Imaging tumor cell movement in vivo

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.

Figures

Figure 19.7.1

Effective excitation spectra of various combinations of fluorophores common in multiphoton microscopy. Excitation by Tsunami, Mai Tai, or Opal are indicated by blue, red, or pink vertical lines, respectively. Excitation spectra of fluorophores are indicated as horizontal bars. (A) Relative output power spectra of Ti:Sapph lasers (Tsunami or Mai Tai Spectra Physics) and optical parametric oscillators (Opal, Spectra Physics). (B) CFP, GFP, and Quantum dots can be combined. The broad excitation spectra of quantum dots make them suitable for on systems employing a single laser. Their wide range of emission spectra makes them particularly useful in combination with each other, as well as with many fluorophores. (C) CFP, GFP, rhodamine and Evans Blue all have overlapping excitation spectra allowing their use with a single laser system. The long Stokes shifts of rhodamine and Evans Blue make them cleanly separable with the filter sets described in this chapter. (D) Use of a second Ti:Sapph femtosecond laser source enables the simultaneous imaging of CFP, Dendra2 green, and Dendra2 red. (E,F) Use of a Ti:Sapph femtosecond laser source in combination with an optical parametric oscillator allows the use of a variety of fluorophores whose emission spectrum lies in the red and far red (>650 nm). For the color version of this figure, go to http://www.currentprotocols.com/protocol/cb1907.

Figure 19.7.2

Diagram of tumor fixturing setup. (A) Supine view of mouse with skin flap surgery indicating placement locations of rubber support pieces used on the microscope stage. Two small (0.5 × 0.75 × 0.25-in.) pieces of rubber placed at the sides of the tumor act as a support for the tissue and prevent over compression of the tumor vasculature. A third thin strip of rubber (0.25 × 0.25 × 1.5-in.) is placed between the body and the tumor to isolate the tumor from breathing motion. (B) Lateral view of mouse mounted on microscope stage. The two small pieces of rubber from (A) can be seen supporting and suspending the skin, with its foam backing, above the coverslip. Two anchoring strips of rubber then are affixed to the tumor with adhesive tape.

Figure 19.7.3

Multiphoton imaging of MTLn3 xenotransplant tumor in mouse. (A) GFP-expressing tumor cells are shown in the green channel, and matrix second harmonic image is shown in both red and blue channels (for contrast). (B) Mix of GFP-expressing cells (green channel) with CFP-expressing cells (red and blue channels) in a single tumor. (C) Simultaneous imaging of GFP-expressing tumor cells (green) and blood vessels (red) following intravenous injection of Texas Red-dextran. (D) Imaging of macrophages labeled with Texas Red-dextran (red) in GFP-expressing MTLn3 tumor. Scale bars for A and C represent 50 μm, those for B and D represent 25 μm. For the color version of this figure, go to http://www.currentprotocols.com/protocol/cb1907.