Imaging Tumor Cell Movement In Vivo

David Entenberg1, Dmitriy Kedrin1, Jeffrey Wyckoff2, Erik Sahai3, John Condeelis2, Jeffrey E. Segall2

1 These authors contributed equally to this work., 2 Albert Einstein College of Medicine, Bronx, New York, 3 London Research Institute, London, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.7
DOI:  10.1002/0471143030.cb1907s58
Online Posting Date:  March, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used both to provide a mammalian system for studying human tumor cells (as xenografts in immunocompromised mice) as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol in this unit describes the standard methods used for generation of mammary tumors and imaging them. Additional protocols for labeling macrophages, blood vessel imaging, and image analysis are also included. Curr. Protoc. Cell Biol. 58:19.7.1‐19.7.19. © 2013 by John Wiley & Sons, Inc.

Keywords: imaging; metastasis; mammary tumor; image analysis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Generation and In Vivo Imaging of Mammary Tumors
  • Support Protocol 1: In Vivo Imaging Microscope Setup
  • Support Protocol 2: Labeling Vasculature and Macrophages
  • Support Protocol 3: Blood Vessel Imaging Using an Indwelling Catheter
  • Support Protocol 4: Second Harmonic Fiber Imaging
  • Basic Protocol 2: Multiphoton Time‐Lapse Image Analysis Using ImageJ and Custom Plugins
  • Support Protocol 5: Separation of Spectral Overlap
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Generation and In Vivo Imaging of Mammary Tumors

  Materials
  • MTLn3 cells (available from authors; )
  • MTLn3 growth medium (see recipe)
  • 2 mM EDTA (Invitrogen, cat. no. 15575‐038) in calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Invitrogen; also see appendix 2A)
  • Dulbecco's phosphate‐buffered saline (with calcium and magnesium; DPBS; Invitrogen; also see appendix 2A)
  • Female 5‐ to 6‐week‐old BALB/c SCID/Ncr mice (National Cancer Institute), Fisher 344 rats (The Jackson Laboratory), or transgenic mouse tumor models (e.g., MMTV‐pyMT; The Jackson Laboratory)
  • 70% (v/v) ethanol
  • Isoflurane, USP (AERRANE, Baxter)
  • 15‐cm tissue culture dishes (Falcon)
  • 15‐ml conical polypropylene centrifuge tubes, sterile
  • Cell scrapers
  • Centrifuge (e.g., Sorvall GLC‐1)
  • 1‐ml syringes with 25‐G needles for cell injections
  • Oxygen/anesthesia apparatus (Forane Vaporizer, model 100, from SurgiVet)
  • 100% compressed oxygen tanks to be used in combination with vaporizer
  • Surgical instruments
  • In vivo imaging microscope setup ( protocol 2)
  • Multiphoton apparatus setup ( protocol 5)
  • Surgical tape (optional)
  • Additional reagents and equipment for tissue culture techniques including counting cells (unit 1.1), housing of immunocompromised mice (Donovan and Brown, ), imaging blood vessels ( protocol 3 or 3; optional), inhalant anesthesia of rodents (Donovan and Brown, ), euthanasia of rodents (Donovan and Brown, ), and image analysis and quantitation ( protocol 6)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be carried out in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Support Protocol 1: In Vivo Imaging Microscope Setup

  Materials
  • Mouse with xenograft‐induced tumor ( protocol 1)
  • 70% (v/v) ethanol
  • 20 mg/ml dye labeled–dextran (70 kDa, Invitrogen) in DPBS (Invitrogen; also see appendix 2A)
  • Mouse restrainer (Donovan and Brown, )
  • 1‐ml syringe with 26‐G needle
  • Superglue
  • Additional reagents and equipment for mouse restraint (Donovan and Brown, )

Support Protocol 2: Labeling Vasculature and Macrophages

  • Mouse with xenograft‐induced tumor ( protocol 1)
  • Heparinized PBS solution (Baxter, cat. no. FKB0953G)
  • Indwelling tail vein catheter (no. MTV‐01, SAI Inc.; http://www.sai‐infusion.com)
  • 1‐ml syringes
  • Waterproof marker (e.g., Sharpie)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ahmed, F., Wyckoff, J., Lin, E.Y., Wang, W., Wang, Y., Hennighausen, L., Miyazaki, J., Jones, J., Pollard, J.W., Condeelis, J.S., and Segall, J.E. 2002. GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res. 62:7166‐7169.
   Donovan, J. and Brown, P. 2001. Anesthesia. Curr. Protoc. Immunol. 27:1.4.1‐1.4.5.
   Donovan, J. and Brown, P. 2006a. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Donovan, J. and Brown, P. 2006b. Handling and restraint. Curr. Protoc. Immunol. 73:1.3.1‐1.3.6.
   Donovan, J. and Brown, P. 2007. Managing immunocompromised animals. Curr. Protoc. Immunol. 77:1.2.1‐1.2.5.
   Drobizhev, M., Tillo, S., Makarov, N.S., Hughes, T.E., and Rebane, A. 2009. Absolute two‐photon absorption spectra and two‐photon brightness of orange and red fluorescent proteins. J. Phys. Chem. B 113:855‐859.
   Entenberg, D., Wyckoff, J., Gligorijevic, B., Roussos, E.T., Verkhusha, V.V., Pollard, J.W., and Condeelis, J. 2011. Setup and use of a two‐laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat. Protoc. 6:1500‐1520.
   Ewald, A.J., Werb, Z., and Egeblad, M. 2011. Dynamic, long‐term in vivo imaging of tumor‐stroma interactions in mouse models of breast cancer using spinning‐disk confocal microscopy. Cold Spring Harb. Protoc. 2011:pdb top97.
   Faust, N., Varas, F., Kelly, L.M., Heck, S., and Graf, T. 2000. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96:719‐726.
   Gligorijevic, B., Kedrin, D., Segall, J.E., Condeelis, J., and van Rheenen, J. 2009. Dendra2 photoswitching through the Mammary Imaging Window. J. Vis. Exp. doi:pii: 1278. 10.3791/1278.
   Jain, R.K., Munn, L.L., and Fukumura, D. 2011. Transparent window models and intravital microscopy: Imaging gene expression, physiological function and drug delivery in tumors. In Tumor Models in Cancer Research, 2nd ed. (B.A. Teicher, ed.) pp. 641‐679. Springer, New York.
   Kawano, H., Kogure, T., Abe, Y., Mizuno, H., and Miyawaki, A. 2008. Two‐photon dual‐color imaging using fluorescent proteins. Nat. Methods 5:373‐374.
   Lin, E.Y., Jones, J.G., Li, P., Zhu, L., Whitney, K.D., Muller, W.J., and Pollard, J.W. 2003. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am. J. Pathol. 163:2113‐2126.
   Meijering, E.H.W., Niessen, W.J., and Viergever, M.A. 2001. Quantitative evaluation of convolution‐based methods for medical image interpolation. Med. Image Anal. 5:111‐126.
   Motoike, T., Loughna, S., Perens, E., Roman, B.L., Liao, W., Chau, T.C., Richardson, C.D., Kawate, T., Kuno, J., Weinstein, B.M., Stainier, D.Y., and Sato, T.N. 2000. Universal GFP reporter for the study of vascular development. Genesis 28:75‐81.
   Neri, A., Welch, D., Kawaguchi, T., and Nicolson, G.L. 1982. Development and biologic properties of malignant cell sublines and clones of a spontaneously metastasizing rat mammary adenocarcinoma. J. Natl. Cancer Inst. 68:507‐517.
   Piatkevich, K.D., Hulit, J., Subach, O.M., Wu, B., Abdulla, A., Segall, J.E., and Verkhusha, V.V. 2010. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. U.S.A. 107:5369‐5374.
   Sahai, E., Wyckoff, J., Philippar, U., Segall, J.E., Gertler, F., and Condeelis, J. 2005. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy. BMC Biotechnol. 5:14.
   Sasmono, R.T., Oceandy, D., Pollard, J.W., Tong, W., Pavli, P., Wainwright, B.J., Ostrowski, M.C., Himes, S.R., and Hume, D.A. 2003. A macrophage colony‐stimulating factor receptor‐green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101:1155‐1163.
   Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. 2012. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9:671‐675.
   Thevenaz, P., Ruttimann, U.E., and Unser, M. 1998. A pyramid approach to subpixel registration based on intensity. IEEE T Image Process 7:27‐41.
   Tillo, S.E., Hughes, T.E., Makarov, N.S., Rebane, A., and Drobizhev, M. 2010. A new approach to dual‐color two‐photon microscopy with fluorescent proteins. BMC Biotechnol. 10:6.
   Wang, W., Wyckoff, J.B., Wang, Y., Bottinger, E.P., Segall, J.E., and Condeelis, J.S. 2003. Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse. BMC Biotechnol. 3:13.
   Wang, W., Goswami, S., Lapidus, K., Wells, A.L., Wyckoff, J.B., Sahai, E., Singer, R.H., Segall, J.E., and Condeelis, J.S. 2004. Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors. Cancer Res. 64:8585‐8594.
   Wang, W., Goswami, S., Sahai, E., Wyckoff, J.B., Segall, J.E., and Condeelis, J.S. 2005. Tumor cells caught in the act of invading: Their strategy for enhanced cell motility. Trends Cell Biol. 15:138‐145.
   Wyckoff, J.B., Jones, J.G., Condeelis, J.S., and Segall, J.E. 2000. A critical step in metastasis: In vivo analysis of intravasation at the primary tumor. Cancer Res. 60:2504‐2511.
   Wyckoff, J., Wang, W., Lin, E.Y., Wang, Y., Pixley, F., Stanley, E.R., Graf, T., Pollard, J.W., Segall, J., and Condeelis, J. 2004. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res. 64:7022‐7029.
   Wyckoff, J., Gligorijevic, B., Entenberg, D., Segall, J., and Condeelis, J. 2011. High‐resolution multiphoton imaging of tumors in vivo. Cold Spring Harb. Protoc. 2011:1167‐1184.
   Xue, C., Wyckoff, J., Liang, F., Sidani, M., Violini, S., Tsai, K.L., Zhang, Z.Y., Sahai, E., Condeelis, J., and Segall, J.E. 2006. Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res. 66:192‐197.
   Zipfel, W.R., Williams, R.M., Christie, R., Nikitin, A.Y., Hyman, B.T., and Webb, W.W. 2003. Live tissue intrinsic emission microscopy using multiphoton‐excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. U.S.A. 100:7075‐7080.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library