An advanced glioma cell invasion assay based on organotypic brain slice cultures

Abstract

Background

The poor prognosis for glioblastoma patients is caused by the diffuse infiltrative growth pattern of the tumor. Therefore, the molecular and cellular processes underlying cell migration continue to be a major focus of glioblastoma research. Emerging evidence supports the concept that the tumor microenvironment has a profound influence on the functional properties of tumor cells. Accordingly, substantial effort must be devoted to move from traditional two-dimensional migration assays to three-dimensional systems that more faithfully recapitulate the complex in vivo tumor microenvironment.

Methods

In order to mimic the tumor microenvironment of adult gliomas, we used adult organotypic brain slices as an invasion matrix for implanted, fluorescently labeled tumor spheroids. Cell invasion was imaged by confocal or epi-fluorescence microscopy and quantified by determining the average cumulative sprout length per spheroid. The tumor microenvironment was manipulated by treatment of the slice with small molecule inhibitors or using different genetically engineered mouse models as donors.

Results

Both epi-fluorescence and confocal microscopy were applied to precisely quantify cell invasion in this ex vivo approach. Usage of a red-emitting membrane dye in addition to tissue clearing drastically improved epi-fluorescence imaging. Preparation of brain slices from of a genetically engineered mouse with a loss of a specific cell surface protein resulted in significantly impaired tumor cell invasion. Furthermore, jasplakinolide treatment of either tumor cells or brain slice significantly reduced tumor cell invasion.

Conclusion

We present an optimized invasion assay that closely reflects in vivo invasion by the implantation of glioma cells into organotypic adult brain slice cultures with a preserved cytoarchitecture. The diversity of applications including manipulation of the tumor cells as well as the microenvironment, permits the investigation of rate limiting factors of cell migration in a reliable context. This model will be a valuable tool for the discovery of the molecular mechanisms underlying glioma cell invasion and, ultimately, the development of novel therapeutic strategies.