4-DIMENSIONAL ANALYSIS OF HUMAN BRAIN-TUMOR SPHEROID INVASION INTO FETAL-RAT BRAIN AGGREGATES USING CONFOCAL SCANNING LASER MICROSCOPY

The advent of confocal microscopy and fluorescence probes has made pos sible the routine visualization of the complex three-dimensional struc tures of thick fixed or live specimens. Four-dimensional (4-D) imaging of biological specimens (three-dimensional image reconstruction of th e same living sample at different time points), remains a seldom-used application of confocal microscopy. In the present study we used 4-D i maging techniques to quantitate the invasion of human brain tumor sphe roids into fetal rat brain aggregates (FRBAs), using the vital fluores cence membrane dyes, 3,3'-Dioctadecyloxacarbocyanine perchlorate (DiO) and -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (D iI) as visualization probes. We found invasion patterns similar to the in vivo behavior of these tumors in the brain. Glioblastoma spheroids showed diffuse and circumscribed infiltration accompanied by cystic d egeneration or necrosis of FRBAs. Spheroids from cerebral metastasis, however, showed a sharp delimitation of the invasive margin, and did n ot penetrate the FRBA beyond a depth of 55 mu m. Measured rates of gli oblastoma invasion varied with the tumor specimens examined. The slope s of the mid-portions of plots of % infiltration vs. time (hours) for four glioblastoma cell lines were 1.7 +/- 0.21 (SD), 0.67 +/- 0.11, 1. 4 +/- 0.22 and 1.3 +/- 0.18. We conclude that confocal microscopy with vital fluorescence probes is a practical method that allows for close monitoring and quantitation of the process of invasion in live tissue preparations, and may be used for assessing the in vitro effects of v arious tumor treatments.