Orthotopic Xenograft Model of Cervical Cancer for Studying Microenvironmental Effects on Metastasis Formation and Response to Drug Treatment

Naz Chaudary1, David W. Hedley1, Richard P. Hill1

1 Ontario Cancer Institute/Princess Margaret Hospital, Campbell Family Institute for Cancer Research and University of Toronto, Toronto, Ontario, Canada
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.19
DOI:  10.1002/0471141755.ph1419s53
Online Posting Date:  June, 2011
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Cancers arising in the uterine cervix are usually squamous cell carcinomas that develop from preneoplastic lesions. They invade locally, and then typically metastasize to the regional lymph nodes and eventually to distant sites. Orthotopically grown xenografts are technically challenging to perform, but recapitulate the clinical situation to a greater extent than xenografts grown at subcutaneous or intramuscular sites. Thus, orthotopic xenografts develop lymphovascular invasion and metastasize to the para‐aortic lymphatic chain in a pattern similar to that seen in patients. The extent of (lymph node) metastases is particularly apparent when the implanted tumor cells are transfected to express a fluorescent marker, such as DsRed, which allows the exposed retroperitoneum to be examined by fluorescence microscopy. Described in this unit is a surgical technique for orthotopic implantation and the use of this model for investigating the effects of novel agents as inhibitors of tumor growth and metastasis. Curr. Protoc. Pharmacol. 53:14.19.1‐14.19.11. © 2011 by John Wiley & Sons, Inc.

Keywords: cervical carcinoma; orthotopic; metastasis; lymph nodes; hypoxia

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • ME180 or SiHa human cervical carcinoma cells (ATCC)
  • Alpha‐minimal essential medium (α‐MEM; Invitrogen)
  • 0.05% trypsin/EDTA
  • 6‐ to 8‐week‐old female, immune‐deficient (SCID) mice (obtained from an in‐house breeding program at the Ontario Cancer Institute animal colony; http://intranet.uhnres.utoronto.ca/resources/arc/)
  • Hair removal cream (e.g., Nair)
  • Distilled water
  • Surgical scrub (e.g., Betadine)
  • Beaker of 70% ethanol
  • Isoflurane
  • Eye gel (e.g., Tear‐Gel; Novartis)
  • Meloxicam diluted in saline (supplied by Animal Research Colony/OCI)
  • Formalin or OCT medium, optional
  • 75‐cm2 flasks
  • Coulter counter (Beckman Coulter)
  • Centrifuge
  • 1‐ml syringes equipped with 27‐G needles
  • Sterile swabs for application
  • Sterile gauze pads
  • Nose cone apparatus‐anesthetic machine (Benson‐Medical)
  • Sterile surgical instruments including:
    • Scissors
    • Forceps
    • Scalpel
    • Disposable scalpel blade (no. 11)
    • Small hemostat
  • 60‐mm2 cell culture dishes
  • Suture material (8‐0 silk or 6‐0 proline)
  • Glass bead sterilizer
  • 9‐mm wound clips, with applier and remover
  • Warming pad
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, )
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Literature Cited

Literature Cited
   Cairns, R.A. and Hill, R.P. 2004a. A fluorescent orthotopic model of metastatic cervical carcinoma. Clin. Exp. Metastasis 21:275‐281.
   Cairns, R.A. and Hill, R.P. 2004b. Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma. Cancer Res. 64:2054‐2061.
   Dewhirst, M.W. 2009. Relationships between cycling hypoxia, HIF‐1, angiogenesis and oxidative stress. Radiat. Res. 172:653‐665.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Fyles, A., Milosevic, M., Hedley, D., Pintilie, M., Levin, W., Manchul, L., and Hill, R.P. 2002. Tumor hypoxia has independent predictor impact only in patients with node‐negative cervix cancer. J. Clin. Oncol. 20:680‐687.
   Fyles, A., Milosevic, M., Pintilie, M., Syed, A., Levin, W., Manchul, L., and Hill, R.P. 2006. Long‐term performance of interstial fluid pressure and hypoxia as prognostic factors in cervix cancer. Radiother. Oncol. 80:132‐137.
   Hockel, M., Schlenger, K., Aral, B., Mitze, M., Schaffer, U., and Vaupel, P. 1996. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res. 56:4509‐4515.
   Milosevic, M., Fyles, A., Hedley, D., Pintilie, M., Levin, W., Manchul, L., and Hill, R. 2001. Interstitial fluid pressure predicts survival in patients with cervix cancer independent of clinical prognostic factors and tumor oxygen measurements. Cancer Res. 61:6400‐6405.
   Nordsmark, M., Loncaster, J., Aquino‐Parsons, C., Chou, S.C., Ladekarl, M., Havsteen, H., Lindegaard, J.C., Davidson, S.E., Varia, M., West, C., Hunter, R., Overgaard, J., and Raleigh, J.A. 2003. Measurements of hypoxia using pimonidazole and polarographic oxygen‐sensitive electrodes in human cervix carcinomas. Radiother. Oncol. 67:35‐44.
   Schwock, J., Dhani, N., Cao, M.P., Zheng, J., Clarkson, R., Radulovich, N., Navab, R., Horn, L.C., and Hedley, D.W. 2009. Targeting focal adhesion kinase with dominant‐negative FRNK or Hsp90 inhibitor 17‐DMAG suppresses tumor growth and metastasis of SiHa cervical xenografts. Cancer Res. 69:4750‐4759.
   Wilhelm, S., Carter, C., Lynch, M., Lowinger, T., Dumas, J., Smith, R.A., Schwartz, B., Simantov, R., and Kelley, S. 2006. Discovery and development of sorafenib: A multikinase inhibitor for treating cancer. Nat. Rev. Drug Discov. 5:835‐844.
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