Hypoxia occurs when limited oxygen supply impairs physiological functions and is a pathological hallmark of many diseases including cancer and ischemia. Thus, detection of hypoxia can guide treatment planning and serve as a predictor of patient prognosis. Unfortunately, current methods suffer from invasiveness, poor resolution and low specificity. To address these limitations, we present Hypoxia Probe 1 (HyP-1), a hypoxia-responsive agent for photoacoustic imaging. This emerging modality converts safe, non-ionizing light to ultrasound waves, enabling acquisition of high-resolution 3D images in deep tissue. HyP-1 features an N-oxide trigger that is reduced in the absence of oxygen by heme proteins such as CYP450 enzymes. Reduction of HyP-1 produces a spectrally distinct product, facilitating identification via photoacoustic imaging. HyP-1 exhibits selectivity for hypoxic activation in vitro, in living cells, and in multiple disease models in vivo. HyP-1 is also compatible with NIR fluorescence imaging, establishing its versatility as a multimodal imaging agent.

BackgroundThe exchange of metabolites and the reprogramming of metabolism in response to shifting microenvironmental conditions can drive subpopulations of cells within colonies toward divergent behaviors. Understanding the interactions of these subpopulations—their potential for competition as well as cooperation—requires both a metabolic model capable of accounting for a wide range of environmental conditions, and a detailed dynamic description of the cells’ shared extracellular space.ResultsHere we show that a cell’s position within an in silicoEscherichia coli colony grown on glucose minimal agar can drastically affect its metabolism: “pioneer” cells at the outer edge engage in rapid growth that expands the colony, while dormant cells in the interior separate two spatially distinct subpopulations linked by a cooperative form of acetate crossfeeding that has so far gone unnoticed. Our hybrid simulation technique integrates 3D reaction-diffusion modeling with genome-scale flux balance analysis (FBA) to describe the position-dependent metabolism and growth of cells within a colony. Our results are supported by imaging experiments involving strains of fluorescently-labeled E. coli. The spatial patterns of fluorescence within these experimental colonies identify cells with upregulated genes associated with acetate crossfeeding and are in excellent agreement with the predictions. Furthermore, the height-to-width ratios of both the experimental and simulated colonies are in good agreement over a growth period of 48 hours.ConclusionsOur modeling paradigm can accurately reproduce a number of known features of E. coli colony growth, as well as predict a novel one that had until now gone unrecognized. The acetate crossfeeding we see has a direct analogue in a form of lactate crossfeeding observed in certain forms of cancer, and we anticipate future application of our methodology to models of tissues and tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0155-1) contains supplementary material, which is available to authorized users.

Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker α V β 3 integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of α V β 3 activity (a radiolabeled version, 64Cu-NOTA-PEG4-cRGD2, for PET imaging, and a fluorescent version, FITC-PEG4-cRGD2, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for α V β 3 in HUVECs (K d ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within α V β 3-positive infarcted myocardium. 64Cu-NOTA-PEG4-cRGD2 demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of α V β 3 expression, both in vitro and in vivo.

Cancer stem cells (CSCs) are progenitor cells that contribute to treatment-resistant phenotypes during relapse. CSCs exist in specific tissue microenvironments that cell cultures and more complex models cannot mimic. Therefore, the development of new approaches that can detect CSCs and report on specific properties (e.g., stem cell plasticity) in their native environment have profound implications for studying CSC biology. Herein, we present AlDeSense, a turn-on fluorescent probe for aldehyde dehydrogenase 1A1 (ALDH1A1) and Ctrl-AlDeSense, a matching nonresponsive reagent. Although ALDH1A1 contributes to the detoxification of reactive aldehydes, it is also associated with stemness and is highly elevated in CSCs. AlDeSense exhibits a 20-fold fluorescent enhancement when treated with ALDH1A1. Moreover, we established that AlDeSense is selective against a panel of common ALDH isoforms and exhibits exquisite chemostability against a collection of biologically relevant species. Through the application of surface marker antibody staining, tumorsphere assays, and assessment of tumorigenicity, we demonstrate that cells exhibiting high AlDeSense signal intensity have properties of CSCs. Using these probes in tandem, we have identified CSCs at the cellular level via flow cytometry and confocal imaging, as well as monitored their states in animal models.

Highlights d Aberrant expression of the non-muscle RBFOX2 40 isoform in hearts of DM1 patients d Forced expression of RBFOX2 40 isoform reproduces DM1like cardiac pathology in mice d RBFOX2 40 isoform induces DM1-related splicing defects in voltage-gated ion channels d Silencing RBFOX2 40 restores the normal cardiac rhythm and splicing of ion channels

The receptor for advanced glycation end-products (RAGE) is central to multiple disease states, including diabetes-related conditions such as peripheral arterial disease (PAD). Despite RAGE's importance in these pathologies, there remains a need for a molecular imaging agent that can accurately assess RAGE levels in vivo. Therefore, we have developed a multimodal nanoparticle-based imaging agent targeted at RAGE with the well-characterized RAGE ligand, carboxymethyllysine (CML)-modified human serum albumin (HSA).Methods: A multimodal tracer (64Cu-Rho-G4-CML) was developed using a generation-4 (G4) polyamidoamine (PAMAM) dendrimer, conjugated with both rhodamine and copper-64 (64Cu) chelator (NOTA) for optical and PET imaging, respectively. First, 64Cu-Rho-G4-CML and its non-targeted analogue (64Cu-Rho-G4-HSA) were evaluated chemically using techniques such as dynamic light scattering (DLS), electron microscopy and nuclear magnetic resonance (NMR). The tracers' binding capabilities were examined at the cellular level and optimized using live and fixed HUVEC cells grown in 5.5-30 mM glucose, followed by in vivo PET-CT imaging, where the probes' kinetics, biodistribution, and RAGE targeting properties were examined in a murine model of hindlimb ischemia. Finally, histological assessment of RAGE levels in both ischemic and non-ischemic tissues was performed.Conclusions: Our RAGE-targeted probe demonstrated an average size of 450 nm, a Kd of 340-390 nM, rapid blood clearance, and a 3.4 times greater PET uptake in ischemic RAGE-expressing hindlimbs than their non-ischemic counterpart. We successfully demonstrated increased RAGE expression in a murine model of hindlimb ischemia and the feasibility for non-invasive examination of cellular, tissue, and whole-body RAGE levels with a molecularly targeted tracer.

Melanoma topology promotes a stem cell phenotype with the propensity to facilitate new vessel growth.

Combinations of novel pulse-echo acquisitions and clutter filtering techniques can improve the sensitivity and the specificity of power Doppler (PD) images, thus reducing the need for exogenous contrast enhancement. We acquire echoes following bursts of Doppler pulse transmissions sparsely applied in regular patterns over long durations. The goal is to increase the sensitivity of the acquisition to slow disorganized patterns of motion from the peripheral blood perfusion. To counter a concomitant increase in clutter signal power, we arrange the temporal echo acquisitions into two data-array axes, combine them with a spatial axis for the tissue region of interest, and apply 3-D singular-value decomposition (SVD) clutter filtering. Successful separation of blood echoes from other echo signal sources requires that we partition the 3-D SVD core tensor. Unfortunately, the clutter and blood subspaces do not completely uncouple in all situations, so we developed a statistical classifier that identifies the core tensor subspace dominated by tissue clutter power. This paper describes an approach to subspace partitioning as required for optimizing PD imaging of peripheral perfusion. The technique is validated using echo simulation, flow-phantom data, and in vivo data from a murine melanoma model. We find that for narrow eigen-bandwidth clutter signals, we can routinely map phantom flows and tumor perfusion signals at speeds less than 3 mL/min. The proposed method is well suited to peripheral perfusion imaging applications.