Proteomic features of gray matter layers and superficial white matter of the rhesus monkey neocortex: comparison of prefrontal area 46 and occipital area 17.

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.

Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

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 pJNK3 B max 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.

Fe-HBED Analogs: A Promising Class of Iron-Chelate Contrast Agents for Magnetic Resonance Imaging.

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.

In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations.

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.

Multiparametric characterization of response to anti-angiogenic therapy using USPIO contrast-enhanced MRI in combination with dynamic contrast-enhanced MRI.

BACKGROUND: Steady state susceptibility contrast (SSC)-MRI provides information on vascular morphology but is a rarely used method. PURPOSE: To 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 TYPE: Prospective preclinical animal model study. ANIMAL MODEL: Mice bearing subcutaneous TOV-21G human ovarian cancer xenografts treated with bevacizumab (n = 9) or saline (n = 9). FIELD STRENGTH/SEQUENCE: Imaging 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 R2*-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). ASSESSMENT: ΔR1 , ΔR2 , and ΔR2* maps were computed from SSC-MRI. DCE-MRI was analysed using the extended Tofts model. STATISTICAL TESTS: Results 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. RESULTS: Already 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 CONCLUSION: SSC-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: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:1589-1600.

Pointwise mutual information quantifies intratumor heterogeneity in tissue sections labeled with multiple fluorescent biomarkers.

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 USPIO GEH121333 for MR imaging of cancer immune responses.

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.

Estimating amounts of iron oxide from gradient echo images.

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 approximately 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 microL, 3-microg iron injection produced signal void volumes of 80 and 210 microL at TE of 9.8 and 25 ms, respectively, giving apparent iron contents of 6 +/- 1 and 10 +/- 2 microg 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 cos(2)theta - 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 microg std, independently of sample orientation. Estimates in empty control regions were -90 and -140 ng. A 1-microg injection indicated 0.5, 1.2, and 1.2 microg.

Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides.

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.

Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery.

In this article, we review recombinant DNA methods for the design and synthesis of amino acid-based biopolymers, and briefly summarize an approach, recursive directional ligation (RDL), that we have employed to synthesize oligomeric genes for such biopolymers. We then describe our ongoing research in the use of RDL to synthesize recombinant polypeptide carriers for the targeted delivery of radionuclides, chemotherapeutics and biomolecular therapeutics to tumors. The targeted delivery system uses a thermally responsive, elastin-like polypeptide (ELP) as the drug carrier to enhance the localization of ELP-drug conjugates within a solid tumor that is heated by regional hyperthermia. In the context of this drug delivery application, we discuss the design of ELPs and their recombinant synthesis, which enables the molecular weight and the thermal properties of the polypeptide to be precisely controlled. Finally, our results pertaining to the in vivo targeting of tumors with ELPs are briefly summarized.

Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair.

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.

Targeted drug delivery by thermally responsive polymers.

This review article summarizes recent results on the development of macromolecular carriers for thermal targeting of therapeutics to solid tumors. This approach employs thermally responsive polymers in conjunction with targeted heating of the tumor. The two thermally responsive polymers that are discussed in this article, poly(N-isopropylacrylamide-co-acrylamide) (poly(NIPAAm)) and an artificial elastin-like polypeptide (ELP), were designed to exhibit a soluble-insoluble lower critical solution transition in response to increased temperature slightly above 37 degrees C. In vivo fluorescent videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately two-fold increase in tumor localization compared to the same polypeptide without hyperthermia. Similar results were also obtained for poly(NIPAAm) though the extent of accumulation was somewhat lesser than observed for the ELP. The endocytotic uptake of a thermally responsive ELP was also observed to be significantly enhanced by the thermally triggered phase transition of the polypeptide in cell culture for three different tumor cell lines. Preliminary cytotoxicity studies of an ELP-doxorubicin conjugate indicate that the ELP-doxorubicin conjugate has near equivalent cytotoxicity as free doxorubicin in a cell culture assay.

Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.

We report a new strategy for the synthesis of genes encoding repetitive, protein-based polymers of specified sequence, chain length, and architecture. In this stepwise approach, which we term "recursive directional ligation" (RDL), short gene segments are seamlessly combined in tandem using recombinant DNA techniques. The resulting larger genes can then be recursively combined until a gene of a desired length is obtained. This approach is modular and can be used to combine genes encoding different polypeptide sequences. We used this method to synthesize three different libraries of elastin-like polypeptides (ELPs); each library encodes a unique ELP sequence with systematically varied molecular weights. We also combined two of these sequences to produce a block copolymer. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of these polypeptides provides an example of the importance of precise control over these parameters that is afforded by RDL.