Enrichment of epithelial cells for molecular studies (original) (raw)

Nature Medicine volume 5, pages 459–462 (1999)Cite this article

Accurate molecular analysis of tumors and their precursor lesions requires the extraction of specific subpopulations of cells (normal, preneoplastic and tumor) from a composite background of multiple cell types. Attempts to obtain pure tumor cell samples have resulted in the genesis of several methods of cell enrichment, including the use of tumor cell lines, xenografted tumors, and microdissection of frozen or paraffin-embedded tissues. All methods now used have advantages and limitations. The first two methods provide self-replicating sources of high-quality reagents but are difficult, expensive and time consuming and the results may not be representative of the parent tumor. The development of laser capture microdissection (LCM) has revolutionized microdissection. However, this technique is limited by the poor quality of nucleic acids and proteins from archival samples and by the drawbacks of working with frozen sections. To enhance the potential of LCM and to overcome some of the limitations of other methods, we have developed a rapid, simple procedure for enrichment of normal and tumor cells called epithelial aggregate separation and isolation (EASI). Smears of tumor tissues are prepared on glass slides, rapidly fixed in methanol and stained. EASI preps yield nearly pure aggregates of epithelial cell populations (malignant, normal and preneoplastic), which are identified by microscopic examination. LCM is then done and the intermediate-quality nucleic acids thus obtained are suitable for many DNA and RNA assays, including allelotyping, mutation analysis, automatic sequencing, RT–PCR and library construction. The EASI method is applicable to most normal, preneoplastic and malignant epithelial tissues, requires minimal expertise and can be conveniently integrated into standard surgical pathology practice. For tumor or epithelial cell enrichment, the method of choice should be based on its relative advantages and limitations, as well as on the available facilities.

Current methods of cell enrichment

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$209.00 per year

only $17.42 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

References

  1. Simone, N.L., Bonner, R.F., Gillespie, J.W., Emmert-Buck, M.R. & Liotta, L.A. Laser-capture microdissection: Opening the microscopic frontier to molecular analysis. Trends Genet. 14, 272–276 (1998).
    Article CAS Google Scholar
  2. Seymour, A.B. et al. Allelotype of pancreatic adenocarcinoma. Cancer Res. 54, 2761–2764 ( 1994).
    CAS PubMed Google Scholar
  3. Going, J.J. & Lamb, R.F. Practical histological microdissection for PCR analysis. J. Pathol. 179, 121– 124 (1996).
    Article CAS Google Scholar
  4. Fujii, H., Zhou, W. & Gabrielson, E. Detection of frequent allelic loss of 6q23-q25.2 in microdissected human breast cancer tissues. Genes Chromosomes Cancer 16, 35–39 ( 1996).
    Article CAS Google Scholar
  5. Shibata, D. et al. Specific genetic analysis of microscopic tissue after selective ultraviolet radiation fractionation and the polymerase chain reaction. Am. J. Pathol. 141, 539–543 (1992).
    CAS PubMed PubMed Central Google Scholar
  6. Bonner, R.F. et al. Laser capture microdissection: Molecular analysis of tissue. Science 278, 1481–1483 (1997).
    Article CAS Google Scholar
  7. Wright, D. & Manos, M. in PCR Protocols: A Guide to Methods and Applications (ed. MA, Innis) 153–158 (Academic, San Diego, 1990).
    Google Scholar
  8. Goelz, S.E., Hamilton, S.R. & Vogelstein, B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem. Biophys. Res. Commun. 130, 118–126 (1985).
    Article CAS Google Scholar
  9. Hung, J. et al. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. J. Am. Med. Assoc. 273, 558–563 (1995).
    Article CAS Google Scholar
  10. Hahn, S.A. et al. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res. 55, 4670–4675 (1995).
    CAS PubMed Google Scholar
  11. Reyes, G. et al. Orthotopic xenografts of human pancreatic carcinomas acquire genetic aberrations during dissemination in nude mice. Cancer Res. 56, 5713–5719 ( 1996).
    CAS PubMed Google Scholar
  12. Thiagalingam, S. et al. Evaluation of the FHIT gene in colorectal cancers. Cancer Res. 56, 2936–2939 (1996).
    CAS PubMed Google Scholar
  13. Gazdar, A.F. & Minna, J.D. NCI series of cell lines: An historical perspective. J. Cell Biochem. Suppl. 24, 1–11 (1996).
    Article CAS Google Scholar
  14. McQueen, H.A., Wyllie, A.H., Piris, J., Foster, E. & Bird, C.C. Stability of critical genetic lesions in human colorectal carcinoma xenografts. Br. J. Cancer 63, 94–96 (1991).
    Article CAS Google Scholar
  15. Wistuba, I.I. et al. Comparison of features of human breast cancer cell lines and their corresponding tumors. Clin. Cancer Res. 4 , 2931–2938. (1998).
    CAS PubMed Google Scholar
  16. Foster, H.M., Tay, D.L. & Whitehead, R.H. Changes in the DNA ploidy patterns of human colorectal carcinomas, subsequent to culture or xenografting. J. Surg. Oncol. 45, 4–9 (1990 ).
    Article CAS Google Scholar
  17. Gazdar, A.F. et al. Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer. Int. J. Cancer 78, 766–774 (1998).
    Article CAS Google Scholar
  18. Stevenson, H. et al. Tumor cell lines established in vitro: An independent prognostic factor for survival in non-small-cell lung cancer. Ann. Intern. Med. 113, 764–770 (1990).
    Article CAS Google Scholar
  19. Bodnar, A.G. et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349– 352 (1998).
    Article CAS Google Scholar
  20. Bar, J.K., Harlozinska, A., Sobanska, E. & Cislo, M. Cytomorphologic characterization of cell subsets isolated by density gradient centrifugation from tumor effusions of ovarian endometrioid carcinoma. Tumori 80, 290–294 ( 1994).
    Article CAS Google Scholar
  21. Racila, E. et al. Detection and characterization of carcinoma cells in the blood. Proc. Natl. Acad. Sci. USA 95, 4589 –4594 (1998).
    Article CAS Google Scholar
  22. Eaton, M.C., Hardingham, J.E., Kotasek, D. & Dobrovic, A. Immunobead RT-PCR: A sensitive method for detection of circulating tumor cells. Biotechniques 22, 100– 105 (1997).
    Article CAS Google Scholar
  23. Sakaguchi, M. et al. Development of a sensitive, specific reverse transcriptase polymerase chain reaction-based assay for epithelial tumor cells in effusions. Br. J. Cancer 79, 416– 422, 1999.
    Article CAS Google Scholar

Download references

Acknowledgements

We thank R.A. Starr and Y. Kohda for RT–PCR on podoplanin mRNA from rat glomeruli. This work was supported by Lung Cancer SPORE P50CA70907 and NIH Fellowship Training grant (A.S.).

Author information

Authors and Affiliations

  1. Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. , Dallas, 75235, Texas, USA
    Anirban Maitra, Ignacio I. Wistuba, Arvind K. Virmani, M. Sakaguchi, Inwon Park, Amy Stucky, John D. Minna & Adi F. Gazdar
  2. Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas , 75235, Texas, USA
    Anirban Maitra, Sara Milchgrub, David Gibbons & Adi F. Gazdar
  3. Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 75235, Texas, USA
    John D. Minna
  4. Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas , 75235, Texas, USA
    John D. Minna

Authors

  1. Anirban Maitra
    You can also search for this author inPubMed Google Scholar
  2. Ignacio I. Wistuba
    You can also search for this author inPubMed Google Scholar
  3. Arvind K. Virmani
    You can also search for this author inPubMed Google Scholar
  4. M. Sakaguchi
    You can also search for this author inPubMed Google Scholar
  5. Inwon Park
    You can also search for this author inPubMed Google Scholar
  6. Amy Stucky
    You can also search for this author inPubMed Google Scholar
  7. Sara Milchgrub
    You can also search for this author inPubMed Google Scholar
  8. David Gibbons
    You can also search for this author inPubMed Google Scholar
  9. John D. Minna
    You can also search for this author inPubMed Google Scholar
  10. Adi F. Gazdar
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toAdi F. Gazdar.

Rights and permissions

About this article

Cite this article

Maitra, A., Wistuba, I., Virmani, A. et al. Enrichment of epithelial cells for molecular studies.Nat Med 5, 459–462 (1999). https://doi.org/10.1038/7458

Download citation

This article is cited by