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Feb 1, 2011 (Vol. 31, No. 3)

Broadening the Focus of In Vivo Imaging

From Single Cell to Whole Organism, Change Is Under Way in Numerous Laboratories

  • Cell Dynamics

    John Condeelis, Ph.D., professor and co-chair of the department of anatomy and structural biology at Albert Einstein College of Medicine of Yeshiva University, spoke about the application of intravital multiphoton microscopy and photoconversion technology to define cell dynamics and cell-cell interactions that drive normal morphogenesis and metastatic tumor progression in the mouse mammary gland. A clearer picture of the early stages of metastatic behavior could lead to the identification of novel targets for chemotherapeutic drug development.

    As single-cell movement is the dominant mode of metastasis, technology is needed to enable the study of cell motility and the tumor microenvironment at single-cell resolution. The microenvironment plays a major role in determining the gene-expression profile and migratory phenotype of tumor cells, he explained.

    The transparent nature of embryos facilitates in vivo imaging studies. To image adult organisms—solid tissues and tumors in particular—and to study the behavior of single cells, Dr. Condeelis utilizes multiphoton imaging technology, which allows for visualization “to depths 10x to 30x greater than one photon excitation.”

    New microscope designs enable scanning of multiple photons simultaneously using probes that fluoresce at different wavelengths across the spectrum detectable by a particular imaging device. Photoconvertible proteins are used to follow cell movement and morphological changes over time. Novel exotic fluorophores and new generations of photoconvertible proteins are contributing to rapid advances in the field.

    To facilitate repeated imaging of mammary tumors in individual mice, Dr. Condeelis’ group developed a technique in which they suture a “window” in place of the epidermal layer of skin that overlies the mammary gland. He showed in vivo images obtained after labeling a subpopulation of mammary cells using photoconvertible probes.

    The images capture the early stages of metastasis, in which streams of cells that have made the decision to migrate utilize extracellular matrix fibers to access the blood supply. The images point to a key difference between the microenvironment in which cell intravasation occurs versus where it does not: the presence of perivascular clusters of invasive, tumor-associated macrophages, which serve as a source of chemotactic growth factors that mediate tumor cell-macrophage interaction. He described this population of macrophages as “doormen” that create an entry point and support the efficient invasion of tumor cells into the bloodstream.

    Similar types of choreographed co-migratory events have been described during normal embryonic development and mammary gland morphogenesis, and these rely on many of the same signaling, steering, and invasive pathways that drive metastatic tumor cell behavior.

  • Drug Effects

    Ralph Weissleder, M.D., Ph.D., professor, department of radiology at Harvard Medical School and director, Center for Molecular Imaging Research at Massachusetts General Hospital, described his research on in vivo imaging of drug effects and the results of experiments designed to understand the biological basis of tumor cell interaction with host cells.

    A primary goal of this work is to translate the use of intravital microscopy and injectable fluorescent probes from the research laboratory to the clinic for the purpose of assessing the effects of chemotherapeutic agents on tumor and normal cells and identifying more targeted cancer therapies.

    Advances in the adaptation of cell-based assays to intravital imaging in the mouse have demonstrated fundamental differences between what is seen in vitro versus in vivo. Dr. Weissleder emphasized three specific benefits that intravital microscopy offers cancer researchers: the potential to observe physiological responses, to study heterogeneity within populations of cancer cells or between cellular processes, and to image drug responses. How best to achieve these benefits—what cell and tumor types to study, how long to image and follow cell populations, and what imaging parameters are optimal, for example—are all questions that require further study.

    Tumors can be imaged in situ by applying various techniques including the use of “stick” lenses, or microscope objectives that enable “keyhole” imaging, as well as imaging modalities such as laser-scanning confocal or multiphoton microscopy. Studying drug responses in tumors requires high spatial resolution to observe nuclear phenotypes and detect nuclear division, explained Dr. Weissleder.

    Whole tumor imaging can be achieved at high resolution by imaging tumor slices and then combining the individual images—a process called “image stitching.”

    As drug responses evolve over time and may be heterogeneous across tumor cell populations, the ability to do time-lapse imaging and sequential studies in the same animal over multiple time points is important. This presents a particular challenge as tumors are dynamic structures; it requires the development of methods and technologies that will allow researchers to image the same group of cells in vivo over a period of hours to days.

    Cost can be a significant barrier to entry into the emerging field of intravital imaging. While microscopy systems are available in a range of complexity, capabilities, and costs, with personal confocal microscopes available for as low as $100,000, and multiphoton imaging systems for $200,000, instrument costs are only one component of the overall expense. Additional infrastructure needs to build and maintain small animal and surgical facilities must also be considered.

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