Preclinical PET Imaging of Tumor Cell Death following Therapy Using Gallium-68-Labeled C2Am.

There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator.

Metabolic Glycan Labeling of Cancer Cells Using Variably Acetylated Monosaccharides.

Methylcyclopropene (Cyoc)-tagged tetra-acetylated monosaccharides, and in particular mannosamine derivatives, are promising tools for medical imaging of cancer using metabolic oligosaccharide engineering and the extremely fast inverse electron-demand Diels-Alder bioorthogonal reaction. However, the potential of these monosaccharide derivatives has yet to be fully explored due to their low aqueous solubility. To address this issue, we sought to vary the extent of acetylation of Cyoc-tagged monosaccharides and probe its effect on the extent of glycan labeling in various cancer cell lines.

Multi-modal imaging of high-risk ductal carcinoma in situ of the breast using C2Am: a targeted cell death imaging agent.

Background: Ductal carcinoma in situ (DCIS) is a non-invasive form of early breast cancer, with a poorly understood natural history of invasive transformation. Necrosis is a well-recognized adverse prognostic feature of DCIS, and non-invasive detection of its presence and spatial extent could provide information not obtainable by biopsy. We describe here imaging of the distribution and extent of comedo-type necrosis in a model of human DCIS using C2Am, an imaging agent that binds to the phosphatidylserine exposed by necrotic cells.

F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography.

Introduction: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET.

Lectins in Cervical Screening.

Cervical screening in low-resource settings remains an unmet need. Lectins are naturally occurring sugar-binding glycoproteins whose binding patterns change as cancer develops. Lectins discriminate between dysplasia and normal tissue in several precancerous conditions.

Site-selective installation of an electrophilic handle on proteins for bioconjugation.

Site-selective protein modification strategies can be used to insert non-natural functional groups into protein structures. Herein, we report on the use of the bis-electrophile 3-bromo-2-bromomethyl-1-propene as a reagent to introduce an electrophilic handle at cysteine residues under mild conditions. This method is demonstrated on a variety of proteins containing a solvent-exposed cysteine residue, including an anti-HER2 nanobody.

Detection of early neoplasia in Barrett's esophagus using lectin-based near-infrared imaging: an ex vivo study on human tissue.

Background And Study Aims: Endoscopic surveillance for Barrett's esophagus (BE) is limited by long procedure times and sampling error. Near-infrared (NIR) fluorescence imaging minimizes tissue autofluorescence and optical scattering. We assessed the feasibility of a topically applied NIR dye-labeled lectin for the detection of early neoplasia in BE in an ex vivo setting.

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context.

Optoacoustic Detection of Early Therapy-Induced Tumor Cell Death Using a Targeted Imaging Agent.

The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU).

Site-Selective Modification of Proteins with Oxetanes.

Oxetanes are four-membered ring oxygen heterocycles that are advantageously used in medicinal chemistry as modulators of physicochemical properties of small molecules. Herein, we present a simple method for the incorporation of oxetanes into proteins through chemoselective alkylation of cysteine. We demonstrate a broad substrate scope by reacting proteins used as apoptotic markers and in drug formulation, and a therapeutic antibody with a series of 3-oxetane bromides, enabling the identification of novel handles (S-to-S/N rigid, non-aromatic, and soluble linker) and reactivity modes (temporary cysteine protecting group), while maintaining their intrinsic activity.

Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I.

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. The capability of near-infrared fluorophore-labeled and Tc- and In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft.

MRI measurements of reporter-mediated increases in transmembrane water exchange enable detection of a gene reporter.

Non-invasive imaging of gene expression can be used to track implanted cells in vivo but often requires the addition of an exogenous contrast agent that may have limited tissue access. We show that the urea transporter (UT-B) can be used as a gene reporter, where reporter expression is detected using H MRI measurements of UT-B-mediated increases in plasma membrane water exchange. HEK cells transfected with the reporter showed an increased apparent water exchange rate (AXR), which increased in line with UT-B expression.

Design and validation of a near-infrared fluorescence endoscope for detection of early esophageal malignancy.

Barrett’s esophagus is a known precursor lesion to esophageal adenocarcinoma. In these patients, early detection of premalignant disease, known as dysplasia, allows curative minimally invasive endoscopic therapy, but is confounded by a lack of contrast in white light endoscopy. Imaging fluorescently labeled lectins applied topically to the tissue has the potential to more accurately delineate dysplasia, but tissue autofluorescence limits both sensitivity and contrast when operating in the visible region.

Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

Glycosylation is a ubiquitous post-translational modification, present in over 50 % of the proteins in the human genome,1 with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases,2 including cancer.3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe.

Detection of colorectal dysplasia using fluorescently labelled lectins.

Colorectal cancer screening using conventional colonoscopy lacks molecular information and can miss dysplastic lesions. We tested here the ability of fluorescently labelled lectins to distinguish dysplasia from normal tissue when sprayed on to the luminal surface epithelium of freshly resected colon tissue from the Apc(min) mouse and when applied to fixed human colorectal tissue sections. Wheat germ agglutinin (WGA) showed significantly decreased binding to adenomas in the mouse tissue and in sections of human colon from 47 patients.

Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

Glycosylation is a ubiquitous post-translational modification, present in over 50% of the proteins in the human genome, with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, including cancer. We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe.

Development of Timd2 as a reporter gene for MRI.

Purpose: To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), using T1- and T2-weighted imaging.

Methods: Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron-loaded or manganese-loaded ferritin, were imaged using T1- and T2-weighted MRI. Mice were also implanted subcutaneously with Timd2-expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2-weighted imaging.

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.

Dual-sugar imaging using isonitrile and azido-based click chemistries.

We report the first account of metabolically labelling N-acetylglucosamine, in conjunction with either N-acetylgalactosamine or N-acetylmannosamine using a combination of isonitrile- and azide-based chemistries. With the appropriately labelled fluorescent probe molecules, that react with either the azido or isonitrile groups, the method enabled co-visualisation of cancer cell glycoproteins.

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.

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.

Analysis of image heterogeneity using 2D Minkowski functionals detects tumor responses to treatment.

Purpose: The acquisition of ever increasing volumes of high resolution magnetic resonance imaging (MRI) data has created an urgent need to develop automated and objective image analysis algorithms that can assist in determining tumor margins, diagnosing tumor stage, and detecting treatment response.

Methods: We have shown previously that Minkowski functionals, which are precise morphological and structural descriptors of image heterogeneity, can be used to enhance the detection, in T1 -weighted images, of a targeted Gd(3+) -chelate-based contrast agent for detecting tumor cell death. We have used Minkowski functionals here to characterize heterogeneity in T2 -weighted images acquired before and after drug treatment, and obtained without contrast agent administration.

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.