Additionally, there was a statistically significant higher level of SCF expression in high-grade gliomas compared to low-grade gliomas. Since high-grade gliomas are characterized by a much greater amount of tumor-associated angiogenesis compared to low-grade gliomas, the positive correlation of SCF expression with increasing glioma grade is consistent with a potential role for SCF in glioma-associated angiogenesis. We have demonstrated that the SCF receptor, c-Kit, is expressed on the surface of all endothelial cells (ECs) examined and that exposure of BMVEC-b, HUVEC and HMVEC-d in basal medium to SCF resulted in thymidine incorporation and cellular proliferation in all 3 EC lines in a dose-dependent manner even at low concentrations in the absence of other cytokines such as VEGF. SCF also induced EC migration and differentiation in an in vitro wound healing assay and capillary tube formation assay. These data demonstrated the ability of SCF to induce proliferation, migration and differentiation of BMVEC-b in vitro. We next subcutaneously implanted Matrigel impregnated with SCF, b-FGF (positive control) or vehicle alone into the adult SCID mice. The data obtained demonstrated that SCF can promote angiogenesis in vivo. By a similar technology we also demonstrated that suppression of SCF in glioma cells results in significant inhibition of glioma-induced angiogenesis in vivo. We next evaluated whether suppression of SCF would effect the survival of animals with intracranial gliomas. U373/as-SCF or U373/vector cells were stereotactically implanted to the cerebral subcortex of adult athymic nu/nu mice. Log-rank analysis of the Kaplan-Meier survival curves demonstrated a significant survival advantage for the U373/as-SCF bearing mice compared to the U373/vector control bearing animals (P<0.05), despite the fact that the growth rate of both cells types in vitro was identical. To confirm these results in actual tumor samples, immunohistochemical analysis of multiple surgical specimens from patients with glioblastoma revealed profound expression of SCF in cerebral cortex infiltrated by glioma cells secondary to both tumor-and neuronal-associated SCF expression. In summary, SCF expression appears to reside most prominently in the invasive front of the infiltrating glioma, suggesting its roles in the tumor progression. Given our data demonstrating the importance of SCF in tumor and host cell-induced angiogenesis, we hypothesize that a previously unrecognized, but major anti-tumor mechanism of Gleevec may be as an anti-angiogenic agent through its ability to potently inhibit c-kit signaling. We have therefore embarked on a series of in vivo experiments to look at the effects of Gleevec on glioma-mediated angiogenesis in our orthotopic glioma models. Thus, we will embark on a series of preclinical studies evaluating the combination of Gleevec with specific VEGF inhibitors (LY317615, Avastin, etc.). The poor penetration of Gleevec through an intact blood-brain barrier, however, will also force us to screen other tyrosine kinase inhibitors that have activity against c-kit but may have more favorable pharmacokinetics.