Summary Exosomes are lipid bilayer-enclosed extracellular vesicles (EVs) that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer cell-derived exosomes in circulation is currently lacking. Using mass spectrometry analyses, we identified a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of cancer patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreas cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreas disease from patients with early and late stage pancreas cancer. Levels of GPC1+ crExos correlate with tumor burden and survival in patients pre- and post-surgical tumor resection. GPC1+ crExos from patients and from mice with spontaneous pancreas tumors driven by oncogenic KRAS contained RNA with specific KRAS mutation, and it emerges as a reliable biomarker for the detection of PanIN lesions despite negative signal by MRI in mice. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreas cancer to facilitate possible curative surgical therapy.

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The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo −/− mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions. The heme-containing enzyme MPO is a key component of the cytotoxic armamentarium of phagocytic white blood cells 1,2 . MPO is by far the most abundant protein product in azurophilic granules of neutrophils (5%), constitutes approximately 1% of monocyte protein and is found in the lysosomes of other polymorphonuclear leukocytes and macrophages. The phagosomal oxidative burst is initiated by a stimulus-dependent assembly of the phagocytic NADPH oxidase (Phox), a multimeric protein complex located on the phagosomal membrane. Phox then reduces molecular oxygen to produce superoxide anion (O 2•− ), which further dismutates to yield the relatively unreactive hydrogen peroxide (H 2 O 2 ) 1 . Upon phagocytic activation, large quantities of active MPO are secreted into phagosomes, catalyzing the production of highly bactericidal hypochlorous acid (HOCl) with H 2 O 2 and chloride ions (Cl − ) as substrates (Fig. 1a) 1 .

An optical imaging probe was synthesized by attaching a near-infrared carbocyanine fluorophore to an affinity group containing two zinc(II) dipicolylamine (Zn-DPA) units. The probe has a strong and selective affinity for the surfaces of bacteria, and it was used to image infections of Gram-positive S. aureus and Gram-negative E. coli bacteria in living nude mice. After intravenous injection, the probe selectively accumulates at the sites of localized bacterial infections in the thigh muscles of the mice.Bacterial imaging is an emerging technology that has many health and environmental applications. 1 For example, there is an obvious need to develop highly sensitive assays that can detect very small numbers of pathogenic bacterial cells in food, drinking water or biomedical samples. In other situations, the goal is to study in vivo the temporal and spatial distribution of bacteria in live animals.Optical imaging of bacteria in vivo is much less developed than methods such as radioimaging and MRI. One approach is to use bacteria that are genetically encoded to produce luciferase or green fluorescent protein. 2 A second strategy, which is the focus of this study, employs a molecular probe with a fluorescent reporter group. An obvious limitation with a live animal is restricted tissue penetration of the light. However, near-infrared (NIR) dyes with emission wavelengths in the region of 650−900 nm can propagate through two or more centimeters of tissue, and may enable deeper tissue imaging if sensitive detection techniques are employed. Molecular imaging probes can often be deconstructed into two structural components, an affinity ligand and a reporter group. In the case of bacterial targeting, previously reported affinity ligands include antibodies, 5 sugars, 6 bacteria binding peptides, 7 antimicrobial peptides, 8 enzyme substrates, 9 and antibiotic drugs. 10 Recently, we discovered that fluorescent molecular probes containing synthetic zinc(II) dipicolylamine (Zn-DPA) coordination complexes as affinity groups are able to selectively stain the surfaces of bacterial cells 11 and apoptotic animal cells. 12 Zn-DPA affinity ligands bind strongly to the anionic surfaces that are a common characteristic of these two cell-types, whereas affinity for the zwitterionic surfaces of healthy animal cells is weak. These in vitro results have motivated us to pursue in vivo studies, and we report that molecular probe 1, which has a NIR fluorophore attached to an affinity group with two Zn-DPA units, can be used for targeted, fluorescence imaging of bacterial infection in a living whole animal.The bacterial imaging probe 1 (λ max abs: 794 nm, em: 810 nm) was prepared in straightforward fashion using a carbocyanine dye as the NIR fluorophore. 13 Researchers have incorporated this fluorophore into probes for other optical imaging applications. 14 In vitro fluorescence microscopy studies proved that probe 1 can effectively stain the periphery of bacterial cells (Figure 1). In contrast, the cells are not stained when treated un...

It's hip to be square: Squaraine rotaxanes have very similar photophysical properties to the commonly used Cy‐5 fluorophore, but are substantially more photostable and resist self‐quenching upon aggregation. Molecular probes containing squaraine rotaxanes (see structure) are shown to be versatile, high‐performance NIR fluorescence stains for in vitro fluorescence imaging of cells (middle) and in vivo whole‐body imaging of living mice (right).

Optical imaging of bacterial infection in living animals is usually conducted with genetic reporters such as light emitting enzymes or fluorescent proteins. However, there are many circumstances where genetic reporters are not applicable, and there is a need for exogenous synthetic probes that can selectively target bacteria. The focus of this study is a fluorescent imaging probe that is composed of a bacterial affinity group conjugated to a near infrared dye. The affinity group is a synthetic zinc (II) coordination complex that targets the anionic surfaces of bacterial cells. The probe allows detection of Staphylococcus aureus infection (5 × 10 7 cells) in a mouse leg infection model using whole animal near infrared fluorescence imaging. Region of interest analysis showed that the signal ratio for infected leg to uninfected leg reaches 3.9 ± 0.5 at 21 h post-injection of the probe. Ex vivo imaging of the organs produced a signal ratio of 8 for infected to uninfected leg. Immunohistochemical analysis confirmed that the probe targeted the bacterial cells in the infected tissue. Optimization of the imaging filter set lowered the background signal due to autofluorescence and substantially improved imaging contrast. The study shows that near infrared molecular probes are amenable to non-invasive optical imaging of localized S. aureus infection.

A wide variety of bioluminescent luciferase proteins are available for use in transcriptional or biochemical reporter assays. However, spectral overlap normally prevents them from being monitored simultaneously. To address this problem, a Java plug-in for ImageJ was written to deconvolute bioluminescent images composed of signals from multiple luciferases. The methodology was validated by testing the program with both simulated and real luciferase images. Bioluminescent images were acquired using a CCD camera equipped with optical filters, and the images were deconvoluted using the ImageJ plug-in. HeLa cells were transfected with either click beetle red luciferase (CBR), click beetle green luciferase (CBG99), or Renilla luciferase (Rluc), and mixed lysates were imaged in varying proportions in a 96-well plate to biochemically validate the methodology. After spectral deconvolution, the predicted, pure luciferase signals could be recovered with maximal cross-talk errors of +/-1.5%. In addition, live cells expressing CBR, CBG99, and Rluc were simultaneously imaged and deconvoluted in 96-well plates to demonstrate the feasibility of applying this methodology to high-throughput applications. Finally, multicolor transcriptional and posttranslational modification reporters were simultaneously imaged and shown to deconvolute normalized IkappaBeta kinase activity in longitudinal assays. Thus, our software provided a rapid, simple, and accurate method for simultaneously measuring multiple bioluminescent reporters in living cells.

Apoptosis is an important process involved in diverse developmental pathways, homeostasis, and response to therapy for a variety of diseases. Thus, noninvasive methods to study regulation and to monitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, a novel cell-permeable activatable caspase substrate, TcapQ647, was synthesized and Km, kcat, and Ki values were biochemically characterized. Specific cleavage of TcapQ647 by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ647 with a kcat 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ647 cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C6-ceramide, TcapQ647 detected apoptosis in individual- and population-based fluorescent cell assays in an effector caspase inhibitor-specific manner. Further, only background fluorescence was observed in cells incubated with dTcapQ647, a noncleavable all d-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ647 to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ647 represents a sensitive, effector caspase-specific far-red "smart" probe to noninvasively monitor apoptosis in vivo.