GLIOMA CELL INVASION VISUALIZED BY SCANNING CONFOCAL LASER MICROSCOPYIN AN IN-VITRO COCULTURE SYSTEM

Confrontation cultures between glioma spheroids and brain cell aggrega tes are well established in glioma research, and the model reflects se veral similarities to the in vivo brain tumour invasive process. The l ipid-binding fluorescent carbocyanine dyes DiO (3,3'-dioctadecyloxacar bocyanine perchlorate) and DiI '-dioctadecyl-3,3,3',3'-tetramethylinoc arbocyanine perchlorate) are widely used in cell biology as tracers fo r studying cell movement. Mature brain cell aggregates grown from feta l rat brain cells, and spheroids initiated from two glioma cell lines (GaMg and D-54Mg) were stained with DiO and DiI, respectively. Penetra tion of DiI and DiO into the tumour spheroids and brain aggregates was studied by confocal laser scanning microscopy (CLSM). After 48 h of d ye exposures, the tracers had almost completely penetrated the tumour spheroids and brain aggregates. Light-microscopic sec tions of the spe cimens indicated that the dye incorporation had little effect on cellu lar morphology. Cell migration from DiI stained D-54Mg and GaMg sphero ids was similar to that observed from unstained spheroids. Growth was also unaffected after 48 h of DiI exposure. Glioma cell invasion was a ssessed by CLSM using co-cultures of DiI-stained spheroids and DiO-sta ined brain cell aggregates. Optical sections revealed a gradual decrea se in remaining brain volume, indicating a progressive invasive proces s. Single tumour cells were identified deep within the brain aggregate s. In addition normal brain cells were also identified in the tumour s pheroids. It is concluded that vital staining can be used to identify both normal cells and tumour cells during tumour cell invasion in vitr o. The method may provide the possibility for studying the kinetics of single normal and tumour cell movement in individual tumour/brain co- cultures.