Analysis of the molecular mechanism of glioma invasion by using a brain slice invasion model

为了定量分析器官型脑片培养中胶质瘤细胞迁移的空间范围，我们借助激光共聚焦扫描显微镜(CLSM)建立了一种新的侵袭模型。CLSM不仅可以在活体脑片上三维显示人T98G胶质瘤细胞的侵袭模式，还可以连续分析几周内的侵袭过程。在T98G胶质瘤球体与脑片共培养24小时后，胶质瘤细胞脱离球体，自发地继续在脑片表面迁移，并通过向更深部位的迁移向切片内扩散侵袭。免疫组织化学分析显示，这些迁移的胶质瘤细胞对基质金属蛋白酶-2和-9的免疫染色比保留在植入部位的肿瘤球体强得多。用MMPs的特异性抑制剂1，10-菲咯啉处理T98G胶质瘤球体，不仅能显著抑制细胞在脑片表面的迁移，而且能显著抑制胶质瘤细胞对脑片的侵袭。目前使用CLSM建立的胶质瘤侵袭模型可以连续几周在活体脑片上对胶质瘤细胞的侵袭程度进行空间和序列分析，这使它成为在与活体正常脑组织最相似的条件下研究胶质瘤侵袭的细胞和分子机制的非常有用的工具。

In order to quantitatively analyze the spatial extent of glioma cell migration in an organotypic brain slice culture, we developed a new invasion model with the aid of confocal laser scanning microscopy (CLSM). CLSM allowed not only for three-dimensional visualization of the invasive pattern of human T98G glioma cells in the living brain slice but also for serial analysis of the invasive process over several weeks. Twenty-four hours after the T98G glioma spheroid was initiated to coculture with a brain slice, the glioma cells detached themselves from the spheroid and spontaneously continued to migrate on the surface of the brain slice, while they diffusely invaded into the slice by migrating to a deeper site. Immunohistochemical analysis revealed that these migrating glioma cells much more strongly immunostained for matrix metalloproteinase (MMP)-2 and -9 than the tumor spheroid which remained at the implanted site. Treatment of the T98G glioma spheroid with 1, 10-phenanthroline, a specific inhibitor of MMPs, significantly inhibited not only the cell migration on the surface of the brain slice but also the invasion of the glioma cells into the slice. The present version of the glioma invasion model using CLSM makes it possible to spatially and serially analyze the extent of glioma cell invasion in the living brain slice for several weeks, making it a very useful tool for investigating the cellular and molecular mechanisms of glioma invasion under conditions most analogous to those of normal brains in vivo.