André A. Neves

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus

Barretts esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barretts esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barretts esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5.

Imaging Cell Death

There is currently a need for imaging methods capable of detecting cell death in tissues and the early onset of tumor cell death resulting from therapy. However, to date, no probe has been approved for routine imaging of cell death in the clinic. The challenge is to identify hallmarks of cell death, which have clinical relevance, and then to develop and validate imaging biomarkers for these hallmarks. We focus here on cell death imaging probes, which either have been trialed in the clinic or have significant promise, based on preclinical studies.

Detection of Cell Death in Tumors by Using MR Imaging and a Gadolinium-based Targeted Contrast Agent

PURPOSE To prospectively determine in an animal model whether an ionic gadolinium (Gd(3+)) chelate conjugate of the C2A domain of synaptotagmin I can be used with magnetic resonance (MR) imaging to detect tumor cell death noninvasively in vivo. MATERIALS AND METHODS Animal experiments were approved by a local ethics review committee. Gd(3+) chelates and fluorescent probes were attached to the lysine epsilon-amino groups of a glutathione-S-transferase-C2A fusion protein. Binding to phosphatidylserine (PS) was characterized by using surface plasmon resonance, and binding to dying cells in vitro was characterized by using flow cytometry and MR imaging. Binding to dying tumor cells in vivo was detected with T1 mapping and T1-weighted MR imaging and compared in drug-treated animals (n = 10); in animals injected with a site-directed mutant, which was inactive in PS binding (PS inactive) and which showed lesser binding to dying cells (n = 6); and in untreated animals injected with PS-active (n = 6) and PS-inactive (n = 6) contrast agents. Among groups, differences that were significant were analyzed by using analysis of variance and Dunnett post hoc analysis. RESULTS The contrast agent had a relatively high affinity for PS (dissociation constant = 333 nmol/L +/- 85 [mean +/- standard error of the mean]; n = 3) and bound to apoptotic and necrotic, but not viable, cells in vitro. There was a greater tumor accumulation of the PS-active contrast agent compared with the PS-inactive contrast agent in drug-treated animals (P < .05) and compared with untreated animals injected with the PS-active and PS-inactive contrast agents (P < .01 for both). CONCLUSION A relatively small (approximately 100 kDa) Gd(3+)-based contrast agent, which gives positive contrast on MR images, can be used to detect tumor cell death in vivo, and future derivatives of it may be used to assess early tumor responses to treatment.

Imaging sialylated tumor cell glycans in vivo

Cell surface glycans are involved in numerous physiological processes that involve cell‐cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido‐labeled N‐acetyl‐man‐nosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5‐azidoneuraminic, or azidosialic acid. Azidosialic acid‐labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far‐red fluorophore or 111In, there was a significant azido‐labeled N‐acetyl‐mannosamine‐dependent increase in tumor‐to‐tissue contrast, which was detected using optical imaging or single‐photon‐emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.—Neves, A. A., Stöckmann, H., Harmston, R. R., Pryor, H. J., Alam, I. S., Ireland‐Zecchini, H., Lewis, D. Y., Lyons, S. K., Leeper, F. J., Brindle, K. M. Imaging sialylated tumor cell glycans in vivo. FASEB J. 25, 2528–2537 (2011). www.fasebj.org

Imaging cell surface glycosylation in vivo using "double click" chemistry.

Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell–cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels–Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse.

Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells

Two reagents have been synthesized for selective labeling of cell surface azidoglycans, an unusually stable version of a dibenzocyclooctyne (TMDIBO) and a third-generation difluorinated cyclooctyne (DIFO3). Both syntheses are efficient with minimal purification, and the dibenzocyclooctyne is stable under basic and acidic conditions. Flow cytometric measurements with azidosugar labeled cancer cells, in which these reagents were linked to the fluorophore Alexa Fluor 647, gave a signal-to-background ratio of up to 35 with TMDIBO as compared to ≈10 for DIFO3 and ≈5 for a phosphine reagent. TMDIBO-based probes should have applications in molecular imaging of cell surface glycans in vivo.

Effects of preculture variability on clavulanic acid fermentation

The production profile of clavulanic acid by Streptomyces clavuligerus was shown to be strongly dependent on inoculum activity. Two sets of fermentations (A and B) were investigated at industrial pilot-plant scale using complex media. Type A fermentations were inoculated using late exponential growth phase mycelia. Type B fermentations were inoculated using mycelia harvested at stationary phase. Productivities throughout type A fermentations were consistently higher than type B, reaching a maximum at about 70 h and then decaying to the same final productivities at 140 h of type B runs. Several scheduling alternatives, based on combinations of the two inocula types and different fermentation lengths, were compared in terms of the overall process economics (fermentation and downstream). An increase of ca. 22% on the overall process profit is predicted using late exponential growth phase inocula and a fermentation duration of only 96 h. A new operating strategy was thus proposed for inoculum production based on the control of preculture activity using off-gas analysis. This method ensures higher productivity and better batch-to-batch reproducibility of clavulanic acid fermentations than traditional methods based on constant age inocula.

Metabolic Glycan Imaging by Isonitrile–Tetrazine Click Chemistry

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.

Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death.

The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell death, and radionuclide-labeled derivatives have undergone clinical trials for detecting tumor cell death in vivo following treatment. We show here that the C2A domain of Synaptotagmin-I, which had been fluorescently labeled at a single cysteine residue introduced by site-directed mutagenesis, detected the same levels of cell death as a similarly labeled Annexin-V derivative, in drug-treated murine lymphoma and human breast cancer cell lines in vitro. However, the C2A derivative showed significantly less binding to viable cells and, as a consequence, up to 4-fold more specific binding to apoptotic and necrotic cells when compared with Annexin-V. C2A offers a potential route for the development of a new generation of more specific imaging probes for the detection of tumor cell death in the clinic.

Functional assessment of tissue-engineered meniscal cartilage by magnetic resonance imaging and spectroscopy.

A perfusion bioreactor system was used to grow bioartificial meniscal cartilage tissue in vitro. Magnetic resonance imaging and magnetic resonance spectroscopy methods were used to characterize the flow and perfusion profiles and the growth, distribution, and bioenergetics of the fibrochondrocytes in the resulting constructs. These measurements were correlated with each other and with subsequent histologic analysis. The study has demonstrated that these noninvasive magnetic resonance methods will be useful for designing bioreactor operation strategies and cell scaffolds that lead to the production of tissue-engineered meniscal cartilage constructs with properties resembling those of the native tissue.