Clonogenic assays

Herzog, E. et al. (2007) A new approach to the toxicity testing of carbon-based nanomaterials - the clonogenic assay. Toxicology Letters, 174 (1-3), 49-60. 

Short-Term Self-Renewal Assay (Clonogenic Assay)... 

The clonogenic assay is normally exploited to analyze the effect of compounds on the symmetry of division in NSCs/CSCs. In this assay, the number of secondary spheres generated from the dissociation of a single sphere reflects the frequency of NSC/CSCs present in the original primary clone. This analysis also returns an estimate of the relative frequencies between symmetric proliferative (two SCs generated at each cycle) and symmetric differentiative (two differentiated and/or dead cells generated after cell division). 

Fig. 5. HuTu80 human colon cancer cells are radiosensitized by FddUrd. HuTu80 cells were irradiated under control conditions (O) or after a 14-h exposure to 100 nmol L FdUrd ( ). They were then assessed doe survival using a clonogenic assay. Data are expresses as the mean (point) SE (bar), which is within the symbol unless indicated.

Pessina, A., Malerba, 1. and Gribaldo, L. (2005) Hematotoxicity testing by cell clonogenic assay in drug development and preclinical trials. Current Pharmaceutical Design, 11, 1055-1065. 

The MTT assay was initially developed as a quantitative assay for cell survival and proliferation, not as an in vitro assay for chemosensitivity testing. Further study was required to ascertain if the method accurately predicted the in vivo antitumor activities of anticancer agents. Shimoyama et al. [189] studied the predictability of the MTT assay with respect to a clonogenic assay (Sect. 4.1.1.3.) and showed excellent reproducibility and a close correlation to the in vivo predictability rate of the clonogenic assay. Another study [190] also showed that the MTT assay closely approximated (90%) the clinical activity of anticancer agents. Many authors have since utilized the MTT assay to determine the efficacy of polymeric anticancer drug conjugates. 

Rakestraw et al. [197] recently demonstrated the efficacy of chlorin e6 immunoconjugates through a clonogenic assay. Other examples of the use of clonogenic assays may be found [198, 199]. 

Tab. 5.11 Potentiation by reserpine and verapamil of anticancer drug cytotoxicity in parental AA8 cells and their MDR T19 subline as determined by the clonogenic assay. (Reprinted from Tab. 1 of ref. 109 with permission from Blackwell Science)... 

Neuse EW, Kanzawa F (1990) Evaluation of the activity of some water-soluble ferrocene and ferricinium compounds against carcinoma of the lung by the human tumor clonogenic assay. Appl Organomet Chem 4 19-26... 

Colony-forming efficiency/clonogenic assays are the most reliable methods for assessing viable cell number. These methods are time-consuming, however, and become impractical when many samples have to be analysed (e.g. in toxicity assessment where several chemicals at a range of concentrations and combinations need to be analysed). 

Parent-Massin D (2001) Relevance of clonogenic assays in hematotoxicology. Cell Biology and Toxicology 17(2) 87-94. 

The number of cells within each colony depends on the number of cell doublings and can be used as an estimate of the effects, if any, of the drug on the cell doubling time. A clonogenic assay can thus discriminate between cytotoxic (cell kill) and cytostatic (decreased growth rate) effects. Because a cytostatic effect may be lost upon removal of the drug a cytotoxicity assay based on colony formation is also described since this allows continuous drug exposure. 

Fig. 1. A typical dose response curve obtained by clonogenic assay. The human colon tumor cell line HT29 in exponential growth was exposed to mitomycin C for 3 h and then plated out at a density of 500 cells/6 cm Petri dish. The mean colony count in the control dishes was 281, which is a cloning efficiency of 56%. Three flasks of cells were used at each dose level and each point is the mean standard error of the mean of the three estimates. Estimation of the IC50 value (the drug concentration required to kill 50% of the cells) is shown by the straight lines.

Fix and stain the colonies and evaluate as for the standard clonogenic assay. 

Weisenthal, L. M., Dill, P. L., Kumick, N. B., and Lippman, M. E. (1983) Comparison of dye exclusion assays with a clonogenic assay in the determination of drug-induced cytotoxicity. Cancer Res. 43, 258-264. 

Salmon, S. E Young, L., Scuderi, P., and Clark, B. Antineoplastic effects of tumor necrosis factor alone and in combination with gamma-interferon on tumor biopsies in clonogenic assay. J. Clin. Oncol. 5, 1816-1821 (1987). 

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