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Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor

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Summary

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (103 to 105 cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 104 total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 104 cells per dish. The CFE for untreated tumors was 18±10% (±S.D.), demonstrating that there is significant biological variation.

The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log10 cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.

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Author notes

  1. Kenneth T. Wheeler

    Present address: Laboratory of Radiobiology, University of California, 94143, San Francisco, Ca

Authors and Affiliations

  1. The Howard C. Naffziger Laboratories, Department of Neurological Surgery, University of California Medical Center, 94143, San Francisco, California

    Mark L. Rosenblum, Kathy D. Knebel, Kenneth T. Wheeler, Marvin Barker & Charles B. Wilson

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  1. Mark L. Rosenblum
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  2. Kathy D. Knebel
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  3. Kenneth T. Wheeler
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  4. Marvin Barker
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  5. Charles B. Wilson
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Additional information

This work was supported by NIH Center Grant CA 13525, the National Phi Beta Psi Sorority, the Joe Gheen Medical Foundation, and the Association for Brain Tumor Research.

Recipient of NIH Individual Postdoctoral Research Fellowship Award 1 F22 CA02196-01

Supported by NIH Career Development Award.

The tumor used in this study was provided by William H. Sweet, Paul L. Kornblith, Jannette L. Messer, and Beverly O. Whitman of Massachusetts General Hospital, Boston, Mass.

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Rosenblum, M.L., Knebel, K.D., Wheeler, K.T. et al. Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor. In Vitro 11, 264–273 (1975). https://doi.org/10.1007/BF02615637

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