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Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

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Summary

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson’s Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best’s carmine, Page-Green method for inclusion bodies, and Snook’s reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.

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References

  1. Metcalf, D. Recent results in cancer research. vol. 61. Hemopoietic colonies. Berlin, Heidelberg, New York: Springer-Verlag; 1977.

    Google Scholar 

  2. Salmon, S. E. Progress in clinical and biological research. vol. 48. Cloning of human tumor stem cells. New York: Alan R. Liss; 1980.

    Google Scholar 

  3. Meyskens, F.; Salmon, S. E. Modulation of human melanoma clonogenic cells by follicle-stimulating hormone, melatonin and nerve growth factor. Br. J. Cancer 43: 111–116; 1981.

    PubMed  CAS  Google Scholar 

  4. Meyskens, F. L.; Soehnlen, B. J.; Saxe, D. F.; Casey, W. J.; Salmon, S. E. In vitro clonal assay for human metastatic malignant melanoma. Stem Cells 1: 61–72; 1981.

    PubMed  Google Scholar 

  5. Hamburger, A. W.; Salmon, S. E. Primary bioassay of human myeloma stem cells. J. Clin. Invest. 60: 846–854; 1977.

    Article  PubMed  CAS  Google Scholar 

  6. Meyskens, F. L.; Moon, T. E.; Dana, B.; Gilmartin, E.; Casey, W. J.; G-Chen, H. S.; Hood Franks, D.; Young, L.; Salmon, S. E. Quantitation of drug sensitivity by human metastatic melanoma colony-forming units. Br. J. Cancer 44: 787–797; 1981.

    PubMed  CAS  Google Scholar 

  7. Salmon, S. E.; Hamburger, A. W.; Soehnlen, B. J.; Durie, B. G. M.; Alberts, D. S.; Moon, T. E. Quantitation of differential sensitivity to human tumor stem cells to anticancer drugs. New Engl. J. Med. 298: 1321–1327; 1978.

    Article  PubMed  CAS  Google Scholar 

  8. Trent, J.; Salmon, S. Human tumor karyology: marked analytic improvement by short-term agar culture. Br. J. Cancer 41: 867–874; 1980.

    PubMed  CAS  Google Scholar 

  9. Buick, R. N.; MacKillop, W. J. Measurement of self-renewal in culture of clonogenic cells from human ovarian carcinoma. Br. J. Cancer 44: 349–355; 1981.

    PubMed  CAS  Google Scholar 

  10. Thomson, S. P.; Meyskens, F. L. A method for measurement of self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. Cancer Res. In press.

  11. Salmon, S. E.; Buick, R. N. Preparation of permanent slides of intact soft-agar colony cultures of hematopoietic and tumor stem cells. Cancer Res. 39: 1133–1136; 1979.

    PubMed  CAS  Google Scholar 

  12. Humphries, R. K.; Eaves, A. C.; Eaves, C. J. Self-renewal of hemopoietic stem cells during mixed colony formation in vitro. Proc. Natl. Acad. Sci. USA 78: 3629–3633; 1981.

    Article  PubMed  CAS  Google Scholar 

  13. Metcalf, D. The clonal culture in vitro of human leukemic cells. Catovsky, D. ed. The leukemic cell. London: Churchill Livingston; 1981: 220–252.

    Google Scholar 

  14. Hamburger, A. W.; Salmon, S. E. Primary bioassay of human tumor stem cells. Science 197: 461–463; 1977.

    Article  PubMed  CAS  Google Scholar 

  15. Sainte-Marie, G. A paraffin embedding technique for studies employing immunofluorescence. Histochem. Cytochem. 10: 250–256; 1962.

    Google Scholar 

  16. Mautner, V.; Hynes, R. O. Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells. J. Cell Biol. 75: 743–768; 1977.

    Article  PubMed  CAS  Google Scholar 

  17. Karnovsky, M. J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27: 137A-138A; 1965.

    Google Scholar 

  18. Venable, J. H.; Coggeshall, R. A simplified lead citrate strain for use in electron microscopy. J. Cell Biol. 25: 407–408; 1965.

    Article  PubMed  CAS  Google Scholar 

  19. Greenlee, T. K.; Ross, R.; Hartman, J. L. The fine structure of elastic fibers. J. Cell Biol. 30: 59–71; 1966.

    Article  PubMed  Google Scholar 

  20. Persky, B.; Brailey, J. L.; Meyskens, F. L. Jr.; Hendrix, M. J. C. Transmission electron microscopic characterization of two morphological variants of primary human melanoma colonies from biopsies of fresh tissue grown in soft agar. J. Cell Biol. 91(2): 149a; 1981.

    Google Scholar 

  21. Sternberger, L. A. Immunocytochemistry. 2nd ed. New York: John Wiley & Sons; 1979.

    Google Scholar 

  22. Engvall, E.; Ruoslahti, E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int. J. Cancer 20: 1–5; 1977.

    Article  PubMed  CAS  Google Scholar 

  23. Pearlstein, E.; Gold, L. I.; Garcia-Pardo, A. Fibronectin: a review of its structure and biological activity. Mol. Cell Biochem. 29: 103–128; 1980.

    Article  PubMed  CAS  Google Scholar 

  24. Stenman, S.; Vaheri, A. Fibronectin in human solid tumors. Int. J. Cancer 27: 427–435; 1981.

    Article  PubMed  CAS  Google Scholar 

  25. Hynes, R. O.; Fox, C. F. Progress in clinical and biological research. vol. 41. Tumor cell surfaces and malignancy. New York: Alan R. Liss, Inc.; 1980.

    Google Scholar 

  26. Mackay, B.; Osborne, B. M. The contribution of electron microscopy to the diagnosis of tumors. Ioachim, H. L. ed. Pathobiology annual. vol. 8. New York: Raven Press; 1978: 359–405.

    Google Scholar 

  27. Durie, B. G. M.; Persky, B.; Soehnlen, B. J.; Grogan, T. M.; Salmon, S. E. Amyloid production in human myeloma stem cell culture with morphologic evidence of “preamyloid” secretion by associated macrophages. New Engl. J. Med. In press.

  28. Jacobs, P.; Dubovsky, D.; Smith, S.; Randall, G.; Bracher, M. Bone marrow culture in vitro. A technique for analysis and permanent recording of cellular composition. Exp. Hematol. 7: 177–182; 1979.

    PubMed  CAS  Google Scholar 

  29. Van Noord, M. J.; Blansjar, N.; Nakeff, A. The processing of mammalian haemopoietic cells in thin layer agar cultures for electron microscopy. Stain Technol. 48: 239–246; 1973.

    PubMed  Google Scholar 

  30. Konwalinka, G.; Glaser, P.; Odavic, R.; Bogusch, E.; Schmalzl, F.; Braunsteiner, H. A new approach to the morphological and cytochemical evaluation of human bone marrow CFUc in agar cultures. Exp. Hematol. 8: 434–440; 1980.

    PubMed  CAS  Google Scholar 

  31. Kubota, K.; Mizoguchi, H.; Miura, Y.; Suda, T.; Takaku, F. A new technique for the cytochemical examination of human hemopoietic cells grown in agar gel. Exp. Hematol. 8: 339–344; 1980.

    PubMed  CAS  Google Scholar 

  32. Harris, G. J.; Zeagler, J.; Hodek, A.; Casper, J.; Von Hoff, D. D. Ultrastructural analysis of colonies growing in a human tumor cloning system. Cancer 50: 722–726; 1982.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Anatomy, Internal Medicine and Cancer Center, College of Medicine, University of Arizona, 85724, Tucson, Arizona

    Bruce Persky, Stephen P. Thomson, Frank L. Meyskens & Mary J. C. Hendrix

Authors
  1. Bruce Persky
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  2. Stephen P. Thomson
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  3. Frank L. Meyskens
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  4. Mary J. C. Hendrix
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Additional information

This work was supported by Grant T32 CA09213, a National Research Service Award from the National Cancer Institute to B. P., by Grants PDT-205 (M. J. C. H.) and PDT-184 (F. M.) from the American Cancer Society, and from the National Cancer Institute (CA-17904).

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Persky, B., Thomson, S.P., Meyskens, F.L. et al. Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar. In Vitro 18, 929–936 (1982). https://doi.org/10.1007/BF02796349

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  • DOI: https://doi.org/10.1007/BF02796349

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