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Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide
  1. Christina Parkes, MSc, BSc(Hons)*,
  2. Areege Kamal, PhD, MBBS*,
  3. Anthony J. Valentijn, MSc, BSc*,
  4. Rafah Alnafakh, MBBS*,
  5. Stephane R. Gross, PhD,
  6. Roger Barraclough, PhD,
  7. Diana Moss, PhD§,
  8. John Kirwan, MBChB, MRCOG and
  9. Dharani K. Hapangama, MD, MBChB, BSc, MRCOG*,
  1. * Department of Women's and Children's Health, Institute of Translational Medicine, University of Liverpool, Liverpool Women's Hospital, Liverpool;
  2. School of Life and Health Sciences, Aston University, Birmingham; and
  3. Institute of Integrative Biology and
  4. § Cellular and Molecular Physiology, University of Liverpool; and
  5. Liverpool Women's Hospital NHS Trust, Liverpool, United Kingdom.
  1. Address correspondence and reprint requests to Dharani K. Hapangama, MD, MBChB, BSc, MRCOG, Department of Women's and Children's Health, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool L8 7SS, United Kingdom. E-mail: dharani{at}liv.ac.uk.

Abstract

Objective Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models.

Methods Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model.

Results Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data.

Conclusions We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

  • Cell lines
  • Endometrial cancer
  • Estrogen
  • AR - androgen receptor
  • CAM - chorioallantoic membrane
  • CSFBS - charcoal-stripped fetal bovine serum
  • E2 - 17β-estradiol
  • EC - endometrial cancer
  • ER - estrogen receptor
  • ISK - Ishikawa
  • NBF - neutral-buffered formalin
  • PBS - phosphate-buffered saline
  • PR - progesterone receptor
  • STR - short tandem repeat

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Footnotes

  • The authors declare no conflicts of interest.

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