DOXORUBICIN DISTRIBUTION IN MULTICELLULAR PROSTATE-CANCER SPHEROIDS EVALUATED BY CONFOCAL LASER-SCANNING MICROSCOPY AND THE OPTICAL PROBE TECHNIQUE

Multicell-mediated drug resistance is a major impediment for the effec tiveness of chemotherapeutic approaches and has been shown to be a fea ture of many solid tumors, We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroid s (MCS) (O150 +/- 50 mu m and 350 +/- 50 mu m). MCS (O150 +/- 50 mu m) solely consist of proliferating cells, whereas in MCS (O350 +/- 50 mu m) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas, A technique was dev eloped which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 mu m in Living MCS, This was achi eved by confocal radial recordings of the mean Dox fluorescence in 600 mu m(2) regions of interest (ROI), equidistantly spaced (10 mu m) fro m the center of MCS towards their periphery, The resulting fluorescenc e intensitiy profiles were subsequently corrected for absorbtion and l ight scattering in the depth of the tissue by a convenient algorithm, A 10 min incubation of MCS (O150 +/- 50 mu m) with Dox (10 mu M) led a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS, In contrast, after Dox treatment of MCS (O350 +/ - 50 mu m) for 2 h, the drug was accumulated within the peripheral pro liferating cell rim of 78 +/- 8 mu m, whereas deeper, quiescent cell l ayers remained unstained, When MCS were incubated with verapamil, cycl osporin A, orthovanadate, and quinidine, which are known to reverse P- glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulate d also in deeper cell layers, Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated p rotein (MRP) were without effects, The optical probe technique proved to be well suited to study MDR in a Living three dimensional tissue co ntext. (C) 1998 Wiley-Liss, Inc.