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Clonogenicity of mammalian cells in hybrid spheroids a new assay method

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Summary

A new in vitro method for determining the clonogenicity of mammalian cells in culture is described. The method is based on packaging clonogens into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates, termed hybrid spheroids, provide an in vivo-like environment for entrapped test cells, offering a realistic system for prospective tumor control studies. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of non-colony forming spheroids, the average number of clonogens per spheroid can be calculated, and the survival of irradiated test cells determined. In this fashion survival curves were obtained for HeLa, B-16 and HEp3 cells which corresponded to survival curves obtained in the conventional manner. The clonogenicity of cells, derived from a human maxillar melanoma surgical specimen was also determined by the hybrid spheroid method. With this method, plating efficiency increased in those cells which normally plate poorly, such as tumor cells, thus enabling survival measurements when this is not practical using conventional methods.

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References

  1. Ajani JA, Baker FL, Spitzer G, Kelly A, Brock W, Tomasovic B, Singletary SE, McMurtrey M, Plager C (1987) Comparison between clinical response and in vitro drug sensitivity of primary human tumors in the adhesive tumor cell culture system. J Clin Oncol 5:1912–1921

    PubMed  Google Scholar 

  2. Arundel C, Bock S, Brock WA, Tofilon PJ (1987) Radiosensitization of primary human tumor cell cultures by N-methylformamide. Int J Radiat Oncol Biol Phys 13:753–757

    PubMed  Google Scholar 

  3. Baker F, Spitzer G, Ajani JA, Brock WA, Lukeman J, Pathak S, Tomasovic B, Thielvoldt D, Williams M, Vines C, Tofilon PJ (1986) Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium. Cancer Res 46:1263–1274

    PubMed  Google Scholar 

  4. Brock WA, Williams M, Bhadkamkar VA, Spitzer G, Baker F (1985) Radiosensitivity testing of primary cultures derived from human tumors. In: Proceedings of the Third International Meeting on Progress in Radio-Oncology. Karcher, Vienna, pp 185–194

    Google Scholar 

  5. Courtenay VD (1984) A replenishable soft agar colony assay for human tumor sensitivity testing. Recent Results Cancer Res 94:17–34

    PubMed  Google Scholar 

  6. Durand RE (1975) Cure, regression and cell survival: a comparison of common radiobiological endpoints using an in vitro tumor model. Br J Radiol 48:556–571

    PubMed  Google Scholar 

  7. Fletcher R (1972) Fortran subroutines for minimization by Quasi-Newton methods. AERE, p 7125

  8. Gershman H, Drumm J, Culp L (1976) Sorting of normal and virus-transformed cells in cellular aggregates. J Cell Biol 68:276–286

    PubMed  Google Scholar 

  9. Gilbert CW (1974) A double minus log transformation of mortality probabilities. Int J Radiat Biol 25:633–634

    Google Scholar 

  10. Gordon JR, Quigley JP (1986) Early spontaneous metastasis in the human epidermoid carcinoma HEp3/chick embryo model: contribution of incidental colonization. Int J Cancer 38:437–444

    PubMed  Google Scholar 

  11. Griffiths P, Hill ID (1985) Applied statistics algorithms. Ellis Hopwood, Chichester

    Google Scholar 

  12. Hahn GM, Rockwell S, Kallman RE, Gordon LF, Frindel E (1974) Repair of potentially lethal damage in vivo in solid tumor cells after x-irradiation. Cancer Res 34:351–354

    PubMed  Google Scholar 

  13. Hamburger AW, Salmon SE, Kim MB, Trent JM, Soehnlen BJ, Alberts DS, Schmidt HJ (1978) Direct cloning of human ovarian carcinoma cells in agar. Cancer Res 38:3438–3447

    PubMed  Google Scholar 

  14. Hill RP, Ng R, Warren BF, Bush RS (1979) The effect of intercellular contact on the radiation sensitivity of KHT sarcoma cells. Radiat Res 77:182–192

    PubMed  Google Scholar 

  15. Hopwood LE (1982) Response of human tumor cells from biopsy specimens to radiation and hyperthermia. Int J Radiat Oncol Biol Phys 8 [Suppl I]:72

    Google Scholar 

  16. Jones AC, Stratford IJ, Wilson PA, Peckham MJ (1982) In vitro cytotoxic drug sensitivity testing of human tumor xenografts grown as multicellular tumor spheroids. Br J Cancer 46:870–879

    PubMed  Google Scholar 

  17. Lange CS, Gilbert CW (1968) Studies on the cellular basis of radiation lethality. III. The measurement of stem cell repopulation probability. Int J Radiat Biol 14:373–388

    Google Scholar 

  18. Munro TR, Gilbert CW (1961) The relation between tumor lethal doses and the radiosensitivity of tumor cells. Br J Radiol 34:246–251

    PubMed  Google Scholar 

  19. Moore JV, Hendry JH (1984) Relationship of clonogenic cells and “tumor-rescuing cells”, modelled in irradiated spheroids in vitro. Br J Radiol 57:935–937

    PubMed  Google Scholar 

  20. O'Neil R (1971) Function minimization using a simplex procedure algorithm. Appl Statist 47:38–400

    Google Scholar 

  21. Pourreau-Schneider N, Malaise E (1981) Relationship between surviving fractions using the colony method, the LD50, and the growth delay after irradiation of human melanoma cells grown as multicellular spheroids. Radiat Res 85:321–332

    PubMed  Google Scholar 

  22. Rofstad EK, Wahl A, Brustad T (1986) Radiation response of human melanoma multicellular spheroids measured as single cell survival, growth delay, and spheroid cure: Comparisons with the parent tumor xenograft. Int J Radiat Oncol Biol Phys 12:975–982

    PubMed  Google Scholar 

  23. Rofstad EK, Wahl A, Tveit KM, Monge OR, Brustad T (1985) Survival curves after x-ray and heat treatments for melanoma cell derived directly from surgical specimens of tumors in man. Radiother Oncol 4:33–44

    PubMed  Google Scholar 

  24. Selby P, Buick RN, Tannock I (1983) A critical appraisal of the “human stem-cell assay”. N Engl J Med 308:129–134

    PubMed  Google Scholar 

  25. Sinclair WK, Morton RA (1964) Recovery following x-irradiation of synchronized Chinese hamster cells. Nature 203:247–250

    PubMed  Google Scholar 

  26. Sutherland RM (1988) Cell and environment interaction in tumor microregions: The multicell spheroid model. Science 240:177–184

    PubMed  Google Scholar 

  27. Sutherland RM, Durand RE (1984) Growth and cellular characteristics of multicell spheroids. Recent Results Cancer Res 95:24–49

    PubMed  Google Scholar 

  28. Tofilon PJ, Buckley N, Deen DF (1981) Effect of cell-cell interactions on drug sensitivity and growth of drug-sensitive and resistant tumor cells in spheroids. Science 226:262–864

    Google Scholar 

  29. Yuhas JM (1983) Clonogenic assays for predicting the response of solid tumors to therapy. In: Fletcher GH, Nervi G, Withers HR (eds) Biological bases and clinical implications of tumors resistance. Masson, New York, pp 383–390

    Google Scholar 

  30. Yuhas JM, Li AP, Martinez AO, Ladman AJ (1977) A simplified method for production and growth of multicellular tumor spheroids. Cancer Res 37:3639–3643

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Radiation Oncology, SUNY Health Sciences Center at Brooklyn, 11203, Brooklyn, NY, USA

    Bozidar Djordjevic & Christopher S. Lange

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  1. Bozidar Djordjevic
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  2. Christopher S. Lange
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Djordjevic, B., Lange, C.S. Clonogenicity of mammalian cells in hybrid spheroids a new assay method. Radiat Environ Biophys 29, 31–46 (1990). https://doi.org/10.1007/BF01211233

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  • DOI: https://doi.org/10.1007/BF01211233

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