Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.

https://cancerres.aacrjournals.org/content/canres/47/4/936.full.pdf

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.

Radiation survival curves were generated for V79 Chinese hamster and two human lung cancer cell lines (NCI-H460 and NCI-H249) with doubling times of 10, 20, and 85 h, respectively, using a standard clonogenic assay, a dye exclusion assay, and a semiautomated colorimetric assay utilizing a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide. Comparable results for D0 and extrapolation number (n) were observed for all assays in the lines with doubling times of 10 and 20 h. In these instances the tumor cell lines had undergone seven or more doublings after radiation. For the tumor line (H249) with an 80-h doubling time the D0S were comparable between the assays while the extrapolation number was increased in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay, a result probably related to the lower number of doublings (less than 4) after radiation. We then tested the ability of the assays to detect radiation protection and sensitization using known agents. We found that cysteamine treatment resulted in radioprotection (by a factor of 8 at 8 Gy) while 5-bromo-2-deoxyuridine incorporation caused enhancement of radiation sensitivity in all three assays. We conclude that, while optimal conditions for each cell line (cell number plated and doubling time) must be established, using characterized tumor cell lines, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay could be automated and thus be of great value in screening large numbers of potential radiosensitizers or protectors.

https://cancerres.aacrjournals.org/content/canres/47/4/943.full.pdf

Evaluation of a Tetrazolium-based Semiautomated Colorimetric Assay: Assessment of Radiosensitivity

Viability measurements in mammalian cell systems.

Three human tumor cell lines of widely differing radiosensitivity were used to examine the characteristics of the 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphery]tetradium bromide (MTT) assay and to select suitable conditions for its use in assessing the response of cells to ionizing radiation. The optimal concentration of MTT and the time of incubation of the cells with MTT were individualized for each cell line. The relationship between absorbance and cell number was not linear over the wide range of cell numbers that were used. A calibration curve of absorbance against cell number for each cell line was therefore used. Using the assay to quantify metabolically viable cells, growth curves of irradiated and unirradiated cells were constructed on days 0-14 after irradiation. Accurate surviving fractions could be calculated only when cells were in exponential growth. Using this modification to its interpretation, the MTT assay was able to provide a reproducible measure of survival, which compared well with clonogenic cell survival measurements. However, the necessity to optimize conditions of the MTT assay for each cell line severely limits its usefulness in determining the radiosensitivity of cells in primary human tumor cultures.

https://cancerres.aacrjournals.org/content/canres/50/5/1392.full.pdf

Use of the Tetrazolium Assay in Measuring the Response of Human Tumor Cells to Ionizing Radiation

In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions

Dissociated cancer cells are exposed to antineoplastic drugs (5 × 10 4 viable cells/drug for 1 hr or continuously) and cultured for 4 to 6 days in liquid medium. Cells are then stained with Fast green dye, sedimented onto slides with a Cytospin centrifuge, and counterstained with a modified hematoxylin and eosin technique. Dead cells stain with Fast green, and living cells stain with hematoxylin and eosin. Cell kill is calculated as percentage of control based on the relative numbers of living tumor cells, living non-tumor cells, and dead cells. Drug sensitivity could be assayed in 125 of 162 specimens of human neoplasms obtained from malignant effusions (16 of 18), excisional biopsies (31 of 44), needle biopsies (34 of 47), endoscopic biopsies (18 of 23), peripheral blood samples (19 of 20), and bone marrow aspirates (five of seven). The assays were successful (median of ten drugs tested) in: 46 of 64 adenocarcinomas; four of 11 squamous cell carcinomas; five of seven lymphomas; six of seven melanomas; two of four sarcomas; 18 of 20 transitional-cell carcinomas; 14 of 15 small-cell carcinomas; seven of eight myelomas; 12 of 12 chronic lymphocytic leukemias; seven of nine acute leukemias, and four of five "undifferentiated" carcinomas. The assay results demonstrated a strong correlation between the in vitro chemosensitivity of different types of tumors and the known clinical response patterns of these tumors. This assay can be used to determine which specific cells are killed in a heterogeneous cell population. Further work is needed to determine if this assay may be useful in blind screening trials for antineoplastic agents or if it may be of clinical use in predicting response to agents which are not cycle specific.

https://cancerres.aacrjournals.org/content/canres/43/2/749.full.pdf

Joye A. Marsden

Papers

A Novel Dye Exclusion Method for Testing in Vitro Chemosensitivity of Human Tumors