Highly sensitive detection of early-stage pancreatic cancer by multimodal near-infrared molecular imaging in living mice.

Pancreatic cancer is a serious disease with poor patient outcome, often as a consequence of late diagnosis in advanced stages. This is in large part due to the lack of diagnostic tools for early detection. To address this deficiency, we have investigated novel molecular near-infrared fluorescent (NIRF) in vivo imaging techniques in clinically relevant mouse models of pancreatic cancer. Genome wide gene expression profiling was used to identify cathepsin cystein proteases and matrix metalloproteinases (MMP) as targets for NIRF imaging. Appropriate protease activatable probes were evaluated for detection of early-stage pancreatic cancer in mice with orthotopically implanted pancreatic cancer cell lines. Mice with pancreatitis served as controls. Whole body in vivo NIRF imaging using activatable cathepsin sensitive probes specifically detected pancreatic tumors as small as 1-2 mm diameter. Imaging of MMP activity demonstrated high specificity for MMP positive tumors. Intravital flexible confocal fluorescence lasermicroscopy of protease activity enabled specific detection of pancreatic tumors at the cellular level. Importantly, topical application of NIRF-probes markedly reduced background without altering signal intensity. Taken together, macroscopic and confocal lasermicroscopic molecular in vivo imaging of protease activity is highly sensitive, specific and allows discrimination between normal pancreatic tissue, inflammation and pancreatic cancer. Translation of this approach to the clinic could significantly improve endoscopic and laparoscopic detection of early-stage pancreatic cancer.

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse.

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.

Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer.

Oncogenic Kras activates a plethora of signaling pathways, but our understanding of critical Ras effectors is still very limited. We show that cell-autonomous phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1), but not Craf, are key effectors of oncogenic Kras in the pancreas, mediating cell plasticity, acinar-to-ductal metaplasia (ADM), and pancreatic ductal adenocarcinoma (PDAC) formation. This contrasts with Kras-driven non-small cell lung cancer, where signaling via Craf, but not PDK1, is an essential tumor-initiating event. These in vivo genetic studies together with pharmacologic treatment studies in models of human ADM and PDAC demonstrate tissue-specific differences of oncogenic Kras signaling and define PI3K/PDK1 as a suitable target for therapeutic intervention specifically in PDAC.

Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.