Technical Reports in 2008

A goal of cancer research is to develop specific and sensitive tumor-imaging techniques for early detection while minimizing background signals from nontarget, 'normal' tissues. The authors have designed a 'pH-activatable' probe, consisting of a targeted macromolecule (monoclonal antibody) and a fluorescence probe, which is activated after internalization in the lysosomes of targeted cancer cells. The utility of this approach for imaging HER2-positive lung cancer cells in mice is shown.

Peter Eirew and his colleagues describe a new assay for detecting, quantifying and characterizing normal human mammary epithelial stem cells. The assay, which combines in vivo transplantation under the kidney capsule of immunodeficient mice and an in vitro colony-forming assay, provides a system for studying the mechanisms regulating normal human mammary stem cell proliferation and differentiation in vivo and in human breast cancer.

In HIV research, new types of reagents are needed to target infected cells and overcome HIV's ability to vary its HLA-I-restricted antigens and escape from host cytotoxic T lymphocytes. Here Varela-Rohena and colleagues use phage display technology to generate high-affinity T-cell antigen receptors that recognize common epitope-escape variants of the immunodominant HLA-A^*02-restricted, HIVgag-specific peptide SLYNTVATL (SL9).

Kelly et al. offer a method for controlling the tropism of replicating viruses. Viral replication in a specific target tissue is inhibited by incorporation of tissue-specific microRNA targets within the viral genome. Using an oncolytic picornavirus that causes myositis in tumor-bearing mice, they show the virus is unable to replicate in muscle but still retains oncolytic activity, with implications for the clinical use of oncolytic viruses and vaccine development.

There is a pressing need for better methods to analyze specific proteins in a high-throughput manner. Current approaches involve studying only a few markers at a time. Here Kattah and his colleagues describe a new technology for multiplexed protein detection called high-throughput immunophenotyping using transcription (HIT). Use of this multianalyte, antibody-based protein array platform is demonstrated for profiling cytokines in serum, intracellular signaling molecules and cell surface markers.

Banaszynski et al. combine genetic manipulation with small-molecule regulation to produce rapid, reversible and tunable regulation of protein expression in vivo. The approach builds on earlier work showing that fusion of a destabilizing domain to a gene of interest confers instability to the expressed protein. Degradation of the protein is then prevented by subsequent addition of the cell-permeable stabilizing ligand, Shield-1, which binds specifically to the destabilizing domains. Three in vivo applications of the technology in mice are described.

De-Xue Fu et al. present a novel approach to radiotherapy of herpesvirus-associated tumors by first inducing the expression of viral thymidine kinase by pretreatment with bortezomib and then by administering a radiopharmaceutical that targets the viral enzyme. The authors show that this approach is effective in lymphoid and epithelial malignancies in several xenograft mouse models of human tumors.

Traykova-Brauch et al. have developed a new approach to modeling renal diseases such as polycystic kidney disease, renal fibrosis and renal cancer in transgenic mice. In contrast to currently available tools, Pax8-rtTA–transgenic mice have high levels of transgene expression in a highly kidney-specific, uniform and tetracycline–dependent manner. The usefulness of the Pax8–rtTA system, which is both inducible and reversible, has been shown in three different settings.

The degree of lymph-node metastasis in prostate cancer is crucial for both staging the disease and planning treatment. Here, Burton and colleagues describe a one-step, non-invasive imaging technology using prostate-specific adenoviral vectors that express imaging reporter genes. This set-up specifically and accurately detects lymph-node metastases in a model of human prostate cancer and eliminates the need for invasive lymphadenectomy required by the current lymphoscintigraphy method.

A major challenge in biomedicine is the rapid and accurate measurement of biomarkers in biological samples. Here, Lee et al. describe a chip-based NMR diagnostic platform that can perform sensitive and selective measurements on small volumes of unprocessed biological samples. This miniaturized biosensing system is high throughput, low cost and portable, and its utility is shown in a number of biomedical applications.

Kuznetsov and his colleagues address a pressing problem in risk assessment for predisposition to breast cancer—whether a particular allele is cancer predisposing or not. Using a two-tiered approach, they have developed a functional assay for the classification of BRCA2 sequence variants of unknown importance. The assay may serve as a model to generate functional assays for other human disease genes.

Noninvasively monitoring immune function by positron emission tomography could affect the diagnosis and treatment evaluation of immunological disorders. Progress, however, has been hampered by the lack of probes with distinct biodistribution patterns. Radu et al. exploit the fact that many immune cells utilize a salvage pathway for nucleotide generation during DNA synthesis to develop [¹⁸F]FAC (1-(2′-deoxy-2′[¹⁸F]fluoroarabinofuranosyl) cytosine), a new probe with increased accumulation in proliferating T cells. Studies in mice show it has advantages over commonly used probes and may be clinically useful.