Summary
This study was undertaken to evaluate the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay as a means of testing the sensitivity of gliomas to chemotherapeutic agents in vitro. Eight human glioma established cell lines were plated in 96-well tissue culture plates and incubated for 4 days with 10 different anti-cancer agents; 5 different concentrations of each drug were tested. The MTT dye was then added to the wells, and the resulting formazan precipitate was solubilized with dimethylsulfoxide (DMSO). The spectrophotometric absorbance (measured at 570 nm) of control and experimental wells was used to calculate the cytotoxicity index (CI). Values with a CI greater than 50% growth inhibition indicated cytotoxic efficacy (sensitivity to the chemotherapeutic drug).
Six of the seven (85.7%) glioma cell lines were highly sensitive at varying concentrations to mitomycin C, cisplatin, and doxorubicin. Four of the seven (57.1%) cell lines demonstrated intermediate sensitivity to mitoxantrone and vinblastine. Five of the seven (71.4%) cell lines exhibited resistance to etoposide, bleomycin, cosmegen, and BCNU. One of the cell lines tested, U-138MG, failed to produce the MTT formazan precipitate, so that the sensitivity of this cell line to the panel chemotherapeutic drugs could not be determined. The variability of the results indicates the need for an in vitro screening method to evaluate the effectiveness of clinical and experimental chemotherapeutic agents. The MTT assay provides a rapid method of screening antineoplastic agents against gliomas for cytotoxicity.
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References
Kornblith PL, Walker M: Chemotherapy for malignant gliomas. J Neurosurg 68: 1–17, 1988
Hill BT: Use of continuous human tumor cell lines to evaluate drugs by clonogenic assays. In: Dendy PP and Hill BT (eds) Human Tumor Drug Sensitivity Testing in vitro, Academic Press, London 129–146, 1983
Courtenay VD, Mills J: An in vitro colony assay for human tumors grown in immune-suppressed mice and treated in vivo with cytotoxic agent. Br J Cancer 37: 261–268, 1978
Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 197: 461–463, 1977
Alley MC, Lieber MM: Improved optical detection of colony enlargement and drug cytotoxicity in primary soft agar cultures of human solid tumor cells. Br J Cancer 49: 225–233, 1984
Bertoncello I, Bradley TR, Campbell JJ, Day AJ, McDonald IA, McLeish GR, Quinn MA, Rome R, Hodgson GS: Limitations of the clonal agar assay for the assessment of primary human ovarian tumor biopsies. Br J Cancer 45: 803–811,1982
Kubota T, Kawamura E, Kurihara H, Ishibiki K, Abe O, Suzuki T, Suzuki Y, Yamada T: Clonogenic assay as the in vitro chemosensitivity test. In: Ishigami J (ed) Recent Advances in Chemotherapy, Anticancer Section, University of Tokyo Press, Tokyo 288–289, 1985
Selby P, Bizzari JP, Buick RN: Therapeutic implications of a stem cell model for human breast cancer: a hypothesis. Cancer Treat Rep 67: 659–663, 1983
Von Hoff DD, Clark GM, Stogdill BJ, Sarodsy MF, O'Brien MT, Casper JT, Mattox DE, Page CP, Cruz AB, Sandbach JF: Prospective clinical trial of a human tumorcloning system. Cancer Res 43: 1926–1931, 1983
Weisenthal LM, Marsden JA, Dill PL, Macaluso CK: A novel dye exclusion method for testing in vitro chemosensitivity of human tumors. Cancer Res 43: 749–757, 1983
Kirkpatrick DL, Duke M, Goh TS: Chemosensitivity testing of fresh human leukemia cells using both a dye exclusion assay and a tetrazolium dye (MTT) assay. Leukemia Res 14 (5):459–466,1990
Pieters R, Huismans DR, Leyva A, Veerman AJP: Comparison of the rapid automated MTT assay with a dye exclusion assay for chemosensitivity testing in childhood leukemia. Br J Cancer 59: 217–220, 1989
Bird MC, Godwin VAJ, Antrobus JH, Bosanquet AG: Comparison of in vitro drug sensitivity by the differential staining cytotoxicity (DiSC) and colony-forming assays. Br J Cancer 55: 429–431, 1987
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB: Evaluation of a tetrazolium-based semi-automatic colorimetric assay. Cancer Res 47: 943–946, 1987
Laurent G, Kuhlein E, Casellas P, Canat X, Carayon P, Poncelet P, Correll S, Rigal F, Jansen FK: Determination of sensitivity of fresh leukaemia cells to immunotoxins. Cancer Res 46: 2289–2294, 1986
Weisenthal LM, Dill PL, Kurnick NB, Lippman ME: Comparison of dye exclusion assays with a clonogenic assay in the determination of drug-induced cytotoxicity. Cancer Res 43:258–264,1983
Bird MC, Bosanquet AG, Forskitt S, Gilby ED: Long-term comparison of results of an in vitro drug sensitivity assay with patient response in lymphatic neoplasms. Cancer 61: 1104–1109,1988
Bosanquet AG, Bird MC, Price WJP, Gilby ED: An assesment of a short-term tumour chemosensitivity assay in chronic lymphocytic leukaemia. Br J Cancer 47: 781–789, 1983
Denizot F, Lang R: Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271–277, 1986
Black MM, Speer FD: Further observations on the effects of cancer chemotherapeutic agents in the in vitro dehydrogenase activity of cancer tissue. J Natl Cancer Inst 14: 1147–1158,1954
Kondo T, Ohkubo K: In vitro test for prediction of side effects of carcinostatic agents. Gann 58 (4): 349–354, 1967
Adams GE, Stratford IJ, Wallace RG, Wardman P, Watts ME: Toxicity of nitro compounds toward hypoxic mammalian cells: dependence upon reduction potential. J Natl Cancer Inst 64: 555–560, 1980
Harker WG, Slade DL, Lund K: In vitro doxorubicin (DOX) sensitivity of human and murine cell lines: Comparison of clonogenic and MTT assays. Proc Am Assoc Cancer Res 28: 420–427, 1987
Kennedy KA, Teicher BA, Rockwell S, Sartorelli A: The hypoxic tumour cell: A target for selective cancer chemotherapy. Biochem Pharmacol 29: 1–7, 1980
Shimoyama Y, Kubota T, Watanabe M, Ishibiki K, Abe O: Predictability of in vivo chemosensitivity by in vitro assay with reference to the clonogenic assay. J Surg Oncol 41: 12–18,1989
Alley MC, Scudiero DA, Monks A, Huyrsey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR: Feasibility of drug screening with panels of human tumour cell lines using a microculture tetrazolium assay. Cancer Res 48: 589–601, 1988
Carmichael J, Mitchell JB, DeGraff WG, Gamson J, Gazdar AF, Johnson BE, Glatstein E, Minna JD: Chemosensitivity testing of human lung cancer cell lines using the MTT assay. Br J Cancer 57 (6): 540–547, 1988
Cole SPC: Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother Pharmacol 17: 259–263, 1986
Finlay GJ, Wilson WR, Baguley BC: Comparison of in vitro activity of cytotoxic drugs towards human carcinoma and leukaemia cell lines. Eur J Cancer Clin Oncol 22: 655–662,1986
Park JG, Kramer BS, Steinberg SM, Carmichael J, Collins JM, Minna JD, Gazdar AF: Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazolium based colorimetric assay. Cancer Res 47: 5875–5879, 1987
Twentyman PR, Luscombe M: A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 56: 279–285, 1987
Mickisch G, Fajta S, Keilhauer G, Schlick E, Tschada R, Alken P: Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT). Urol Res 18 (2): 131–136, 1990
Suto A, Kubota T, Shimoyama Y, Ishibiki K, Abe O: MTT assay with reference to the clinical effect of chemotherapy. J Surg Oncol42: 28–32, 1989
Wilson JK, Sargent JM, Elgie AW, Hill JG, Taylor CG: A feasibility study of the MTT assay for chemosensitivity testing in ovarian malignancy. Br J Cancer 62: 189–194, 1990
Hongo T, Fujii Y, Igarashi Y: An in vitro chemosensitivity test for the screening of anti-cancer drugs in childhood leukemia. Cancer 65: 1263–1271, 1990
Sargent JM, Taylor CG: Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. Br J Cancer 60: 206–210, 1989
Twentyman PR, Fox NE, Rees JKH: Chemosensitivity testing of fresh leukaemia cells using the MTT colorimetric assay. Br J Haematol 71: 19–24, 1989
Boyd MR: Status of the NCI preclinical antitumor drug discovery screen. Principles & Practices of Oncology 3 (10): 1–12,1989
Weislow OS, Kiser R, Fine DL, Bader J, Shoemaker RH, Boyd MR: New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening of syn thetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst 81(8): 577–586, 1989
Sasaoka N: Chemosensitivity assays for malignant gliomas. Gan-To-Kagaku-Ryoho 17 (11): 2247–2252, 1990
Yung WK: In vitro chemosensitivity testing and its clinical application in human gliomas. Neurosurgical Review 12 (3):197–203,1989
Hubbell HR, Liu RS, Maxwell BL: Independent sensitivity of human tumor cell lines to interferon and double-stranded RNA. Cancer Res 44 (8): 3252–3257, 1984
Dick RS, Hubbell HR: Sensitivities of human glioma cell lines to interferons and double-stranded RNAs individually and in synergistic combinations. J Neuro Oncol 5: 331–338, 1987
Giard DJ, Aaronson SA: In vitro cultivation of human tumors: Establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51: 1417–1423, 1973
Gross JL, Behrens DL, Mullins DE, Kornblith PL, Dexter DL: Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. Cancer Res 48: 291–296, 1988
Ponten J, MacIntyre EH: Long term culture of normal and neoplastic human glia. Acta Path Microbiol Scand 74: 465–486,1968
Tepe P, Hassenbusch SJ, Benoit R, Anderson JH: BCNU stability as a function of ethanol concentration and temperature. J Neuro-Oncol 10: 121–127, 1991
Levin VA, Levin EM: Dissolution and stability of carmustine in the absence of ethanol. Selective Cancer Therapeutics 5 (1): 33–35, 1989
Metcalfe SA: A review of methods for estimating clinically achievable antitumour drug levels and their association with studies in vitro. In: Dendy PP, Hill BT (eds) Human Tumour Drug Sensitivity Testing In Vitro. Academic Press: London 213–224,1983
Durkin WJ, Ghanta VK, Hiramoto RN: Results obtained using a vital dye exclusion assay and clinical correlations. In: Dendy PP and Hill BT (eds) Human Tumor Drug Sensitivity Testing In Vitro. Academic Press, London 259–268, 1983
Green LM, Reade JL, Ware CF: Rapid colorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J Immunol Methods 70:257–268,1984
Kornblith PL, Szypko PE: Variations in response of human brain tumors to BCNU in vitro. J Neurosurg 48: 580–586, 1978
Kimmel DW, Shapiro JR, Shapiro WR: In vitro drug testing in human gliomas. J Neurosurg 66: 161–171, 1987
Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63, 1983
Yung WK, Shapiro JR, Shapiro WR: Heterogeneous chemosensitivities of subpopulations of human glioma cells in culture. Cancer Res 42: 992–998, 1982
Vistica DT, Skehan P, Schudiero DA, Monks A, Boyd MR: Tetrazolium-based assays for cellular viability: A critical examination of parameters which affect formazan production. Proc Am Assoc Cancer Res 30: 2437, 1989
Packer RJ: Prognostic factors in patients with brain tumors. In: Salcman M (ed) Neurobiology of Brain Tumors, Vol 4, Williams & Wilkins, Baltimore, 275, 1991
Chang CH, Horton J, Schoenfeld D, Salazer O, Perez-Tamayo R, Kramer S, Weinstein A, Nelson JS, Tsukada Y: Comparison of post-operative radiotherapy and combined post-operative radiotherapy and chemotherapy in the multidisciplinary management of malignant gliomas. Cancer 52:997–1007,1983
Kristiansen K, Hagen S, Kollevold T, Torvik A, Holme I, Nesbakken R, Hatlevoll R, Lindgren M, Brun A, Lindgren S, Notter G, Andersen AP, Elgen K: Combined modality therapy of operated astrocytomas grade III and IV. Confirmation of the value of post-operative irradiation and lack of potentiation of bleomycin on survival time: a prospective multicentered trial of the Scandinavian glioblastoma study group. Cancer 47: 649–652, 1981
Walker MD, Green SB, Byar DP, Alexander E, Batzdorf U, Brooks WH, Hunt WE, MacCarty CS, Mahaley MS, Mealey J, Owens G, Ransohoff J, Robertson JT, Shapiro WR, Smith KR, Wilson CB, Strike TA: Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. N Engl J Med 303: 1323–1329, 1980
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Jordan, J.P., Hand, C.M., Markowitz, R.S. et al. Test for chemotherapeutic sensitivity of cerebral gliomas: use of colorimetric MTT assay. J Neuro-Oncol 14, 19–35 (1992). https://doi.org/10.1007/BF00170942
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DOI: https://doi.org/10.1007/BF00170942