Novel application of multicellular layers culture for in situ evaluation of cytotoxicity and penetration of paclitaxel

Limited drug penetration into tumor tissue is one of the major factors causing clinical drug resistance in human solid tumors. The multicellular layers (MCL) of human cancer cells have been successfully used to study tissue pharmacokinetics of anticancer drugs. The purpose of this study was to develop a direct and simple method to evaluate vitality changes in situ within MCL using calcein-AM. Human colorectal (DLD-1, HT-29) and bladder (HT-1376, J-82) cancer cells were grown in Transwell inserts to form MCL and subjected to paclitaxel exposure. The drug distribution was evaluated using paclitaxel-rhodamine. Photonic attenuation and limited penetration of calcein-AM prevented cellular vitality evaluation on optical sections under confocal microscopy in DLD-1 MCL. However, direct measurement of the fluorescence intensity on frozen sections of MCL allowed successful vitality assessment in more than 80% depth for HT-29 and J-82 MCL and in the upper 40% depth for DLD-1 and HT-1376 MCL. The penetration of paclitaxel-rhodamine was greater in HT-29 than DLD-1 and its distribution pattern was correlated to the spatial profile of vitality deterioration in both MCL, suggesting that tissue penetration may be an important determinant of drug effect in tumors. In conclusion, a novel method for vitality evaluation in situ within MCL was developed using calcein-AM. This method may provide clinically relevant data regarding the spatial pharmacodynamics of anticancer agents within avascular regions of solid tumors. ( Cancer Sci 2008; 99: 423–431)