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

正常情况下，正常大脑中很少或没有检测到 SCF 表达； 然而，我们发现与非肿瘤脑相比，它在神经胶质瘤细胞系和神经胶质瘤中都以高水平表达。此外，与低级别神经胶质瘤相比，高级别神经胶质瘤中的 SCF 表达水平具有统计学意义的更高水平。由于与低级别神经胶质瘤相比，高级别神经胶质瘤的特点是肿瘤相关血管生成量大得多，因此 SCF 表达与神经胶质瘤级别增加的正相关性与 SCF 在神经胶质瘤相关血管生成中的潜在作用一致。我们已经证明，SCF 受体 c-Kit 在所有检查的内皮细胞 (EC) 表面上表达，并且将基础培养基中的 BMVEC-b、HUVEC 和 HMVEC-d 暴露于 SCF 会导致所有 3 个 EC 系中的胸苷掺入和细胞增殖，呈剂量依赖性，即使在不存在其他细胞因子（例如 VEGF）的情况下，即使在低浓度下也是如此。 SCF还在体外伤口愈合试验和毛细管形成试验中诱导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，我们将浸渍有 SCF、b-FGF（阳性对照）或单独载体的基质胶植入成年 SCID 小鼠体内。获得的数据表明SCF可以促进体内血管生成。通过类似的技术，我们还证明了抑制神经胶质瘤细胞中的 SCF 会显着抑制神经胶质瘤诱导的体内血管生成。接下来我们评估了抑制 SCF 是否会影响患有颅内神经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无胸腺nu/nu小鼠的大脑皮层下。 Kaplan-Meier 生存曲线的对数秩分析表明，与 U373/载体对照小鼠相比，U373/as-SCF 小鼠具有显着的生存优势 (P<0.05)，尽管两种细胞类型的体外生长速率相同。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的免疫组织化学分析揭示了继发于肿瘤和神经元相关 SCF 表达的神经胶质瘤细胞浸润的大脑皮层中 SCF 的深度表达。总之，SCF 表达似乎最突出地存在于浸润性神经胶质瘤的侵袭性前沿，表明其在肿瘤进展中的作用。鉴于我们的数据证明了 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但主要的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，我们开展了一系列体内实验，以研究格列卫对原位神经胶质瘤模型中神经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VEGF抑制剂（LY317615、Avastin等）的联合用药。然而，格列卫对完整血脑屏障的渗透性较差，也将迫使我们筛选其他具有抗 c-kit 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&lt;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.