Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non‐Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In Situ: Applications in Evaluating Novel Therapeutic Agents

Denise C. Connolly1, Harvey H. Hensley1

1 Fox Chase Cancer Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.12
DOI:  10.1002/0471141755.ph1412s45
Online Posting Date:  June, 2009
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Abstract

Epithelial ovarian cancer (EOC) is the most commonly fatal gynecologic malignancy in developed countries. Most EOC patients are diagnosed at an advanced stage when disease has spread beyond the ovary. While many patients initially respond to surgery and chemotherapy, the long‐term prognosis is generally unfavorable, with recurrence and development of drug‐resistant disease. There is a critical need to identify new therapeutic agents that prolong disease‐free intervals and effectively manage recurrent disease. Murine models of ovarian carcinoma are excellent models to study tumor biology in the search for new treatments for EOC. Described in this unit are methods for establishing xenograft or allograft models of EOC using ovarian carcinoma cell lines, in vivo imaging strategies for detection and quantification of EOC in transgenic and in xenograft/allograft models, and procedures for necropsy and pathological evaluation of experimental animals. Curr. Protoc. Pharmacol. 45:14.12.1‐14.12.26. © 2009 by John Wiley & Sons, Inc.

Keywords: epithelial ovarian cancer (EOC); xenograft; orthotopic; transgenic mice; metastasis; in vivo imaging; magnetic resonance imaging; bioluminescent imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Xenograft or Allograft Implantation of Ovarian Carcinoma Cells in Mice
  • Basic Protocol 2: Magnetic Resonance Imaging (MRI) of Ovarian Tumors
  • Basic Protocol 3: Bioluminescent Imaging (BLI) of Ovarian Tumor Xenografts/Allografts
  • Basic Protocol 4: Necropsy and Pathology Evaluation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Xenograft or Allograft Implantation of Ovarian Carcinoma Cells in Mice

  Materials
  • Ovarian cancer cell line (human and murine ovarian carcinoma cell lines available from ATCC and individual investigators)
  • Cell culture medium (e.g., DMEM and RPMI)
  • Fetal bovine serum (FBS)
  • Penicillin/streptomycin (P/S)
  • L‐Glutamine
  • 100× insulin/transferrin/selenium (Invitrogen or Cellgro)
  • Phosphate buffered saline (PBS; appendix 2A), sterile
  • 0.25% Trypsin/EDTA
  • 0.4% Trypan Blue
  • Ca2+‐ and Mg2+‐free PBS (optional)
  • Immunocompromised (e.g., Nod/SCID) or syngeneic (e.g., C57Bl/6) recipient female mice (8 to 10 weeks of age; Taconic, Charles River, and Jackson Labs)
  • Alcohol prep pads (70% isopropyl alcohol; Fisher Scientific)
  • CO 2 source
  • 10 mg/ml ketamine hydrochloride and 1 mg/ml xylazine hydrochloride in sterile saline (Fisher Scientific)
  • Betadine (Fisher Scientific)
  • Surgical tissue adhesive (Nexaband, Abbot Laboratories)
  • Sterile physiologic saline
  • Buprenorphine (Fisher Scientific)
  • 37°C, 5% CO 2 incubator (Fisher Scientific)
  • 75‐ or 175‐cm2 cell culture flasks (Fisher Scientific)
  • Platform shaker
  • 37°C water bath (Fisher Scientific)
  • 15‐ml conical tubes (Fisher Scientific)
  • Refrigerated centrifuge
  • Hemacytometer (Fisher Scientific)
  • Inverted microscope
  • Electric hair clippers (Fisher Scientific or Roboz Surgical Instrument Co.)
  • Sterile 1‐ml tuberculin syringes (Fisher Scientific)
  • 26‐, 29½‐, and 30‐G needles (Fisher Scientific)
  • Scale (Fisher Scientific)
  • Calipers (Fisher Scientific)
  • Sterile surgical instruments (scissors, forceps, tissue clips, and surgical wound staples; Roboz Surgical Instrument Co.)
  • Gauze pads
  • Dissection microscope
  • 6‐0 silk or Vicryl sutures (Roboz Surgical Instrument Co.)
  • Surgical staples or wound clips
  • Liquid infant heel warmer (optional, Fisher Scientific cat. no. 22‐024‐646), heating pad, or infrared heat lamp (Fisher Scientific)

Basic Protocol 2: Magnetic Resonance Imaging (MRI) of Ovarian Tumors

  Materials
  • Gadolinium‐diethylenepentaacetic acid (Gd‐DTPA; Magnevist, Berlex Labs)
  • 1× filter‐sterilized PBS
  • GEM models of EOC or mice with orthotopically implanted ovarian carcinoma cell lines (see protocol 1)
  • Isoflurane/oxygen‐based anesthesia system
  • 70% isopropyl alcohol prep pads (Fisher Scientific)
  • Plastic tubing (2‐ to 3‐mm i.d.) and caps
  • Induction chamber (Summit Medical Equipment, Molecular Imaging Products, http://store.mipcompany.com/)
  • Infrared heat lamp
  • 1‐ml tuberculin syringe
  • 30‐G needle
  • Vertical or horizontal bore magnetic resonance imaging scanner (field strength ≥7 Tesla) with 25‐ to 30‐mm birdcage coil
  • Shareware programs: Bru2analyzer (http://www.sph.sc.edu/comd/rorden/bru2anz.html) and MRIcro (http://www.psychology.nottingham.ac.uk/staff/cr1/mricro.html)

Basic Protocol 3: Bioluminescent Imaging (BLI) of Ovarian Tumor Xenografts/Allografts

  Materials
  • Ovarian carcinoma cell lines expressing a luminescent reporter gene, such as firefly luciferase, e.g., the human ovarian carcinoma cell line, SKOV3‐luc‐D3 Bioware cell line (Caliper Life Sciences)
  • Luciferin substrate (Caliper LifeSciences)
  • 1× sterile filtered PBS
  • Ovarian tumor‐bearing mice:
    • Mice xenografted or allografted with ovarian carcinoma cells expressing a luminescent reporter gene (see protocol 1) or
    • Transgenic mice with a spontaneous tumor that expresses a luminescent reporter gene
  • Isoflurane/oxygen‐based anesthesia system
  • 96‐well cell culture dish
  • 37°C water bath or heating block
  • In vivo bioluminescent imaging system (e.g., IVIS Spectrum, Caliper Life Sciences)
  • Induction chamber (e.g., Summit Medical Equipment, Molecular Imaging Products, http://store.mipcompany.com)
  • Portable hair clippers or depilatory lotion
  • 1‐ml tuberculin syringes
  • 30‐G needle
  • Additional reagents and equipment for i.p. or intrabursal injection (see protocol 1)

Basic Protocol 4: Necropsy and Pathology Evaluation

  Materials
  • Mice
  • CO 2 for euthanasia
  • 70% ethanol
  • 10% neutral buffered formalin (NBF; Thermo Scientific, Fisher)
  • Paraffin, optional
  • Hematoxylin & eosin, optional
  • Liquid nitrogen
  • Sterile dissection scissors and forceps
  • Specimen containers
  • 15‐ or 50‐ml conical tubes
  • Sterile transfer pipets
  • Necropsy report form (Fig. )
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Figures

Videos

Literature Cited

Literature Cited
   Adams, A.T. and Auersperg, N. 1981. Transformation of cultured rat ovarian surface epithelial cells by Kirsten murine sarcoma virus. Cancer Res. 41:2063–2072.
   Auersperg, N., Maines‐Bandiera, S.L., and Dyck, H.G. 1997. Ovarian carcinogenesis and the biology of ovarian surface epithelium. J. Cell. Physiol. 173:261‐265.
   Bao, R., Connolly, D.C., Murphy, M., Green, J., Weinstein, J.K., Pisarcik, D.A., and Hamilton, T.C. 2002. Activation of cancer‐specific gene expression by the survivin promoter. J. Natl. Cancer Inst. 94:522‐528.
   Callaghan, P. 1994. Principles of Nuclear Magnetic Resonance Microscopy. Oxford University Press, New York.
   Choy, G., O'Connor, S., Diehn, F.E., Costouros, N., Alexander, H.R., Choyke, P., and Libutti, S.K. 2003. Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging. Biotechniques 35:1022‐1026, 1028‐1030.
   Clark‐Knowles, K.V., Garson, K., Jonkers, J., and Vanderhyden, B.C. 2007. Conditional inactivation of Brca1 in the mouse ovarian surface epithelium results in an increase in preneoplastic changes. Exp. Cell Res. 313:133‐145.
   Connolly, D.C., Bao, R., Nikitin, A.Y., Stephens, K.C., Poole, T.W., Hua, X., Harris, S.S., Vanderhyden, B.C., and Hamilton, T.C. 2003. Female mice chimeric for expression of the SV40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer. Cancer Res. 63:1389‐1397.
   Coppola, D., Saunders, B., Fu, L., Mao, W., and Nicosia, S.V. 1999. The insulin‐like growth factor 1 receptor induces transformation and tumorigenicity of ovarian mesothelial cells and down‐regulates their Fas‐receptor expression. Cancer Res. 59:3264‐3270.
   Crum, C.P., Drapkin, R., Miron, A., Ince, T.A., Muto, M., Kindelberger, D.W., and Lee, Y. 2007. The distal fallopian tube: A new model for pelvic serous carcinogenesis. Curr. Opin. Obstet. Gynecol. 19:3‐9.
   Davies, B.R., Auersperg, N., Worsley, S.D., and Ponder, B.A. 1998. Transfection of rat ovarian surface epithelium with erb‐B2/neu induces transformed phenotypes in vitro and the tumorigenic phenotype in vivo. Am. J. Pathol. 152:297‐306.
   Dickins, R.A., Hemann, M.T., Zilfou, J.T., Simpson, D.R., Ibarra, I., Hannon, G.J., and Lowe, S.W. 2005. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet. 37:1289‐1295.
   Dinulescu, D.M., Ince, T.A., Quade, B.J., Shafer, S.A., Crowley, D., and Jacks, T. 2005. Role of K‐ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer. Nat. Med. 11:63‐70.
   DiSaia, P.J., Morrow, M., Kanabus, J., Piechal, W., and Townsend, D.E. 1975. Two new tissue culture lines from ovarian cancer. Gynecol. Oncol. 3:215‐219.
   Dubeau, L. 1999. The cell of origin of ovarian epithelial tumors and the surface epithelium dogma: Does the emperor have no clothes? Gynecol. Oncol. 72:437‐442.
   Dutertre, M., Gouedard, L., Xavier, F., Long, W.Q., di Clemente, N., Picard, J.Y., and Rey, R. 2001. Ovarian granulosa cell tumors express a functional membrane receptor for anti‐Mullerian hormone in transgenic mice. Endocrinology 142:4040‐4046.
   Flesken‐Nikitin, A., Choi, K.C., Eng, J.P., Shmidt, E.N., and Nikitin, A.Y. 2003. Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. Cancer Res. 63:3459‐3463.
   Fredrickson, T.N. 1987. Ovarian tumors of the hen. Environ. Health Perspect. 73:35‐51.
   Freedman, R.S., Pihl, E., Kusyk, C., Gallager, H.S., and Rutledge, F. 1978. Characterization of an ovarian carcinoma cell line. Cancer 42:2352‐2359.
   Fu, X. and Hoffman, R.M. 1993. Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically‐intact patient specimens. Anticancer Res. 13:283‐286.
   Godwin, A.K., Testa, J.R., Handel, L.M., Liu, Z., Vanderveer, L.A., Tracey, P.A., and Hamilton, T.C. 1992. Spontaneous transformation of rat ovarian surface epithelial cells: Association with cytogenetic changes and implications of repeated ovulation in the etiology of ovarian cancer. J. Natl. Cancer Inst. 84:592‐601.
   Hamilton, T.C., Young, R.C., McKoy, W.M., Grotzinger, K.R., Green, J.A., Chu, E.W., Whang‐Peng, J., Rogan, A.M., Green, W.R., and Ozols, R.F. 1983. Characterization of a human ovarian carcinoma cell line (NIH:OVCAR‐3) with androgen and estrogen receptors. Cancer Res. 43:5379‐5389.
   Hamilton, T.C., Young, R.C., Louie, K.G., Behrens, B.C., McKoy, W.M., Grotzinger, K.R., and Ozols, R.F. 1984. Characterization of a xenograft model of human ovarian carcinoma which produces ascites and intraabdominal carcinomatosis in mice. Cancer Res. 44:5286‐5290.
   Hensley, H., Quinn, B.A., Wolf, R.L., Litwin, S.L., Mabuchi, S., Williams, S.J., Williams, C., Hamilton, T.C., and Connolly, D.C. 2007. Magnetic resonance imaging for detection and determination of tumor volume in a genetically engineered mouse model of ovarian cancer. Cancer Biol. Ther. 6:1717‐1725.
   Ioachim, H.L., Dorsett, B.H., Sabbath, M., and Barber, H.R. 1975. Electron microscopy, tissue culture, and immunology of ovarian carcinoma. Natl. Cancer Inst. Monogr. 42:45‐62.
   Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T., and Thun, M.J. 2008. Cancer statistics, 2008. CA Cancer J. Clin. 58:71‐96.
   Kananen, K., Markkula, M., Rainio, E., Su, J.G., Hsueh, A.J., and Huhtaniemi, I.T. 1995. Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha‐subunit promoter/simian virus T‐antigen fusion gene: Characterization of ovarian tumors and establishment of gonadotropin‐responsive granulosa cell lines. Mol. Endocrinol. 9:616‐627.
   Keri, R.A., Lozada, K.L., Abdul‐Karim, F.W., Nadeau, J.H., and Nilson, J.H. 2000. Luteinizing hormone induction of ovarian tumors: Oligogenic differences between mouse strains dictates tumor disposition. Proc. Natl. Acad. Sci. U.S.A. 97:383‐387.
   Kiguchi, K., Kubota, T., Aoki, D., Udagawa, Y., Yamanouchi, S., Saga, M., Amemiya, A., Sun, F.X., Nozawa, S., Moossa, A.R., and Hoffman, R.M. 1998. A patient‐like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer. Clin. Exp. Metastasis 16:751‐756.
   Kolfschoten, G.M., Pinedo, H.M., Scheffer, P.G., Schluper, H.M., Erkelens, C.A., and Boven, E. 2000. Development of a panel of 15 human ovarian cancer xenografts for drug screening and determination of the role of the glutathione detoxification system. Gynecol. Oncol. 76:362‐368.
   Kumar, T.R., Palapattu, G., Wang, P., Woodruff, T.K., Boime, I., Byrne, M.C., and Matzuk, M.M. 1999. Transgenic models to study gonadotropin function: The role of follicle‐stimulating hormone in gonadal growth and tumorigenesis. Mol. Endocrinol. 13:851‐865.
   Liu, J., Yang, G., Thompson‐Lanza, J.A., Glassman, A., Hayes, K., Patterson, A., Marquez, R.T., Auersperg, N., Yu, Y., Hahn, W.C., Mills, G.B., and Bast, R.C. Jr. 2004. A genetically defined model for human ovarian cancer. Cancer Res. 64:1655‐1663.
   Mabuchi, S., Altomare, D.A., Connolly, D.C., Klein‐Szanto, A., Litwin, S., Hoelzle, M.K., Hensley, H.H., Hamilton, T.C., and Testa, J.R. 2007. RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer. Cancer Res. 67:2408‐2413.
   Massazza, G., Tomasoni, A., Lucchini, V., Allavena, P., Erba, E., Colombo, N., Mantovani, A., D'Incalci, M., Mangioni, C., and Giavazzi, R. 1989. Intraperitoneal and subcutaneous xenografts of human ovarian carcinoma in nude mice and their potential in experimental therapy. Int. J. Cancer 44:494‐500.
   Molpus, K.L., Koelliker, D., Atkins, L., Kato, D.T., Buczek‐Thomas, J., Fuller, A.F. Jr., and Hasan, T. 1996. Characterization of a xenograft model of human ovarian carcinoma which produces intraperitoneal carcinomatosis and metastases in mice. Int. J. Cancer 68:588‐595.
   Orsulic, S., Li, Y., Soslow, R.A., Vitale‐Cross, L.A., Gutkind, J.S., and Varmus, H.E. 2002. Induction of ovarian cancer by defined multiple genetic changes in a mouse model system. Cancer Cell 1:53‐62.
   Ozols, R.F., Louie, K.G., Plowman, J., Behrens, B.C., Fine, R.L., Dykes, D., and Hamilton, T.C. 1987. Enhanced melphalan cytotoxicity in human ovarian cancer in vitro and in tumor‐bearing nude mice by buthionine sulfoximine depletion of glutathione. Biochem. Pharmacol. 36:147‐153.
   Parkin, D.M., Bray, F., Ferlay, J., and Pisani, P. 2005. Global cancer statistics, 2002. CA Cancer J. Clin. 55:74‐108.
   Rahman, N.A. and Huhtaniemi, I.T. 2001. Ovarian tumorigenesis in mice transgenic for murine inhibin alpha subunit promoter‐driven Simian Virus 40 T‐antigen: Ontogeny, functional characteristics, and endocrine effects. Biol. Reprod. 64:1122‐1130.
   Resnicoff, M., Ambrose, D., Coppola, D., and Rubin, R. 1993. Insulin‐like growth factor‐1 and its receptor mediate the autocrine proliferation of human ovarian carcinoma cell lines. Lab. Invest. 69:756‐760.
   Risma, K.A., Clay, C.M., Nett, T.M., Wagner, T., Yun, J., and Nilson, J.H. 1995. Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc. Natl. Acad. Sci. U.S.A. 92:1322‐1326.
   Roby, K.F., Taylor, C.C., Sweetwood, J.P., Cheng, Y., Pace, J.L., Tawfik, O., Persons, D.L., Smith, P.G., and Terranova, P.F. 2000. Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogenesis 21:585‐591.
   Rorden, C. and Brett, M. 2000. Stereotaxic display of brain lesions. Behav. Neurol. 12:191‐200.
   Rose, G.S., Tocco, L.M., Granger, G.A., DiSaia, P.J., Hamilton, T.C., Santin, A.D., and Hiserodt, J.C. 1996. Development and characterization of a clinically useful animal model of epithelial ovarian cancer in the Fischer 344 rat. Am. J. Obstet. Gynecol. 175:593‐599.
   Scully, R.E. 1995. Pathology of ovarian cancer precursors. J. Cell. Biochem. 23:208‐218.
   Shih, I.M. and Kurman, R.J. 2005. Molecular pathogenesis of ovarian borderline tumors: New insights and old challenges. Clin. Cancer Res. 11:7273‐7279.
   Testa, J.R., Getts, L.A., Salazar, H., Liu, Z., Handel, L.M., Godwin, A.K., and Hamilton, T.C. 1994. Spontaneous transformation of rat ovarian surface epithelial cells results in well to poorly differentiated tumors with a parallel range of cytogenetic complexity. Cancer Res. 54:2778‐2784.
   Wadghiri, Y.Z., Schneider, A.E., Gray, E.N., Aristizabal, O., Berrios, C., Turnbull, D.H., and Gutstein, D.E. 2007. Contrast‐enhanced MRI of right ventricular abnormalities in CX43 mutant mouse embryos. NMR Biomed. 20:366‐374.
   Ward, B.G., Wallace, K., Shepherd, J.H., and Balkwill, F.R. 1987. Intraperitoneal xenografts of human epithelial ovarian cancer in nude mice. Cancer Res. 47:2662‐2667.
   Woods, L.K., Morgan, R.T., Quinn, L.A., Moore, G.E., Semple, T.U., and Stedman, K.E. 1979. Comparison of four new cell lines from patients with adenocarcinoma of the ovary. Cancer Res. 39:4449‐4459.
   Wu, R., Hendrix‐Lucas, N., Kuick, R., Zhai, Y., Schwartz, D.R., Akyol, A., Hanash, S., Misek, D.E., Katabuchi, H., Williams, B.O., Fearon, E.R., and Cho, K.R. 2007. Mouse model of human ovarian endometrioid adenocarcinoma based on somatic defects in the Wnt/beta‐catenin and PI3K/Pten signaling pathways. Cancer Cell 11:321‐333.
   Xing, D. and Orsulic, S. 2005. A genetically defined mouse ovarian carcinoma model for the molecular characterization of pathway‐targeted therapy and tumor resistance. Proc. Natl. Acad. Sci. U.S.A. 102:6936‐6941.
   Xing, D. and Orsulic, S. 2006. A mouse model for the molecular characterization of brca1‐associated ovarian carcinoma. Cancer Res. 66:8949‐8953.
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