Cancer Chemother Pharmacol (1989) 24:295-301 ancer hemotherapyand harmacology © Springer-Verlag 1989 Comparison of tetrazolium colorimetric and [3H]-uridine assays for in vitro chemosensitivity testing* Christopher H. J. Ford, Vernon J. Richardson, and Georgia Tsaltas Oncology Research, Memorial University and the Newfoundland Cancer Treatment and Research Foundation, St. John's, Newfoundland, Canada A1B 3V6 Summary. We have routinely used a [3H]-uridine micro- plate assay for assessing chemosensitivity. A colorimetric assay with the advantages of safety, cost and simplicity has previously been described and relies on the ability of liv- ing cells to reduce a soluble tetrazolium salt, 3-4,5-di- methylthiazol-2,5-diphenyl-tetrazolium bromide (MMT), into an insoluble formazan precipitate. We compared the chemosensitivity of 14 human tumour cell lines of colonic, lung and cervical carcinoma origin to doxorubicin, vin- desine or vindesine immunoconjugates in both the [3H]-uridine assay and a modified MTT assay to evaluate whether we could change to the non-radiolabelled method. Correlation between the concentration of drug causing 50% inhibition of cell growth (ICs0) for these agents be- tween the two assays was very poor. However, taking ac- count of recent reports in the literature, we modified the MTT assay by removing serum-containing medium and using dimethyl sulphoxide to solubilise the formazan pre- cipitate. This considerably improved the correlation be- tween the assays for doxorubicin (r = 0.871; P= 0.001) and vindesine (r = 0.981; P <0.001). Our data indicates that the MTT assay can be used to replace the [3H]-uridine assay for chemosensitivity screening, but further modifica- tions are necessary to improve the sensitivity and decrease the problem of cell loss after washing, which was noted with some adherent cell lines. Introduction Since the report of a rapid colorimetric assay that uses the ability of viable cells to reduce a soluble tetrazolium salt, 3-4,5-dimethythiazol-2,5-diphenyl-tetrazolium bro- mide (MTT), into an insoluble formazan precipitate [6], a number of laboratories have investigated its potential for in vitro drug screening with panels of human tumour cell lines [1-3, 7, 10]. In an attempt to improve its sensitivity and reproducibility, factors affecting the technical perfor- mance of the MTT assay have also been examined [1, 4, * Supported by grants from the Newfoundland Lung Association, the Faculty of Medicine Research and Development Committee and the Newfoundland Cancer Foundation Offprint requests to: C. H.J. Ford, Oncology Research, Health Sciences Centre, Memorial University, St. John's, Newfoundland, Canada AIB 3V6 11]. The MTT assay has been compared with dye exclu- sion, clonogenic and cellular protein assays, and although there have been comparisons with [3H]-thymidine uptake for the assessment of cell viability, these have involved rel- atively few cell lines of non-human origin [4, 6]. We have used [3H]-leucine incorporation for the assessment of human tumour chemosensitivity in vitro [12] or [3H]-uri- dine incorporation for the in vitro evaluation of monoclo- nal antibody-drug immunoconjugates with the vinca alka- loids [5] or doxorubicin [8]. In this report we describe the results of our direct comparison of the MTT and [3H]-uri- dine assays for the assessment of the in vitro sensitivity of a panel of human tumour cell lines to vindesine, vindesine immunoconjugates and doxorubicin. Materials and methods Tumour cell lines A total of 14 human tumour cell lines were studied: colon carcinomas COLO320DM, COLO201, SWlll6, SW837, WIDR, HT29, LOVO, LS174T and SKCO1 ; lung carcino- mas BENN, CALU-3 and CALU-6; and cervical carcino- mas C33A and MS751. BENN was obtained from Dr. M. Ellison at the Ludwig Institute for Cancer Research (Sut- ton, U.K.); all other cell lines were obtained from the American Type Culture Collection (Rockville, Md, USA). Cells were grown as monolayers in medium containing 10%-16% foetal calf serum (FCS). Media used included Eagle's MEM (SKCO1, LS174T, WIDR, CALU3, C33A and MS751), Dulbecco's Modified Eagle Medium (CALU6), RPMI 1640 (COLO320DM and COLO201), LI5 (SW1116 and SW837), Medium 199 (BENN), McCoy's (HT29) and Harem's F10 (LOVO). Microcytostasis assays [3H]-uridine assay. A terminal uridine assay was used to determine the sensitivity of the cell lines to drug, conjugate or antibody [5]. Cell lines were grown until they were sub- confluent, then trypsinised, washed in PBS and resuspend- ed in fresh media. A total of 100 gl medium containing 10 4 viable cells were plated per well into 96-well microtitre plates. Cells were allowed to attach to the plates for 24 h at 37°C in a 5% CO2 atmosphere. Doxorubicin (DOX; Adria Laboratories Inc., Columbus, Ohio) or vindesine (VDS) (Eli Lilly & Company Inc., Toronto, Ontario) dissolved in sterile saline at 1 mg/ml or vindesine anti-carcinoemb-