Pre‐Clinical Mouse Models of Primary and Metastatic Pleural Cancers of the Lung and Breast and the Use of Bioluminescent Imaging to Monitor Pleural Tumor Burden

Elliot L. Servais1, Christos Colovos1, Stefan S. Kachala1, Prasad S. Adusumilli1

1 Center for Cell Engineering, Memorial Sloan‐Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.21
DOI:  10.1002/0471141755.ph1421s54
Online Posting Date:  September, 2011
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Malignant pleural disease (MPD) results in an estimated 150,000 cases of malignant pleural effusions (MPE) annually. The most common malignancies associated with MPD are primary malignant pleural mesothelioma (MPM) and metastatic lung cancer, breast cancer, and lymphoma. MPM is a rare, regionally aggressive malignancy whose incidence is increasing secondarily to the latency of disease progression. MPD is characteristic of advanced‐stage pleural disease and portends a grave clinical prognosis with a median survival between 3 and 12 months. Preclinical investigations conducted in flank and intraperitoneal tumor models do not fully recapitulate the pleural tumor microenvironment, and the results are not directly translatable to the clinical setting. The protocol described herein provides a mouse model of MPM and MPD from nonhematogenous tumors, resulting in reproducible tumor location, tumor progression, animal survival, and histopathology. Pleural tumor growth in this model resembles the regionally aggressive clinical course and tumor microenvironment of human pleural cancers and provides an optimal animal model to investigate MPD biology and therapies. Curr. Protoc. Pharmacol. 54:14.21.1‐14.21.18. © 2011 by John Wiley & Sons, Inc.

Keywords: malignant pleural disease; malignant pleural effusion; mesothelioma; breast cancer; orthotopic; mouse model; bioluminescent imaging

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Tumor Cell Suspension
  • Basic Protocol 2: Pleural Tumor Cell Injection
  • Basic Protocol 3: Bioluminescent Imaging (BLI) of MPD Mouse Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of Tumor Cell Suspension

  • Tumor cell line (see Strategic Planning for selection and sources)
  • Medium for cell growth: RPMI‐1640 for the MSTO‐211H, H1299, and 4T1 cell lines; DMEM for the AB12 and LLC1 cell lines; and Leibovitz's L‐15 for the MDA‐MB‐231 cell line
  • Sterile fetal bovine serum (FBS)
  • Penicillin‐streptomycin (e.g., Invitrogen; to be added for a final 1× concentration)
  • 1× Dulbecco's phosphate‐buffered saline (DPBS; e.g., Invitrogen)
  • 0.05% trypsin‐EDTA (e.g., Invitrogen)
  • 0.04% trypan blue (see Phelan, )
  • 10‐cm tissue culture plates
  • 50‐ml or 15‐ml conical centrifuge tubes
  • Refrigerated centrifuge
  • Hemacytometer (see Phelan, )
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (Phelan, )

Basic Protocol 2: Pleural Tumor Cell Injection

  • Chlorine dioxide disinfectant solution (Clidox‐S; Pharmacal Research Labs,
  • Mice of appropriate strain (6‐ to 8 week‐old pathogen‐free mice; see Strategic Planning).
  • Ophthalmic ointment
  • Oxygen
  • Isoflurane
  • 10% povidone‐iodine
  • Sterile alcohol prep pads (70% isopropyl alcohol)
  • Tumor cell suspension ( protocol 1)
  • Moistened food for mice
  • Plastic‐backed absorbent paper
  • Dry bead sterilizer (e.g., Germinator 500; SouthPointe Surgical,
  • Inhalational anesthesia vaporizer system with induction chamber (VetEquip, cat. no. 901805;
  • Anesthesia scavengers (e.g., VaporGuard, VetEquip,
  • Additional tubing with nose cone
  • Electric hair clippers
  • Sterile cotton‐tipped applicators
  • Axillary roll (0.8 cm diameter; constructed from 0.8 × 5.0–cm tubing)
  • Bench‐top halogen lamp
  • Fenestrated sterile drape
  • Dissecting scissors: fine iris scissors (10.5 cm) curved or straight
  • Adson forceps, toothed 1 × 2 (12 cm)
  • Tuberculin syringe with 27.5‐G needle
  • Wound clip applicator and 9‐mm wound clips (e.g., Autoclip Kit; Braintree Scientific)
  • Mouse cages and bedding
  • Wound clip remover (autoclip suture remover; Braintree Scientific, cat. no. ACS‐RMV)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (Phelan, ), euthanasia of mice (Donovan and Brown, ), and bioluminescent imaging ( protocol 3)

Basic Protocol 3: Bioluminescent Imaging (BLI) of MPD Mouse Model

  • Mice of appropriate strain (6‐ to 8 week‐old pathogen‐free mice; see Strategic Planning)
  • Tumor cell lines expressing a luminescent reporter gene, such as firefly luciferase: any tumor cell line of interest may be utilized (see Strategic Planning); luciferase vectors are available commercially from a number of vendors (e.g., Promega)
  • 15 mg/ml D‐luciferin (see recipe)
  • Isoflurane
  • Oxygen
  • In vivo bioluminescent imaging system (e.g., Xenogen IVIS 200, Caliper Life Sciences)
  • 1‐ml tuberculin syringes and 27.5‐G needles
  • Inhalational anesthesia vaporizer system with induction chamber (VetEquip, cat. no. 901805;
  • Living Image software for IVIS 100 (Caliper Life Sciences)
  • Computer running Microsoft Excel and GraphPad Prism software
  • Additional reagents and equipment for pleural tumor cell injection ( protocol 2) and intraperitoneal injection of mice (Donovan and Brown, )
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  •   FigureFigure 14.21.1 Surgical stage arrangement, mouse positioning, and initial surgical exposure. (A) The surgical area consists of a surgical stage, anesthetic tubing, and nose cone (taped securely to the stage), and an axillary roll (0.8 cm diameter) positioned underneath the left axilla to maximize exposure of thoracic landmarks. (B) After the initial incision, care must be taken to avoid avulsion of a superficial vein consistently located near the incision site.
  •   FigureFigure 14.21.2 Schematic view of anatomic landmarks for orthotopic pleural injections. Dissection to the right chest wall exposes the ribs (solid lines), diaphragm (dotted line), lungs (immediately superior to the diaphragm), and liver (inferior to the diaphragm). (A) The injection site is identified by a circle. (B) The needle enters through the circle, between the diaphragm and lung, into the pleural space, with the tumor cell suspension injected into the pleural cavity.
  •   FigureFigure 14.21.3 Pleural cancer model noninvasive imaging, macroscopic and microscopic findings. MRI (left) and BLI (right) of MPM tumor (MSTO‐211H, 1 × 105 cells/200 µl). (A) 6 days after implantation showing pleural involvement. (B) 29 days after tumor implantation showing encasement of mediastinal structures and pleura. (C) Orthotopic injection of LLC1 (1 × 105 cells/200 µl) results in pleural tumor and bloody pleural effusions. (D) H&E section of pleural tumor along the chest wall.
  •   FigureFigure 14.21.4 Orthotopic model of pleural malignancy permits evaluation of therapies. In an experiment where athymic nude mice were inoculated with MPM cells (1 × 106 MSTO‐211H) intrapleurally and then treated 10 days later, (A) BLI signal from a representative mouse receiving a single dose of cisplatin (4 mg/kg; solid circles) and an untreated mouse (empty circles) shows disease progression in the absence of treatment. (B) A single dose of chemotherapy prolongs survival and maintains body weight. (empty circles = untreated; solid circles = cisplatin 4 mg/kg).


Literature Cited

Literature Cited
   Adusumilli, P., Eisenberg, D., Stiles, B.M., Chung, S., Chan, M.K., Rusch, V.W., and Fong, Y. 2006a. Intraoperative localization of lymph node metastases with a replication‐competent herpes simplex virus. J. Thorac. Cardiovasc. Surg. 132:1179‐1188.
   Adusumilli, P.S., Stiles, B.M., Chan, M.K., Mullerad, M., Eisenberg, D.P., Ben‐Porat, L., Huq, R., Rusch, V.W., and Fong, Y. 2006b. Imaging and therapy of malignant pleural mesothelioma using replication‐competent herpes simplex viruses. J. Gene Med. 8:603‐615.
   Antunes, G. Neville, E., Duffy, J, Ali, N., and Pleural Diseases Group, Standards of Care Committee, British Thoracic Society. 2003. BTS guidelines for the management of malignant pleural effusions. Thorax 58:ii29‐38.
   Donovan, J. and Brown, P. 2006a. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Donovan, J. and Brown, P. 2006b. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Fenton, K.N. and Richardson, J.D. 1995. Diagnosis and management of malignant pleural effusions. Am. J. Surg. 170:69‐74.
   Hoogstraten‐Miller, S.L. and Brown, P.A. 2008. Techniques in aseptic rodent surgery. Curr. Protoc. Immunol. 82:1.12.1‐1.12.14.
   Manac'h, D., Riquet, M., Medioni, J., Le Pimpec‐Barthes, F., Dujon, A., and Danel, C. 2001. Visceral pleura invasion by non‐small cell lung cancer: An underrated bad prognostic factor. Ann. Thorac. Surg. 71:1088‐1093.
   Michailova, K.N. and Usunoff, K.G. 2006. Serosal membranes (pleura, pericardium, peritoneum). Normal structure, development and experimental pathology. Adv. Anat. Embryol. Cell Biol. 183:i‐vii, 1‐144, back cover.
   Mishra, E. and Davies, R.J.O. 2010. Advances in the investigation and treatment of pleural effusions. Exp. Rev. Resp. Med. 4:123‐133.
   Newell, J. 2007. Guidelines for Survival Rodent Surgery. NIH‐OACU, Bethesda, Md.
   Osaki, T., Nagashima, A., Yoshimatsu, T., Yamada, S., and Yasumoto, K. 2004. Visceral pleural involvement in nonsmall cell lung cancer: Prognostic significance. Ann. Thorac. Surg. 77:1769‐1773.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Sahn, S.A. 2001. Malignant pleural effusions. Semin. Respir. Crit. Care Med. 22:607‐616.
   Shimizu, K., Yoshida, J., Nagai, K., Nishimura, M., Ishii, G. Morishita, Y., and Nishiwaki, Y. 2005. Visceral pleural invasion is an invasive and aggressive indicator of non‐small cell lung cancer. J. Thorac. Cardiovasc. Surg. 130:160‐165.
   Stathopoulos, G.T. and Kalomenidis, I. 2009. Animal models of malignant pleural effusion. Curr. Opin. Pulm. Med. 15:343‐352.
   Stiles, B.M., Adusumilli, P.S., Bhargava, A., Stanziale, S.F., Kim, T.H., Chan, M.K., Huq, R., Wong, R., Rusch, V.W., and Fong, Y. 2006. Minimally invasive localization of oncolytic herpes simplex viral therapy of metastatic pleural cancer. Cancer Gene Ther. 13:53‐64.
   Zocchi, L. 2002. Physiology and pathophysiology of pleural fluid turnover. Eur. Respir. J. 20:1545‐1558.
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