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The presence of short-range chemical order can be a key factor in determining the mechanical behavior of metals, but directly and unambiguously determining its distribution in complex concentrated alloy systems can be challenging. Here, we directly identify and quantify chemical order in the globally single phase BCC-TiVNbHf(Al) system using aberration corrected scanning transmission electron microscopy (STEM) paired with spatial statistics methods. To overcome the difficulties of short-range order (SRO) quantification with STEM when the components of an alloy exhibit large atomic number differences and near equiatomic ratios, “null hypothesis” tests are used to separate experiment from a random chemical distribution. Experiment is found to deviate from both the case of an ideal random solid solution and a fully ordered structure with statistical significance. We also identify local chemical order in TiVNbHf and confirm and quantify the enhancement of SRO with the addition of Al. These results provide insight into local chemical order in the promising TiVNbHf(Al) refractory alloys while highlighting the utility of spatial statistics in characterizing nanoscale SRO in compositionally complex systems.

Figure S1. CD133 expression correlated with PSA expression. Figure S2. CD133 expression in xenografts. Figure S3. Characterization of labeled HA10. Table S1. Ex vivo biodistribution data.

Fig. S1. Plasmid map of A.TSTAPEG101KB in pGL3- Basic Fig. S2. PEG10 staining in positive control and healthy tissue Fig. S3. TSTA Schematic Fig. S4. Luciferase expression in empty pGL3 vector Fig. S5. PEG10 expression by IHC in HT-29 xenografts Fig. S6. HSV-TK and PEG10 expression in CWR-R1 tumors Table S1. Primers for PEG10 promoter cloning.

Purpose:Aggressive variant prostate cancer (AVPC) is a nonandrogen receptor–driven form of disease that arises in men in whom standard-of-care therapies have failed. Therapeutic options for AVPC are limited, and the development of novel therapeutics is significantly hindered by the inability to accurately quantify patient response to therapy by imaging. Imaging modalities that accurately and sensitively detect the bone and visceral metastases associated with AVPC do not exist.Experimental Design:This study investigated the transmembrane protein CD133 as a targetable cell surface antigen in AVPC. We evaluated the expression of CD133 by microarray and IHC analysis. The imaging potential of the CD133-targeted IgG (HA10 IgG) was evaluated in preclinical prostate cancer models using two different imaging modalities: near-infrared and PET imaging.Results:Evaluation of the patient data demonstrated that CD133 is overexpressed in a specific phenotype of AVPC that is androgen receptor indifferent and neuroendocrine differentiated. In addition, HA10 IgG was selective for CD133-expressing tumors in all preclinical imaging studies. PET imaging with [89Zr]Zr-HA10 IgG revealed a mean %ID/g of 24.30 ± 3.19 in CD133-positive metastatic lesions as compared with 11.82 ± 0.57 in CD133-negative lesions after 72 hours (P = 0.0069). Ex vivo biodistribution showed similar trends as signals were increased by nearly 3-fold in CD133-positive tumors (P < 0.0001).Conclusions:To our knowledge, this is the first study to define CD133 as a targetable marker of AVPC. Similarly, we have developed a novel imaging agent, which is selective for CD133-expressing tumors, resulting in a noninvasive PET imaging approach to more effectively detect and monitor AVPC.

The retrotransposon-derived paternally expressed gene 10 (PEG10) protein is ordinarily expressed at high levels in the placenta. Recently, it was discovered that PEG10 isoforms promote the progression of prostate cancer to a highly lethal androgen receptor (AR)-negative phenotype. The presence of PEG10 in other subtypes of prostate cancer has not been explored and a utility for PEG10 overexpression has not been developed. Here, we found that in addition to AR-null disease, PEG10 was also expressed in prostate cancer with constitutively active AR-splice variants. A molecular genetic imaging strategy for noninvasive imaging of AR-splice variant prostate cancer was developed by utilizing the cancer specificity of the PEG10 promoter to drive the expression of reporter genes. Plasmid insertion of a PEG10 promoter sequence optimized for enhanced output upstream of a reporter gene allowed detection of prostate cancer by near-infrared and positron emission tomography imaging after systemic administration of the plasmid in vivo. PEG10 expressing subcutaneous xenograft and intratibial tumor models were imaged by both modalities using this molecular genetic imaging strategy. This study demonstrates a preclinical proof-of-concept that the PEG10 promoter is a powerful and specific tool that can be utilized for noninvasive detection of aggressive prostate cancer subtypes.Significance:PEG10 is expressed by prostate cancer with constitutively active AR-splice variants that can be exploited for noninvasive molecular imaging of this aggressive prostate cancer subytpe.

PDF file - 165K, Additional SPECT/CT images of the uPAR probes imaging the MDA-MB-231 CDM

Components of the plasminogen activation system, which are overexpressed in aggressive breast cancer subtypes, offer appealing targets for development of new diagnostics and therapeutics. By comparing gene expression data in patient populations and cultured cell lines, we identified elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) in highly aggressive breast cancer subtypes and cell lines. Recombinant human anti-uPAR antagonistic antibodies exhibited potent binding in vitro to the surface of cancer cells expressing uPAR. In vivo these antibodies detected uPAR expression in triple negative breast cancer (TNBC) tumor xenografts using near infrared imaging and 111In single-photon emission computed tomography. Antibody-based uPAR imaging probes accurately detected small disseminated lesions in a tumor metastasis model, complementing the current clinical imaging standard 18F-fluorodeoxyglucose at detecting non-glucose-avid metastatic lesions. A monotherapy study using the antagonistic antibodies resulted in a significant decrease in tumor growth in a TNBC xenograft model. In addition, a radioimmunotherapy study, using the anti-uPAR antibodies conjugated to the therapeutic radioisotope 177Lu, found that they were effective at reducing tumor burden in vivo. Taken together, our results offer a preclinical proof of concept for uPAR targeting as a strategy for breast cancer diagnosis and therapy using this novel human antibody technology. Cancer Res; 73(7); 2070–81. ©2013 AACR.

PDF file - 38K, Determining the half-life of the 111In-labeled antibodies in vivo (n = 3 mice / antibody)

PDF file - 62K, FDG avid lesion not detected by 111In-2G10

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Supplemental Figures S1-S6. U33 Fab Sequence (S1); Inhibition constant calculation of U33 Fab (S2); U33 does not bind to the uPA-PAI1 complex (S3); Selectivity of U33 IgG for active uPA versus inactive uPA (S4); Dependence of U33 IgG binding on an accessible uPA active site (S5); Selectivity of U33 IgG for human uPA compared to highly related serine proteases (S6).

PDF file - 41K, The affinity of 2G10 and 3C6 Fabs and IgGs for MDA-MB-231 cells was determined by equilibrium binding flow cytometry. Isotherms were fit using non-linear regression methods to calculate KDs

PDF file - 101K, Mouse model recreating the inflammation observed in Figure 4C. A nude mouse was injected subcutaneously with 100�l of turpentine oil. After 72hrs, the animal was injected with 258°Ci of FDG and imaged 60 minutes later. Probe uptake by the inflammatory cells is only observed with FDG and not with the uPAR probe

PDF file - 71K, Scrambled matched isotype control IgGs labeld with AlexaFlupr 680 at 72hrs in MDA-MB-231 xenografts imaged in the Cy 5.5 channel at 72hr

PDF file - 40K, Time activity curves for 111In-labeled 2G10 and 3C6 in MDA-MB-231 orthotopic xenograft tumor-bearing mice

PDF file - 58K, 177Lu-2G10 SPECT/CT imaging of a mouse enrolled in the RIT study. The mouse was injected with 2.5�g of 2G10 labeled with 75°Ci of 177Lu. The image shown is a coronal view acquitted at 24hrs post-injection. The represented data was processed using AMIDE software

The ability to recover the oxygen reduction reaction of poisoned metal oxide surfaces, central to many energy related applications, is demonstrated by controlling relative surface acidity.

Metal oxides are an important class of functional materials, and for many applications, ranging from solid oxide fuel/electrolysis cells, oxygen permeation membranes, and oxygen storage materials to gas sensors (semiconducting and electrolytic) and catalysts, the interaction between the surface and oxygen in the gas phase is central. Ubiquitous Si-impurities are known to impede this interaction, commonly attributed to the formation of glassy blocking layers on the surface. Here, we examine the surface oxygen exchange coefficient (k

Metastatic castration-resistant prostate cancer (mCRPC) has been largely resistant to immunotherapy. Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill transformed cells without prior sensitization, and their infiltration into prostate tumors corresponds with an increased overall survival among patients with mCRPC. We sought to harness this knowledge to develop an approach to NK-cell based immunotherapy for mCRPC. We engineered an NK cell line (NK-92MI) to express CD64, the sole human high-affinity IgG Fcγ receptor (FcγR1), and bound these cells with antibodies to provide interchangeable tumor-targeting elements. NK-92MICD64 cells were evaluated for cell-activation mechanisms and antibody-dependent cell-mediated cytotoxicity (ADCC). A combination of mAbs was used to target the prostate tumor antigen tumor-associated calcium signal transducer 2 (TROP2) and the cancer-associated fibroblast marker fibroblast activation protein alpha (FAP). We found that CD64, which is normally expressed by myeloid cells and associates with the adaptor molecule FcRγ, can be expressed by NK-92MI cells and mediate ADCC through an association with CD3ζ. Cytotoxicity from the combination approach was two-fold higher compared to treatment with NK-92MICD64 cells and either mAb alone, and seven-fold higher than NK-92MICD64 cells alone at an effector–target cell ratio of 20:1. The cytotoxic effect was lost when using isotype control antibodies, indicating a selective targeting mechanism. The combination approach demonstrated efficacy in vivo as well and significantly reduced tumor growth compared with the saline control. This combination therapy presents a potential approach for treating mCRPC and could improve immunotherapy response.

Design and synthesis of low-cost and efficient bifunctional catalysts for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) in Zn-air batteries are essential and challenging. We report a facile method to synthesize heterostructure carbon consisting of graphitic and amorphous carbon derived from the agricultural waste of red bean pods. The heterostructure carbon possesses a large surface area of 625.5 m2 g-1 , showing ORR onset potential of 0.89 V vs. RHE and OER overpotential of 470 mV at 5 mA cm-2 . Introducing hollow FeCo nanoparticles and nitrogen dopant improves the bifunctional catalytic activity of the carbon, delivering ORR onset potential of 0.93 V vs. RHE and OER overpotential of 360 mV. Electron energy-loss spectroscopy (EELS) O K-edge map suggests the presence of localized oxygen on the FeCo nanoparticles, suggesting the oxidation of the nanoparticles. Zn-air battery with these carbon-based catalysts exhibits a peak power density as high as 116.2 mW cm-2 and stable cycling performance over 210 discharge/charge cycles. This work contributes to the advancement of bifunctional oxygen electrocatalysts while converting agricultural waste into value-added material.

Single-phase multiferroic materials that allow the coexistence of ferroelectric and magnetic ordering above room temperature are highly desirable, motivating an ongoing search for mechanisms for unconventional ferroelectricity in magnetic oxides. Here, we report an antisite defect mechanism for room temperature ferroelectricity in epitaxial thin films of yttrium orthoferrite, YFeO3, a perovskite-structured canted antiferromagnet. A combination of piezoresponse force microscopy, atomically resolved elemental mapping with aberration corrected scanning transmission electron microscopy and density functional theory calculations reveals that the presence of YFe antisite defects facilitates a non-centrosymmetric distortion promoting ferroelectricity. This mechanism is predicted to work analogously for other rare earth orthoferrites, with a dependence of the polarization on the radius of the rare earth cation. Our work uncovers the distinctive role of antisite defects in providing a mechanism for ferroelectricity in a range of magnetic orthoferrites and further augments the functionality of this family of complex oxides for multiferroic applications., Ferroelectricity in orthoferrite perovskites has stimulated intense research, but the mechanism remains unclear. Here, the authors propose an antisite defect mechanism for introducing ferroelectricity in magnetically ordered YFeO3 and the family of rare earth orthoferrites.

Summary The dynamics of polarization evolution and rotation in 0.68PbMg1/3Nb2/3O3-0.32PbTiO3 relaxor ferroelectric thin films are studied via in operando synchrotron-based X-ray diffraction with AC electric fields. A frequency-limited suppression of polarization rotation was observed above ultrasonic frequencies ( ≳ 20 kHz). The nature of this suppression is informed by scanning transmission electron microscopy in the zero-field state, where a high density of nanoscale, low-angle domain walls was observed. In combination with switching dynamics studies, the results suggest that the suppression of polarization rotation at ultrasonic frequencies is due to the large activation field needed to move the domain walls when the polarization rotates between different monoclinic phases. These results are critical in understanding piezoelectric relaxation phenomena in relaxor ferroelectrics.

Analysis of nanoscale short-range chemical and/or structural order via (scanning) transmission electron microscopy (S/TEM) imaging is fundamentally limited by projection of the three dimensional sample, which averages informational along the beam direction. Extracting statistically significant spatial correlations between the structure and chemistry determined from these two-dimensional datasets thus remains challenging. Here, we apply methods commonly used in Geographic Information Systems (GIS) to determine the spatial correlation between measures of local chemistry and structure from atomic-resolution STEM imaging of a compositionally complex relaxor, Pb(Mg$_{1/3}$Nb$_{2/3}$)O$_{3}$ (PMN). The approach is used to determine the type of ordering present and to quantify the spatial variation of chemical order, oxygen octahedral distortions, and oxygen octahedral tilts. The extent of autocorrelation and inter-feature correlation among these short-range ordered regions are then evaluated through a spatial covariance analysis, showing correlation as a function of distance. The results demonstrate that integrating GIS tools for analyzing microscopy datasets can serve to unravel subtle relationships among chemical and structural features in complex materials that can be hidden when ignoring their spatial distributions.

Exsolution synthesizes self-assembled metal nanoparticle catalysts via phase precipitation. An overlooked aspect in this method thus far is how exsolution affects the host oxide surface chemistry and structure. Such information is critical as the oxide itself can also contribute to the overall catalytic activity. Combining X-ray and electron probes, we investigated the surface transformation of thin-film SrTi

Background: Fibroblast Activation Protein (FAP) is a protein expressed by cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) of epithelial cancers. There is potential interest in the use of FAP as a way to selectively target cancers, including colorectal cancer (CRC). Here we examine the expression of FAP in human colorectal cancers across stages and compare that expression to expression in normal colon and liver tissue. Additionally, we examine the correlation of FAP expression with key CRC clinical and biologic features to aid in potentially determining which CRC patients would be the most likely to benefit from FAP-directed therapies. Methods: CRC and adjacent normal tissue samples spanning all stages of disease were collected across 243 patients. Immunohistochemistry (IHC) was used to evaluate FAP, MMP2, αSMA, and PDPN expression and quantified on an intensity scale from 0-3+. These scores were split into low (average score /=2) groups. IHC was used to determine mismatch repair status by staining for MLH1, MSH2, MSH6, and PMS2. Separate samples from these patients were sequenced with the Qiagen Comprehensive Cancer Panel and analyzed for APC, KRAS, BRAF, PIK3CA, and TP53 mutations. Results: Of 149 normal colon tissue sections, 99% had low FAP expression and 94% of those had no FAP expression. Of the 87 normal liver tissue sections, 99% had low FAP expression. In CRC sections, 58% had high FAP expression. Those patients >50 years of age had cancers with a higher rate of FAP expression (p=0.02). There was no correlation with FAP expression and sex, stage of disease, or sidedness. In cancers with BRAF V600 mutations, 91% have high FAP expression (x2 = 0.02). There is a trend for higher FAP expression in cancers possessing APC, KRAS, PIK3CA, or TP53 mutations (x2=NS). In correlating FAP with other CAF markers, MMP2 had the greatest correlation (Spearman’s Rho = 0.56). FAP is moderately associated with αSMA and PDPN (Spearman’s Rho = 0.33, 0.38). Conclusion: FAP is an exciting therapeutic target for CRC due to its cancer selectivity. Here, we demonstrate that FAP marks a rather independent subset of CRCs, though there is particular interest in the BRAF V600 mutant cancers given the high expression and poor prognosis of this subtype. Further investigation is needed to further develop FAP-directed therapies. Citation Format: Anna L. Lippert, Katherine A. Johnson, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Aaron M. LeBeau, Dustin A. Deming. Fibroblast activation protein (FAP) expression as a target for colorectal cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2357.

Hybrid molecular beam epitaxy (MBE) growth of Sn-modified BaTiO3 films was realized with varying domain structures and crystal symmetries across the entire composition space. Macroscopic and microscopic structures and the crystal symmetry of these thin films were determined using a combination of optical second harmonic generation (SHG) polarimetry and scanning transmission electron microscopy (STEM). SHG polarimetry revealed a variation in the global crystal symmetry of the films from tetragonal ( P4 mm) to cubic [Formula: see text] across the composition range, x = 0 to 1 in BaTi1− xSn xO3 (BTSO). STEM imaging shows that the long-range polar order observed when the Sn content is low ( x = 0.09) transformed to a short-range polar order as the Sn content increased ( x = 0.48). Consistent with atomic displacement measurements from STEM, the largest polarization was obtained at the lowest Sn content of x = 0.09 in Sn-modified BaTiO3 as determined by SHG. These results agree with recent bulk ceramic reports and further identify this material system as a potential replacement for Pb-containing relaxor-based thin film devices.

Purpose: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. Experimental Design: We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. Results: Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAP-positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 ± 1.99 versus 6.12 ± 2.03 (n = 3/group; P = 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG by 72 hours. Conclusions: Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [89Zr]Zr-B12 IgG as a selective imaging probe for FAP-expressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.

Relaxor ferroelectrics, which can exhibit exceptional electromechanical coupling, are some of the most important functional materials, with applications ranging from ultrasound imaging to actuators. Since their discovery, their complex nanoscale chemical and structural heterogeneity has made the origins of their electromechanical properties extremely difficult to understand. Here, we employ aberration-corrected scanning transmission electron microscopy to quantify various types of nanoscale heterogeneities and their connection to local polarization in the prototypical relaxor ferroelectric system Pb(Mg1/3Nb2/3)O3–PbTiO3. We identify three main contributions that each depend on Ti content: chemical order, oxygen octahedral tilt and oxygen octahedral distortion. These heterogeneities are found to be spatially correlated with low-angle polar domain walls, indicating their role in disrupting long-range polarization and leading to nanoscale domain formation and the relaxor response. We further locate nanoscale regions of monoclinic-like distortion that correlate directly with Ti content and electromechanical performance. Through this approach, the connections between chemical heterogeneity, structural heterogeneity and local polarization are revealed, validating models that are needed to develop the next generation of relaxor ferroelectrics. Relaxor ferroelectric systems exhibit exceptional electromechanical coupling that arises from a variety of nanoscale chemical ordering. Here, scanning transmission electron microscopy is used to quantify this structural complexity directly.