Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.

Tumors contain many components in addition to the cancer cells, including blood vessels, fibroblasts, and immune cells. Genetic studies and tumor biopsies have generated insights into the importance of these stromal components for cancer progression. However, it remains a challenge to reveal the dynamic interactions among the distinct tumor components within live animals. Studies involving multiphoton microscopy allow direct imaging of cellular movement in live mice, but multiphoton microscopy is expensive, complex, and usually relies on a single excitation wavelength for all fluorophores. This article describes a method for intravital imaging using a microlensed spinning-disk confocal microscope. Although tissue penetration with spinning-disk confocal microscopy is lower than with multiphoton microscopy, image acquisition with this method is very rapid, so artifacts from respiratory motion are avoided. Photobleaching and phototoxicity are low, and multicolor acquisition is cheaper and easier than with multiphoton microscopy. This article discusses various aspects of experimental setup, as well as methods for addressing technical barriers, such as generating and working with multiple tumor microenvironments within individual live mice, image collection, and long-term anesthesia.

[1]  Z. Werb,et al.  Preparation of mice for long-term intravital imaging of the mammary gland. , 2011, Cold Spring Harbor protocols.

[2]  Z. Werb,et al.  Monitoring of vital signs for long-term survival of mice under anesthesia. , 2011, Cold Spring Harbor protocols.

[3]  Z. Werb,et al.  Tumors as organs: complex tissues that interface with the entire organism. , 2010, Developmental cell.

[4]  Jacco van Rheenen,et al.  Intravital imaging of metastatic behavior through a mammary imaging window , 2008, Nature Methods.

[5]  Mikala Egeblad,et al.  Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy , 2008, Disease Models & Mechanisms.

[6]  Z. Werb,et al.  GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model. , 2008, Cancer cell.

[7]  Zhiyuan Hu,et al.  Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors , 2007, Genome Biology.

[8]  P. Ross,et al.  Full developmental potential of mammalian preimplantation embryos is maintained after imaging using a spinning-disk confocal microscope. , 2006, BioTechniques.

[9]  Jeffrey Wyckoff,et al.  Probing the Microenvironment of Mammary Tumors Using Multiphoton Microscopy , 2006, Journal of Mammary Gland Biology and Neoplasia.

[10]  John S. Condeelis,et al.  ROCK- and Myosin-Dependent Matrix Deformation Enables Protease-Independent Tumor-Cell Invasion In Vivo , 2006, Current Biology.

[11]  M. Dewhirst,et al.  Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. , 2006, Journal of the National Cancer Institute.

[12]  L. Coussens,et al.  Paradoxical roles of the immune system during cancer development , 2006, Nature Reviews Cancer.

[13]  C. Halin,et al.  In vivo imaging of lymphocyte trafficking. , 2005, Annual review of cell and developmental biology.

[14]  Z. Werb,et al.  Coevolution of cancer and stromal cellular responses. , 2005, Cancer cell.

[15]  D. Proia,et al.  Stroma: Tumor Agonist or Antagonist , 2005, Cell cycle.

[16]  Vladimir P Torchilin,et al.  Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo , 2005, Nature Medicine.

[17]  A. Rudensky,et al.  Regulatory T cell lineage specification by the forkhead transcription factor foxp3. , 2005, Immunity.

[18]  H. Moses,et al.  Tumor-stroma interactions. , 2005, Current opinion in genetics & development.

[19]  C. Sumen,et al.  ReviewIntravital Microscopy : Visualizing Immunity in Context , 2004 .

[20]  J. Mora,et al.  In vivo imaging of leukocyte trafficking in blood vessels and tissues. , 2004, Current opinion in immunology.

[21]  Jeffrey W Pollard,et al.  Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. , 2003, The American journal of pathology.

[22]  Brian Seed,et al.  Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation , 2003, Nature Medicine.

[23]  M. Dewhirst,et al.  A novel rodent mammary window of orthotopic breast cancer for intravital microscopy. , 2003, Microvascular research.

[24]  Michael C. Ostrowski,et al.  A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. , 2003, Blood.

[25]  Jeffrey Wyckoff,et al.  GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. , 2002, Cancer research.

[26]  A. Nakano.  Spinning-disk confocal microscopy -- a cutting-edge tool for imaging of membrane traffic. , 2002, Cell structure and function.

[27]  Mina J. Bissell,et al.  Putting tumours in context , 2001, Nature Reviews Cancer.

[28]  R. B. Campbell,et al.  In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy , 2001, Nature Medicine.

[29]  R. B. Campbell,et al.  Role of tumor–host interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Messmer,et al.  Dorsal skinfold chamber technique for intravital microscopy in nude mice. , 1993, The American journal of pathology.

[31]  R. Cardiff,et al.  Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease , 1992, Molecular and cellular biology.