The advent of confocal microscopy and fluorescence probes has made possible the routine visualization of the complex three-dimensional structures of thick fixed or live specimens. Four-dimensional (4-D) imaging of
biological specimens (three-dimensional image reconstruction of the same living sample at different time points), remains a seldom-used application of confocal microscopy. In the present study we used 4-D imaging techniques to quantitate the invasion of human
brain tumor spheroids into fetal rat brain aggregates (FRBAs), using the vital fluorescence membrane
dyes, 3, 3'-Dioctadecyloxacarbocyanine
perchlorate (DiO) and
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) 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 degeneration or
necrosis of FRBAs. Spheroids from cerebral
metastasis, however, showed a sharp delimitation of the invasive margin, and did not penetrate the FRBA beyond a depth of 55 microns. Measured rates of
glioblastoma invasion varied with the
tumor specimens examined. The slopes 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 various
tumor treatments.