Utility and Applications of Orthotopic Models of Human Non‐Small Cell Lung Cancer (NSCLC) for the Evaluation of Novel and Emerging Cancer Therapeutics

Verline Justilien1, Alan P. Fields1

1 Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.27
DOI:  10.1002/0471141755.ph1427s62
Online Posting Date:  October, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Lung cancer is a leading cause of cancer deaths worldwide. Despite advances in chemotherapy, radiation therapy, and surgery, lung cancer continues to have a low 5‐year survival rate, highlighting a dire need for more effective means of prevention, diagnosis, prognosis, and treatment. Mouse models that recapitulate the clinical features of advanced human lung cancer are critical for testing novel therapeutic approaches. This unit describes a highly reproducible, easy‐to‐establish orthotopic murine model of lung cancer, provides methods for in vivo imaging and monitoring of tumor growth, and discusses the usefulness of this model for translational lung cancer research and the development of therapeutic strategies. Curr. Protoc. Pharmacol. 62:14.27.1‐14.27.17. © 2013 by John Wiley & Sons, Inc.

Keywords: non‐small cell lung cancer; orthotopic model; bioluminescent in vivo imaging

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Orthotopic NSCLC Mouse Model
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Orthotopic NSCLC Mouse Model

  • H1299 lung carcinoma cell line (ATCC) or other cell line suitable for the generation of lung orthotopic tumors (see Table 14.27.1)
  • Complete cell culture medium: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin
  • Lipofectamine 2000 reagent (Invitrogen)
  • pGL4.51 vector containing a firefly luciferase gene constitutively driven by a CMV promoter and containing a neomycin resistance cassette (Promega, cat. no. E1320)
  • G418 (Invitrogen)
  • 15 mg/ml D‐luciferin, firefly (Caliper Life Sciences, cat. no. 122796)
  • 0.25% trypsin‐EDTA
  • 0.4% trypan blue
  • 1× phosphate‐buffered saline (PBS; Invitrogen)
  • Matrigel, growth factor reduced (BD)
  • Male athymic nude mice (6 to 8 weeks old; Harlan Laboratories) or other suitable mice
  • Ketamine (90 to 120 mg/kg, Cardinal Health)/xylazine (10 mg/kg, Patterson Veterinary Supply) or other appropriate anesthetic
  • 10% povidone‐iodine (Betadine)
  • Ophthalmic ointment (containing neomycin sulfate, polymyxin B sulfate, and bacitracin zinc) (Patterson Veterinary Supply)
  • Buprenorphine (Cardinal Health)
  • Compound(s) for experimental treatment of mice, and vehicle (for control)
  • Isoflurane (100%) (Abbott)
  • 10% buffered formalin (for paraffin embedding) or OCT freezing medium (for storage in liquid nitrogen)
  • Tissue culture flasks and plates [T150 (150 cm2) flasks, 35‐mm plates]
  • In vivo bioluminescent imaging system including inhalational anesthesia vaporizer system with induction chamber (e.g., Xenogen IVIS Spectrum, Caliper Life Sciences)
  • Living Image software for IVIS Spectrum (Caliper Life Sciences)
  • 50‐ml conical centrifuge tubes
  • Sterile surgical towels
  • Heating pad
  • Ear punch
  • 0.5‐ml syringes and needles (27‐G × ½‐in. length; BD Medical)
  • Sterile gloves
  • Sterile alcohol prep pads (70% isopropyl alcohol)
  • 3/10‐cc insulin syringe with 30‐G needle (BD)
  • Sterile surgical scissors and forceps
  • Sterile cotton‐tipped applicators
  • Dry bead sterilizer
  • Wound‐closing surgical metal clips, stapler, and remover (e.g., AutoClip applier, AutoClip remover, and 9‐mm EZ Clip; BD Medical)
  • Heating lamp
  • 25‐G needle
  • 10‐ml syringes
  • 20‐G feeding needle (Roboz)
  • Additional reagents and equipment for fixation in paraffin ( appendix 3D) or by freezing ( appendix 3E)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Boehle, A.S., Dohrmann, P., Leuschner, I., Kalthoff, H., and Henne‐Bruns, D. 2000. An improved orthotopic xenotransplant procedure for human lung cancer in SCID bg mice. Ann. Thorac. Surg. 69:1010‐1015.
  Doki, Y., Murakami, K., Yamaura, T., Sugiyama, S., Misaki, T., and Saiki, I. 1999. Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice. Br. J. Cancer 79: 1121‐1126.
  Erdogan, E., Lamark, T., Stallings‐Mann, M., Jamieson, L., Pellechia, M., Thompson, E.A., Johansen, T, and Fields, A.P. 2006. Aurothiomalate inhibits transformed growth by targeting the PB1 domain of protein kinase Ciota. J. Biol. Chem. 38:28450‐28459.
  Frederick, L.A., Matthews, J.A., Jamieson, L., Justilien, V., Thompson, E.A., Radisky, D.C., and Fields, A.P. 2008. Matrix metalloproteinase‐10 is a critical effector of protein kinase Ciota‐Par6alpha‐mediated lung cancer. Oncogene 27:4841‐4853.
  Goto, H., Yano, S., Zhang, H., Matsumori, Y., Ogawa, H., Blakey, D.C., and Sone, S. 2002. Activity of a new vascular targeting agent, ZD6126, in pulmonary metastases by human lung adenocarcinoma in nude mice. Cancer Res. 62:3711‐3715.
  Grossman, C.E., Pickup, S., Durham, A., Wileyto, E.P., Putt, M.E., and Busch, T.M. 2011. Photodynamic therapy of disseminated non‐small cell lung carcinoma in a murine model. Lasers Surg. Med. 43:663‐675.
  Janne, P.A., Shaw, A.T., Pereira, J.R., Jeannin, G., Vansteenkiste, J., Barrios, C., Franke, F.A., Grinsted, L., Zazulina, V., Smith, P., Smith, I., and Crino, L. 2013. Selumetinib plus docetaxel for KRAS‐mutant advanced non‐small‐cell lung cancer: A randomised, multicentre, placebo‐controlled, phase 2 study. Lancet Oncol. 14:38‐47.
  Jin, H., Yang, R., Ross, J., Fong, S., Carano, R., Totpal, K., Lawrence, D., Zheng, Z., Koeppen, H., Stern, H., Schwall, R., and Ashkenazi, A. 2008. Cooperation of the agonistic DR5 antibody apomab with chemotherapy to inhibit orthotopic lung tumor growth and improve survival. Clin. Cancer Res. 14:7733‐7740.
  Justilien, V. and Fields, A. P. 2009. Ect2 links the PKCι–Par6α complex to Rac1 activation and cellular transformation. Oncogene 41:3597‐3607.
  Justilien, V., Regala, R.P., Tseng, I.C., Walsh, M.P., Batra, J., Radisky, E.S., Murray, N.R., and Fields, A.P. 2012. Matrix metalloproteinase‐10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential. PLoS One 7:e35040.
  Killion, J.J., Radinsky, R., and Fidler, I.J. 1998. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev. 17:279‐284.
  Liu, X., Liu, J., Guan, Y., Li, H., Huang, L., Tang, H., and He, J. 2012. Establishment of an orthotopic lung cancer model in nude mice and its evaluation by spiral CT. J. Thorac. Dis. 4:141‐145.
  Madero‐Visbal, R.A., Colon, J.F., Hernandez, I.C., Limaye, A., Smith, J., Lee, C.M., Arlen, P.A., Herrera, L., and Baker, C.H. 2012. Bioluminescence imaging correlates with tumor progression in an orthotopic mouse model of lung cancer. Surg. Oncol. 21:23‐29.
  McLemore, T.L., Liu, M.C., Blacker, P.C., Gregg, M., Alley, M.C., Abbott, B.J., Shoemaker, R.H., Bohlman, M.E., Litterst, C.C., Hubbard, W.C., Brennan, R.H., McMahon, J.B., Fine, D.L., Eggleston, J.C., Mayo, J.G., and Boyd, M.R. 1987. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res. 47: 5132‐5140.
  McLemore, T.L., Eggleston, J.C., Shoemaker, R.H., Abbott, B.J., Bohlman, M.E., Liu, M.C., Fine, D. L., Mayo, J.G., and Boyd, M.R. 1988. Comparison of intrapulmonary, percutaneous intrathoracic, and subcutaneous models for the propagation of human pulmonary and nonpulmonary cancer cell lines in athymic nude mice. Cancer Res. 48:2880‐2886.
  Moodie, S.A., Willumsen, B.M., Weber, M.J., and Wolfman, A. 1993. Complexes of Ras.GTP with Raf‐1 and mitogen‐activated protein kinase kinase. Science 260:1658‐1661.
  Mordant, P., Loriot, Y., Lahon, B., Castier, Y., Leseche, G., Soria, J.C., Vozenin, M.C., Decraene, C., and Deutsch, E. 2011. Bioluminescent orthotopic mouse models of human localized non‐small cell lung cancer: feasibility and identification of circulating tumour cells. PLoS One 6:e26073.
  Onn, A., Isobe, T., Itasaka, S., Wu, W., O'Reilly, M.S., Ki Hong, W., Fidler, I.J., and Herbst, R.S. 2003. Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice. Clin. Cancer Res. 9:5532‐5539.
  Perez‐Soler, R., Kemp, B., Wu, Q.P., Mao, L., Gomez, J., Zeleniuch‐Jacquotte, A., Yee, H., Lee, J.S., Jagirdar, J., and Ling, Y.H. 2000. Response and determinants of sensitivity to paclitaxel in human non‐small cell lung cancer tumors heterotransplanted in nude mice. Clin. Cancer Res. 6:4932‐4938.
  Ramalingam, S. and Belani, C. 2008. Systemic chemotherapy for advanced non‐small cell lung cancer: recent advances and future directions. Oncologist 13:S5‐S13.
  Regala, R.P., Weems, C., Jamieson, L., Khoor, A., Edell, E.S., Lohse, C.M., and Fields, A.P. 2005. Atypical protein kinase C iota is an oncogene in human non‐small cell lung cancer. Cancer Res. 65:8905‐8911.
  Shimkin, M.B. and Stoner, G.D. 1975. Lung tumors in mice: application to carcinogenesis bioassay. Adv. Cancer Res. 21:1‐58.
  Siegel, R., Naishadham, D., and Jemal, A. 2012. Cancer statistics, 2012. CA Cancer J. Clin. 62:10‐29.
  Stallings‐Mann, M., Jamieson, L., Regala, R.P., Weems, C., Murray, N.R., and Fields, A.P. 2006. A novel small‐molecule inhibitor of protein kinase Cl blocks transformed growth of non–small‐cell lung cancer cells. Cancer Res. 66:1767‐1774.
  Takahashi, O., Komaki, R., Smith, P.D., Jurgensmeier, J.M., Ryan, A., Bekele, B.N., Wistuba, I.I., Jacoby, J.J., Korshunova, M.V., Biernacka, A., Erez, B., Hosho, K., Herbst, R.S., and O'Reilly, M.S. 2012. Combined MEK and VEGFR inhibition in orthotopic human lung cancer models results in enhanced inhibition of tumor angiogenesis, growth, and metastasis. Clin. Cancer Res. 18:1641‐1654.
  Wang, H.Y., Ross, H.M., Ng, B., and Burt, M.E. 1997. Establishment of an experimental intrapulmonary tumor nodule model. Ann. Thorac. Surg. 64:216‐219.
PDF or HTML at Wiley Online Library