In this novel large-scale multiplexed immunofluorescence study we comprehensively characterized and compared layer-specific proteomic features within regions of interest of the widely divergent dorsolateral prefrontal cortex (A46) and primary visual cortex (A17) of adult rhesus monkeys. Twenty-eight markers were imaged in rounds of sequential staining, and their spatial distribution precisely quantified within gray matter layers and superficial white matter. Cells were classified as neurons, astrocytes, oligodendrocytes, microglia, or endothelial cells. The distribution of fibers and blood vessels were assessed by quantification of staining intensity across regions of interest. This method revealed multivariate similarities and differences between layers and areas. Protein expression in neurons was the strongest determinant of both laminar and regional differences, whereas protein expression in glia was more important for intra-areal laminar distinctions. Among specific results, we observed a lower glia-to-neuron ratio in A17 than in A46 and the pan-neuronal markers HuD and NeuN were differentially distributed in both brain areas with a lower intensity of NeuN in layers 4 and 5 of A17 compared to A46 and other A17 layers. Astrocytes and oligodendrocytes exhibited distinct marker-specific laminar distributions that differed between regions; notably, there was a high proportion of ALDH1L1-expressing astrocytes and of oligodendrocyte markers in layer 4 of A17. The many nuanced differences in protein expression between layers and regions observed here highlight the need for direct assessment of proteins, in addition to RNA expression, and set the stage for future protein-focused studies of these and other brain regions in normal and pathological conditions.

The c-Jun N-terminal kinase 3 (JNK3) is a stress-activated kinase primarily expressed in the brain and implicated as an early mediator of neuronal apoptosis. We sought to develop a PET tracer to visualize pathological JNK3 activation. Because regional JNK3 activation precedes apoptosis, such an imaging agent might enable the detection of “at risk” brain regions prior to neuronal death. We prepared a set of 19F-containing compounds on the basis of the reported aminopyrazoles. The candidate, F3, was tritiated and used in autoradiography experiments to demonstrate regional and temporal changes in JNK3 activation in a mouse model of Parkinson’s disease. A significant increase in pJNK3Bmax versus control animals in multiple brain regions was observed at 8 months, including the ventral midbrain. Pathological activation of JNK3 in these regions preceded statistically significant neuron loss. Analyses of brain concentrations of [18F]-F3 in naïve rats following intravenous injection revealed a small but detectable signal over the background, but was likely not sufficient to support PET imaging.

Degeneration of the primary motor cortex is a defining feature of amyotrophic lateral sclerosis (ALS), which is associated with the accumulation of microscopic protein aggregates in neurons and glia. However, little is known about the quantitative burden and pattern of motor cortex proteinopathies across ALS genotypes. We combined quantitative digital image analysis with multi-level generalized linear modelling in an independent cohort of 82 ALS cases to explore the relationship between genotype, total proteinopathy load and cellular vulnerability to aggregate formation. Primary motor cortex phosphorylated (p)TDP-43 burden and microglial activation were more severe in sporadic ALS-TDP disease than C9-ALS. Oligodendroglial pTDP-43 pathology was a defining feature of ALS-TDP in sporadic ALS, C9-ALS and ALS with OPTN, HNRNPA1 or TARDBP mutations. ALS-FUS and ALS-SOD1 showed less cortical proteinopathy in relation to spinal cord pathology than ALS-TDP, where pathology was more evenly spread across the motor cortex-spinal cord axis. Neuronal pTDP-43 aggregates were rare in GAD67+ and Parvalbumin+ inhibitory interneurons, consistent with predominant accumulation in excitatory neurons. Finally, we show that cortical microglia, but not astrocytes, contain pTDP-43. Our findings suggest divergent quantitative, genotype-specific vulnerability of the ALS primary motor cortex to proteinopathies, which may have implications for our understanding of disease pathogenesis and the development of genotype-specific therapies.

Contrast-enhanced magnetic resonance imaging is an essential tool for disease diagnosis and management; all marketed clinical magnetic resonance imaging (MRI) contrast agents (CAs) are gadolinium (Gd) chelates and most are extracellular fluid (ECF) agents. After intravenous injection, these agents rapidly distribute to the extracellular space and are also characterized by low serum protein binding and predominant renal clearance. Gd is an abiotic element with no biological recycling processes; low levels of Gd have been detected in the central nervous system and bone long after administration. These observations have prompted interest in the development of new MRI contrast agents based on biotic elements such as iron (Fe); Fe-HBED (HBED = N,N '-bis(2-hydroxyphenyl)ethylenediamine-N,N '-diacetic acid), a coordinatively saturated iron chelate, is an attractive MRI CA platform suitable for modification to adjust relaxivity and biodistribution. Compared to the parent Fe-HBED, the Fe-HBED analogs reported here have lower serum protein binding and higher relaxivity as well as lower relative liver enhancement in mice, comparable to that of a representative gadolinium-based contrast agent (GBCA). Fe-HBED analogs are therefore a promising class of non-Gd ECF MRI CA.

We present a method for automatic cell classification in tissue samples using an automated training set from multiplexed immunofluorescence images. The method utilizes multiple markers stained in situ on a single tissue section on a robust hyperplex immunofluorescence platform (Cell DIVE™, GE Healthcare) that provides multi-channel images allowing analysis at single cell/sub-cellular levels. The cell classification method consists of two steps: first, an automated training set from every image is generated using marker-to-cell staining information. This mimics how a pathologist would select samples from a very large cohort at the image level. In the second step, a probability model is inferred from the automated training set. The probabilistic model captures staining patterns in mutually exclusive cell types and builds a single probability model for the data cohort. We have evaluated the proposed approach to classify: i) immune cells in cancer and ii) brain cells in neurological degenerative diseased tissue with average accuracies above 95%.

Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre-clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150 mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration-time profiles (t0-240 min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R2 = 0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model.

BackgroundSteady state susceptibility contrast (SSC)‐MRI provides information on vascular morphology but is a rarely used method.PurposeTo investigate the utility of the ultrasmall superparamagnetic iron oxide particles (USPIOs) GEH121333 for measuring tumor response to bevacizumab and compare this with gadolinium‐based DCE‐MRI.Study TypeProspective preclinical animal model study.ANIMAL MODELMice bearing subcutaneous TOV‐21G human ovarian cancer xenografts treated with bevacizumab (n = 9) or saline (n = 9).Field Strength/SequenceImaging was performed on a 7T Bruker Biospec. For SSC‐MRI with GEH121333 we acquired R1‐maps (RARE‐sequence with variable TR), R2‐maps (multi‐spin echo), and ‐maps (multi‐gradient echo). Additionally, R1 and R2 maps were measured on the days after USPIO injection. For DCE‐MRI with gadodiamide we acquired 200 T1‐weighted images (RARE‐sequence).AssessmentΔR1, ΔR2, and maps were computed from SSC‐MRI. DCE‐MRI was analysed using the extended Tofts model.Statistical TestsResults from pre‐ and 3 days posttreatment SSC‐MRI were compared using paired‐sample t‐tests. Treatment and control groups were compared using independent sample t‐tests. Performance of SSC‐ and DCE‐MRI was compared using multivariate partial least squares discriminant analysis.ResultsAlready one day after treatment and USPIO injection, R1 and R2 values were lower in treated (R1 = 0.49 ± 0.03s−1, R2 = 23.07 ± 1.49s−1) compared with control tumors (R1 = 0.52 ± 0.02s−1, R2 = 24.98 ± 1.01s−1), indicating lower USPIO accumulation. Posttreatment SSC‐MRI displayed significantly decreased tumor blood volume (change in ΔR2 = ‐0.43 ± 0.26s−1, P = 0.001) and vessel density (change in Q = ‐0.032 ± 0.020s−1/3, P = 0.002). DCE‐MRI showed among others lower Ktrans in treated tumors (control = 0.064 ± 0.011min−1, tx = 0.046 ± 0.008min−1, P = 0.002). Multivariate analysis suggests that SSC‐MRI was slightly inferior to DCE‐MRI in distinguishing treated from control tumors (accuracy = 75%, P = 0.058 versus 80%, P = 0.028), but a combination of both was best (accuracy = 85%; P = 0.003).Data ConclusionSSC‐MRI with GEH121333 is sensitive to early (<24 h) and late changes in tumor vasculature. SSC‐MRI and DCE‐MRI provide complementary information and can be used to assess different aspects of vascular responses to anti‐angiogenic therapies.Level of Evidence: 1Technical Efficacy: Stage 2J. Magn. Reson. Imaging 2018;47:1589–1600.

We report an automated method for characterization of microvessel morphology in micrographs of brain tissue sections to enable the facile, quantitative analysis of vascular differences across large datasets consisting of hundreds of images with thousands of blood vessel objects. Our objective is to show that virtual 3D parametric models of vasculature are adequately capable of representing the morphology of naturally acquired data in neuropathology. In this work, we focus on three distinct morphologies that are most frequently observed in formalin-fixed, paraffin-embedded (FFPE) human brain tissue samples: single blood vessels showing no (or collapsed) significant lumen (“RoundLumen-”); single blood vessels with distinct lumen (“RoundLumen+”); two blood vessels bundled together in close proximity (“Twins”). The analysis involves extraction of features using pre-trained convolutional neural networks. A hierarchical classification is performed to distinguish single blood vessels (RoundLumen) from Twins; followed by a more granular classification between RoundLumen- and RoundLumen+. A side-by-side comparison of the virtual and natural data models is presented. We observed that classification models built on the virtual data perform well achieving accuracies of 92.8% and 98.3% for the two aforementioned classification tasks respectively.

Background: Measures of spatial intratumor heterogeneity are potentially important diagnostic biomarkers for cancer progression, proliferation, and response to therapy. Spatial relationships among cells including cancer and stromal cells in the tumor microenvironment (TME) are key contributors to heterogeneity. Methods: We demonstrate how to quantify spatial heterogeneity from immunofluorescence pathology samples, using a set of 3 basic breast cancer biomarkers as a test case. We learn a set of dominant biomarker intensity patterns and map the spatial distribution of the biomarker patterns with a network. We then describe the pairwise association statistics for each pattern within the network using pointwise mutual information (PMI) and visually represent heterogeneity with a two-dimensional map. Results: We found a salient set of 8 biomarker patterns to describe cellular phenotypes from a tissue microarray cohort containing 4 different breast cancer subtypes. After computing PMI for each pair of biomarker patterns in each patient and tumor replicate, we visualize the interactions that contribute to the resulting association statistics. Then, we demonstrate the potential for using PMI as a diagnostic biomarker, by comparing PMI maps and heterogeneity scores from patients across the 4 different cancer subtypes. Estrogen receptor positive invasive lobular carcinoma patient, AL13-6, exhibited the highest heterogeneity score among those tested, while estrogen receptor negative invasive ductal carcinoma patient, AL13-14, exhibited the lowest heterogeneity score. Conclusions: This paper presents an approach for describing intratumor heterogeneity, in a quantitative fashion (via PMI), which departs from the purely qualitative approaches currently used in the clinic. PMI is generalizable to highly multiplexed/hyperplexed immunofluorescence images, as well as spatial data from complementary in situ methods including FISSEQ and CyTOF, sampling many different components within the TME. We hypothesize that PMI will uncover key spatial interactions in the TME that contribute to disease proliferation and progression.

Evaluation of the novel SPIO GEH121333 for monitoring changes in tumor vascularity and vascular permeability after antiangiogenic treatment using susceptibility contrast and T1-mapping Else Marie Huuse, Jana Cebulla, Dan E Meyer, Karina Langseth, Siver Andre Moestue, and Tone Frost Bathen Department of medical Imaging, St. Olavs University Hospital, Trondheim, Norway, MI lab and Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway, Biomedical Imaging & Physiology Laboratory, GE Global Research Center, Niskayuna, NY, United States, GE Healthcare AS, Oslo, Norway

Tumor‐associated macrophages (TAM) maintain a chronic inflammation in cancers, which is associated with tumor aggressiveness and poor prognosis. The purpose of this study was to: (1) evaluate the pharmacokinetics and tolerability of the novel ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) compound GEH121333; (2) assess whether GEH121333 can serve as a MR imaging biomarker for TAM; and (3) compare tumor MR enhancement profiles between GEH121333 and ferumoxytol. Blood half‐lives of GEH121333 and ferumoxytol were measured by relaxometry (n = 4 each). Tolerance was assessed in healthy rats injected with high dose GEH121333, vehicle or saline (n = 4 each). Animals were monitored for 7 days regarding body weight, complete blood counts and serum chemistry, followed by histological evaluation of visceral organs. MR imaging was performed on mice harboring MMTV‐PyMT‐derived breast adenocarcinomas using a 7 T scanner before and up to 72 h post‐injection (p.i.) of GEH121333 (n = 10) or ferumoxytol (n = 9). Tumor R1, R2* relaxation rates were compared between different experimental groups and time points, using a linear mixed effects model with a random effect for each animal. MR data were correlated with histopathology. GEH121333 showed a longer circulation half‐life than ferumoxytol. Intravenous GEH121333 did not produce significant adverse effects in rats. All tumors demonstrated significant enhancement on T1, T2 and T2*‐weighted images at 1, 24, 48 and 72 h p.i. GEH121333 generated stronger tumor T2* enhancement than ferumoxytol. Histological analysis verified intracellular compartmentalization of GEH121333 by TAM at 24, 48 and 72 h p.i. MR imaging with GEH121333 nanoparticles represents a novel biomarker for TAM assessment. This new USPIO MR contrast agent provides a longer blood half‐life and better TAM enhancement compared with the iron supplement ferumoxytol. Copyright © 2013 John Wiley & Sons, Ltd.

Rat legs directly injected with superparamagnetic iron oxide (SPIO) were studied by dual‐echo, gradient‐echo imaging. The amount of iron injected was estimated using a point dipole model for the SPIO injection site. Saturation magnetization of 6:1 PEG/amino modified silane‐coated iron oxide particles with 5‐ to 6‐nm core and 20–25 hydrodynamic diameter was ∼110 emu/g of iron. Estimates of the amount of iron injected made from signal void volumes surrounding SPIO centers yielded erroneous results varying with sample orientation in the scanner and echo time (TE). For example, a 10 μL, 3‐μg iron injection produced signal void volumes of 80 and 210 μL at TE of 9.8 and 25 ms, respectively, giving apparent iron contents of 6 ± 1 and 10 ± 2 μg respectively. A more effective approach uses the phase difference between two gradient recalled echo images. To estimate iron content, this approach fits the expected (3 cos2θ − 1)/|r|3 spatial phase distribution to the observed phase differences. Extraneous phase effects made fitting phase at a single TE ineffective. With the dual echo method, 18 independent estimates were 2.48 ± 0.26 μg std, independently of sample orientation. Estimates in empty control regions were −90 and −140 ng. A 1‐μg injection indicated 0.5, 1.2, and 1.2 μg. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.

Elastin-like polypeptides (ELPs) are environmentally responsive biopolymers based on the elastinderived pentapeptide repeat Val-Pro-Gly-Val-Gly. ELPs undergo a reversible phase transition termed an “inverse temperature transition” (Urry 1992, 1997). Below their transition temperature (Tt), the polypeptides are highly soluble in aqueous solutions. However, when the temperature is raised above Tt, the hydrated polypeptide chains hydrophobically collapse and aggregate, forming a separate, ELP-rich phase. The Tt of an ELP can be conveniently controlled at several different levels. For example, the Tt can be adjusted over a wide range of temperatures through control of the amino acid sequence. The transition can also be triggered isothermally by modulation of environmental conditions, in particular by the type and concentration of added co-solutes. Significantly, ELP fusion proteins, which are produced by joining the gene encoding a protein of interest with an ELP gene segment, can also undergo a reversible phase transition similar to that of the free ELP (Meyer and Chilkoti 1999). Thus, the environmental responsiveness of ELPs

At a specific temperature, elastin-like polypeptides (ELPs) undergo a sharp solubility transition that can be exploited in a variety of applications in biotechnology and medicine. The temperature of the transition varies with ELP sequence, molecular weight, and concentration. We present a single equation of three parameters that quantitatively predicts the transition temperature as a function of ELP length and concentration for an ELP of a fixed composition. This model should be useful both for the design of new ELP sequences that exhibit a desired transition temperature and for the selection of variables to trigger the phase transition of an ELP for a given application.

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.

The sensitivity of an elastin-like polypeptide (ELP) to environmental stimuli is used to reversibly immobilize a fusion partner, thioredoxin (TRX), onto a hydrophobic surface. An ELP, fused to TRX at its C-terminus, adsorbs onto a hydrophobic self-assembled monolayer (SAM) on gold above its inverse phase transition temperature (T-c) and is resolubilized from the surface when the solution temperature is lowered below T-c. We show that the adsorbed fusion partner TRX is recognized by an antibody specific to TRX and that the complex is also resolubilized from the surface below T-c. Adsorption of TRX-ELP is inhibited by hydrophilic surfaces that are defect-free. In situ ellipsometry shows that the ELP-anchored TRX adsorbs above T-c up to a well-defined layer thickness that is greater than a monolayer and that the adsorption and desorption cycle can be repeated. These results are confirmed by atomic force microscopy and by surface plasmon resonance spectroscopy. The preferential adsorption of TRX-ELP on hydrophobic surfaces is used to create a pattern of adsorbed protein on a surface composed of a pattern of hydrophobic and hydrophilic SAMs on gold. We term this method to reversibly address an ELP fusion protein to chemically distinct regions of a patterned surface by an external stimulus "thermodynamically reversible addressing of proteins" (TRAP).

Elastin-like polypeptides (ELPs) are artificial polypeptides with unique properties that make them attractive as a biomaterial for tissue-engineered cartilage repair. ELPs are composed of a pentapeptide repeat, Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro), that undergo an inverse temperature phase transition. They are soluble in aqueous solution below their transition temperature (T(t)) but aggregate when the solution temperature is raised above their T(t). This study investigates the rheological behavior of an un-cross-linked ELP, below and above its T(t), and also examines the ability of ELP to promote chondrogenesis in vitro. A thermally responsive ELP with a T(t) of 35 degrees C was synthesized using recombinant DNA techniques. The complex shear modulus of the ELP increased by 3 orders of magnitude as it underwent its inverse temperature phase transition, forming a coacervate, or gel-like, ELP phase. Values for the complex shear moduli of the un-cross-linked ELP coacervate are comparable to those reported previously for collagen, hyaluronan, and cross-linked synthetic hydrogels. Cell culture studies show that chondrocytes cultured in ELP coacervate maintain a rounded morphology and their chondrocytic phenotype, characterized by the synthesis of a significant amount of extracellular matrix composed of sulfated glycosaminoglycans and collagen. These results suggest that ELPs demonstrate great potential for use as in situ forming scaffolds for cartilaginous tissue repair.