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

在正常情况下，SCF在正常大脑中几乎没有表达；然而，我们发现与非肿瘤大脑相比，它在胶质瘤细胞系和胶质瘤中都有高水平的表达。此外，与低级别胶质瘤相比，高级别胶质瘤中SCF的表达水平显著高于高级别胶质瘤。由于高级别胶质瘤比低级别胶质瘤具有更多的肿瘤相关血管生成的特征，SCF的表达与胶质瘤分级的增加正相关，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表达于所有被检测的内皮细胞(ECs)的表面，并且基础培养液中的BMVEC-b、HUVEC和HMVEC-d暴露于SCF后，即使在没有其他细胞因子如VEGF的情况下，也以剂量依赖的方式导致所有3种EC的胸腺嘧啶核苷掺入和细胞增殖。在体外伤口愈合实验和毛细血管形成实验中，SCF还能诱导EC迁移和分化。这些结果表明，SCF在体外具有诱导BMVEC-b增殖、迁移和分化的能力。接下来，我们将干细胞因子、b-成纤维细胞生长因子(阳性对照)或单纯赋形剂的Matrigel植入成年SCID小鼠皮下。结果表明，SCF能促进体内血管生成。通过类似的技术，我们还证明了抑制胶质瘤细胞中的SCF会在体内显着抑制胶质瘤诱导的血管生成。接下来，我们评估了抑制干细胞因子是否会影响患有脑胶质瘤的动物的生存。将U373/AS-SCF或U373/载体细胞立体定向植入成年裸鼠大脑皮质下。Kaplan-Meier生存曲线的对数秩次分析表明，与U373/载体对照动物相比，U373/AS-SCF小鼠具有显著的生存优势(P&lt；0.05)，尽管这两种细胞在体外的生长速度是相同的。为了在实际的肿瘤标本中证实这些结果，来自胶质母细胞瘤患者的多个手术标本的免疫组织化学分析显示，SCF在继发于肿瘤相关和神经元相关的SCF表达的胶质瘤细胞浸润的大脑皮层中表达水平很高。综上所述，SCF的表达似乎最显著地位于浸润性胶质瘤的侵袭前沿，提示其在肿瘤进展中的作用。鉴于我们的数据表明干细胞因子在肿瘤和宿主细胞诱导的血管生成中的重要性，我们假设格列卫的一个先前未被认识的但主要的抗肿瘤机制可能是通过其有效抑制c-kit信号的能力而作为抗血管生成剂。因此，我们开展了一系列体内实验，以观察格列卫在我们的原位胶质瘤模型中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，评估格列卫与特定的血管内皮生长因子抑制剂(LY317615、阿瓦斯丁等)的组合。然而，格列卫对完整的血脑屏障的渗透性很差，这也将迫使我们筛选其他对c-kit有活性但可能具有更有利的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.