Technical Reports in 2009

One of the major challenges of metastasis research is being able to track the fate of individual metastasizing cancer cells over time. Here, Kienast et al. describe an animal model in which multiphoton laser scanning microscopy is used to image the steps involved in the establishment of brain metastasis in vivo. The movement of systemically injected, red fluorescent protein–labeled tumor cells is monitored over several weeks, revealing potential targets for therapy.

Kwakkenbos et al. describe an in vitro method to generate antibody-secreting B cell lines from human peripheral blood memory B cells by transducing them with retroviral vectors encoding Bcl-6 and Bcl-xL. The approach can be used to stably and simultaneously produce high levels of B cell receptor (BCR) on the cell surface and secreted immunoglobulins, useful for studying BCR signaling and producing antigen-specific antibodies.

Adverse events stemming from the use of retroviral vectors in humans has prompted the search for methods predicting the fate and biological consequences of gene-modified cells after vector insertion. Methods of integration site analysis, such as linear amplification-mediated PCR (LAM-PCR), rely on use of restriction enzymes and identify only a fraction of all genomic integrants. This report describes a non–restriction enzyme–based LAM-PCR technique that provides comprehensive, unbiased integration site analysis.

Nonhuman primates are key preclinical models for infectious disease, vaccine development and transplantation research, but their use has been hampered by the complexity and diversity of their major histocompatibility complex (MHC) class I genotypes. Wiseman and his colleagues provide a cost-effective solution to this problem using a next-generation pyrosequencing approach to high-resolution MHC genotyping in various nonhuman primates, identifying both known and new MHC class I alleles.

Despite progress in the biosensor field, a platform that allows the sensitive detection of disease-specific proteins in a diverse range of clinical samples such as saliva, serum and urine has proved elusive. Here, Richard Gaster and his colleagues introduce a magnetic nanosensing protein detection platform that offers quantitative multiplex protein detection at attomolar concentrations over a large linear dynamic range and in a range of biological fluids.

In an effort to develop safer therapeutic agents and to limit unintended side effects, Sabah Oney and her colleagues have designed a set of antidote molecules for a series of aptamers exhibiting anticoagulant activities. These so-called universal antidotes are shown to sequester circulating aptamers and reverse their activity, irrespective of the primary sequence and folded structure of the aptamer.

Optical frequency domain imaging (OFDI) is a wide-field, three-dimensional intravital imaging technique that provides information on the entire tumor vasculature and surrounding tissue microenvironment, allowing visualization of angiogenesis and lymphangiogenesis during tumor growth and with therapy. Here, Vakoc et al. show that, in contrast to multiphoton microscopy, OFDI can image at greater tissue depths with a wider field of view and without the need for exogenous contrast agents.

Jin and colleagues introduce a new chip-based system for the rapid identification and isolation of single antigen-specific antibody–secreting cells from human peripheral blood lymphocytes. The approach can be used to detect antibody-secreting cells for multiple antigens on the same chip and should have advantages over current technologies for isolating and producing human monoclonal antibodies of clinical significance.

Although the TGF-β signaling pathway has been implicated in breast cancer metastasis, studies are hampered by a lack of animal models for in vivo analysis of metastasis signaling pathways. Here a noninvasive xenograft model is described that uses a dual bioluminescence reporter system to study TGF-β signaling in bone metastasis. Disruption of TGF-β signaling in early—not late—stage metastasis is shown to markedly reduce bone metastasis burden.

Caspases are intracellular proteases and key initiators and effectors of apoptosis. Here the authors describe fluorescently labeled activity-based probes that allow the noninvasive in vivo monitoring of the kinetics of caspase activity. Approaches to optimize the probes to enhance their specificity and increase uptake into apoptotic cells are outlined, and their use in tracking the early stages of apoptosis in two mouse models (dexamethasone and the monoclonal antibody Apomab) is demonstrated.

The development of a long-term intestinal culture system has, until recently, eluded researchers. Here the authors describe a method allowing long-term culture of both small intestine and colon that incorporates an air-liquid interface coupled with a three-dimensional matrix scaffold. The cultures show epithelial cell proliferation and multilineage differentiation to the major cell types and accurately recapitulate the Wnt- and Notch-dependent intestinal stem cell niche.

Vanneste and her colleagues describe an array-based approach for scoring genome-wide DNA copy number variations and loss of heterozygosity in single cells. They show that chromosome instability patterns, reminiscent of those seen in human cancers, are also common in cleavage-stage in vitro–fertilized embryos. Such findings during early human embryogenesis could provide a basis for the low fecundity and high miscarriage rate in humanspages 490–491..

In this study, Galbán and his colleagues describe a voxel-wise approach for the quantification of tumor microvasculature properties from perfusion magnetic resonance imaging data. When compared to the standard method of using region of interest analysis of changes in relative cerebral blood flow and volume, the parametric response map approach was found to be more predictive of treatment outcomes and overall survival in individuals with high-grade glioma.

Here Fan et al. describe a protein analysis platform for the sensitive, nanoscale diagnosis and investigation of clinical specimens, including monitoring the response to targeted therapeutics. The nanofluidic proteomic immunoassay can be used to quantify total and phosphorylated forms of oncoproteins in small tumor samples and has been validated in vivo in mouse tumors and in clinical specimens from blood, surgical biopsies and fine-needle aspirates.

The leukocyte enzyme myeloperoxidase (MPO) is key to normal host defense mechanisms. Dysregulated MPO, however, is linked to acute and chronic inflammatory conditions, such as atherosclerosis and cancer. The authors describe a luminol-based bioluminescence imaging system that provides an optical readout of physiological levels of MPO activity in vivo. The system is demonstrated in animal models of acute dermatitis, focal arthritis and spontaneous large granular lymphocytic tumors.

Viral-mediated gene therapy presents many challenges in the clinic, including the potential for physiological effects that overshoot the intended goals. In a new report by Matthew During and his colleagues, the authors devise a scheme by which packaging of a microRNA into the virus, expressed under the control of a physiological response induced by the viral transgene, allows coordinated dampening of the transgene expression when the therapeutic response achieves a certain threshold.

A research team led by Renier Brentjens offers a new approach to monitor in vivo trafficking of T cells by tagging them for bioluminescence imaging using a membrane-anchored form of the humanized Gaussia luciferase enzyme. The set-up provides a high bioluminescent signal and should be useful for studying in vivo T cell function in mouse models of disease investigating mouse or human primary T cells.

By capitalizing on copy number variation, Wu and his colleagues offer an approach for detecting cellular chimerism with fluorescent in situ hybridization probes that target polymorphic deletion loci. These probes can determine the fate of donor cells in situ, irrespective of gender, and should prove useful in understanding the dynamics of cellular chimerism after solid organ, bone marrow and hematopoietic stem cell transplantation.

There is a need for mouse tumor models that more closely recapitulate the pathophysiology of human cancers. Here, a mouse model of glioblastoma multiforme (GBM) is generated with Cre-loxP controlled, lentiviral-mediated delivery of the oncogenes H-Ras and AKT. Transduction of the oncogenes in a small number of cells in adult immunocompetent mice led to the formation of GBM-like tumors, particularly when combined with loss of p53.