Glioma cell invasion visualized by scanning confocal laser microscopy in an in vitro co-culture system.

Confrontation cultures between glioma spheroids and brain cell aggregates are well established in glioma research, and the model reflects several similarities to the in vivo brain tumour invasive process. The lipid-binding fluorescent carbocyanine dyes DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) and DiI (1,1'-dioctadecyl-3,3,3,'3,'-tetramethylinocarbocyanine perchlorate) are widely used in cell biology as tracers for studying cell movement. Mature brain cell aggregates grown from fetal rat brain cells, and spheroids initiated from two glioma cell lines (GaMg and D-54Mg) were stained with DiO and DiI, respectively. Penetration of DiI and DiO into the tumour spheroids and brain aggregates was studied by confocal laser scanning microscopy (CLSM). After 48 h of dye exposures, the tracers had almost completely penetrated the tumour spheroids and brain aggregates. Light-microscopic sections of the specimens indicated that the dye incorporation had little effect on cellular morphology. Cell migration from DiI stained D-54Mg and GaMg spheroids was similar to that observed from unstained spheroids. Growth was also unaffected after 48 h of DiI exposure. Gioma cell invasion was assessed by CLSM using co-cultures of DiI -stained spheroids and DiO-stained brain cell aggregates. Optical sections revealed a gradual decrease in remaining brain volume, indicating a progressive invasive process. Single tumour cells were identified deep within the brain aggregates. In addition normal brain cells were also identified in the tumour spheroids. It is concluded that vital staining can be used to identify both normal cells and tumour cells during tumour cell invasion in vitro. The method may provide the possibility for studying the kinetics of single normal and tumour cell movement in individual tumour/brain co-cultures.