Methods for Evaluating the Morphological and Immunohistochemical Properties of Human Tumor Colonies Grown in Soft Agar

Overview

Journal In Vitro

Specialties Anatomy & Histology
Cell Biology

Date 1982 Nov 1

PMID 6759369

Citations 1

Authors

B Persky

S P Thomson

F L Meyskens Jr

M J Hendrix

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

The human tumour cloning assay in the management of breast cancer patients.

Dittrich C, Jakesz R, Wrba F, Havelec L, HAAS O, Spona J Br J Cancer. 1985; 52(2):197-203.

PMID: 4027163 PMC: 1977099. DOI: 10.1038/bjc.1985.178.

References

Durie B, Persky B, Soehnlen B, Grogan T, Salmon S . Amyloid production in human myeloma stem-cell culture, with morphologic evidence of amyloid secretion by associated macrophages. N Engl J Med. 1982; 307(27):1689-92. DOI: 10.1056/NEJM198212303072706. View

Mackay B, Osborne B . The contribution of electron microscopy to the diagnosis of tumors. Pathobiol Annu. 1978; 8:359-405. View

Greenlee Jr T, Ross R, HARTMAN J . The fine structure of elastic fibers. J Cell Biol. 1966; 30(1):59-71. PMC: 2106991. DOI: 10.1083/jcb.30.1.59. View

Salmon S, Buick R . Preparation of permanent slides of intact soft-agar colony cultures of hematopoietic and tumor stem cells. Cancer Res. 1979; 39(3):1133-6. View

Pearlstein E, Gold L, Garcia-Pardo A . Fibronectin: a review of its structure and biological activity. Mol Cell Biochem. 1980; 29(2):103-28. DOI: 10.1007/BF00220304. View

Buick R, Mackillop W . Measurement of self-renewal in culture of clonogenic cells from human ovarian carcinoma. Br J Cancer. 1981; 44(3):349-55. PMC: 2010772. DOI: 10.1038/bjc.1981.191. View

Humphries R, Eaves A, Eaves C . Self-renewal of hemopoietic stem cells during mixed colony formation in vitro. Proc Natl Acad Sci U S A. 1981; 78(6):3629-33. PMC: 319624. DOI: 10.1073/pnas.78.6.3629. View

Thomson S, Meyskens Jr F . Method for measurement of self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. Cancer Res. 1982; 42(11):4606-13. View

Trent J, Salmon S . Human tumour karyology: marked analytic improvement by short-term agar culture. Br J Cancer. 1980; 41(6):867-74. PMC: 2010341. DOI: 10.1038/bjc.1980.162. View

10.

Meyskens Jr F, MOON T, DANA B, Gilmartin E, Casey W, Chen H . Quantitation of drug sensitivity by human metastatic melanoma colony-forming units. Br J Cancer. 1981; 44(6):787-97. PMC: 2010878. DOI: 10.1038/bjc.1981.277. View

11.

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. 1980; 8(3):339-44. View

12.

Meyskens Jr F, Salmon S . Modulation of clonogenic human melanoma cells by follicle-stimulating hormone, melatonin, and nerve growth factor. Br J Cancer. 1981; 43(1):111-5. PMC: 2010506. DOI: 10.1038/bjc.1981.17. View

13.

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. 1980; 8(4):434-40. View

14.

Mautner V, Hynes R . Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells. J Cell Biol. 1977; 75(3):743-68. PMC: 2111579. DOI: 10.1083/jcb.75.3.743. View

15.

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. 1979; 7(4):177-82. View

16.

Harris G, Zeagler J, Hodach A, Casper J, Harb J, Von Hoff D . Ultrastructural analysis of colonies growing in a human tumor cloning system. Cancer. 1982; 50(4):722-6. DOI: 10.1002/1097-0142(19820815)50:4<722::aid-cncr2820500416>3.0.co;2-7. View

17.

Hamburger A, Salmon S . Primary bioassay of human myeloma stem cells. J Clin Invest. 1977; 60(4):846-54. PMC: 372433. DOI: 10.1172/JCI108839. View

18.

Van Noord M, Blansjar N, Nakeff A . The processing of mammalian haemopoietic cells in thin layer agar cultures for electron microscopy. Stain Technol. 1973; 48(5):239-46. DOI: 10.3109/10520297309116631. View

19.

Salmon S, Hamburger A, Soehnlen B, Durie B, Alberts D, MOON T . Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs. N Engl J Med. 1978; 298(24):1321-7. DOI: 10.1056/NEJM197806152982401. View

20.

VENABLE J, COGGESHALL R . A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965; 25:407-8. PMC: 2106628. DOI: 10.1083/jcb.25.2.407. View