Ultrasound­targeted microbubble destruction technology delivering ß­klotho to the heart enhances FGF21 sensitivity and attenuates heart remodeling post­myocardial infarction.

Fibroblast growth factor (FGF)21 is a peptide hormone that improves mitochondrial function and energy metabolism, and the deficiency of its co­receptor ß­klotho (KLB) causes decreased FGF21 sensitivity. The present study examined whether the cardiac delivery of plasmids containing the KLB gene via ultrasound­targeted microbubble destruction (UTMD) enhances the efficacy of FGF21 against heart failure post­acute myocardial infarction (AMI). For this purpose, the levels of FGF21 in patients and rats with heart dysfunction post­infarction were determined using ELISA. Sprague­Dawley rats received the 3X UTMD­mediated delivery of KLB@cationic microbubbles (KLB@CMBs) 1 week following the induction of AMI. Echocardiography, histopathology and biochemical analysis were performed at 4 weeks following the induction of AMI. The results revealed that patients with heart failure post­infarction had higher serum FGF21 levels than the healthy controls. However, the downstream signal, KLB, but not α­klotho, was reduced in the heart tissues of rats with AMI. As was expected, treatment with FGF21 did not substantially attenuate heart remodeling post­infarction. It was found that decreased receptors KLB in the heart may result in the insensitivity to FGF21 treatment. In vivo, the UTMD technology­mediated delivery of KLB@CMBs to the heart significantly enhanced the effects of FGF21 administration on cardiac remodeling and mitochondrial dysfunction in the rats following infarction. The delivery of KLB to the heart by UTMD and the administration of FGF21 attenuated mitochondrial impairment and oxidative stress by activating nuclear factor erythroid 2­related factor 2 signals. On the whole, the present study demonstrates that the cardiac delivery of KLB significantly optimizes the cardioprotective effects of FGF21 therapy on adverse heart remodeling. UTMD appears a promising interdisciplinary approach with which to improve heart failure post­myocardial infarction.

JUNO high purity nitrogen plant.

The Jiangmen Underground Neutrino Observatory (JUNO) is a 20 kt low level radioactivity liquid scintillator detector in a laboratory 650 m underground. An excellent energy resolution and a large volume offer exciting opportunities for addressing many important topics in neutrino physics. High purity nitrogen is an important factor to ensure the low background of the JUNO detector. High Purity Nitrogen (HPN) is used for detector purging, pipe cleaning, and scintillator purification, among other things in JUNO. According to JUNO's requirements, the radon concentration in HPN should be less than 10 µBq/m3. To meet this requirement, A high-purity nitrogen plant with 100 Nm3/h maximum rate was designed and constructed. Low-temperature adsorption technology is used to remove radioactive impurities in nitrogen. High purification efficiency was ensured by using an activated carbon column with high column height-to-diameter ratio. Electrostatic collection and low-temperature enrichment methods are combined to measure radon in nitrogen. After ten days of continuous operation at 50 Nm3/h flux rate, the plant can to reduce the radon concentration in nitrogen from 37.4±1.8µBq/m3 to less than 1.33 µBq/m3. After HPN with flow rate of 50 Nm3/h passing through low-background pipeline (About 1.3 km), the radon concentration of HPN is 5.6±0.6µBq/m3.

[System Construction and the Function Improvement of Ecological Carbon Sink in Coal Mining Areas Under the Carbon Neutral Strategy].

The loss of ecological carbon sinks often occurs in the process of coal resource development. Under the carbon neutral strategy, it is of great significance to explore technologies and models for improving ecological carbon sinks in coal mining areas. This study firstly addressed the system construction framework of the ecological carbon sink in coal mining areas, which included two levels of management mode and technical methods; three main categories of soil carbon sink, vegetation carbon sink, and wetland carbon sink; and several technical contents such as ecological carbon sink planning, carbon sink monitoring and investigation, carbon sink function improvement, and carbon sink loss prevention. The study analyzed the main types of ecological carbon sink (mainly involving soil carbon sinks and vegetation carbon sinks, whereas wetland carbon sinks were mainly related to coal mining subsidence areas with high groundwater level) and circumstances of carbon sink losses (including coal mining activities, the process of ecological vegetation construction, and ecological stability risk under long-term conditions) and proposed methods to improve ecological carbon sinks and prevent carbon sink losses for soil carbon sinks and vegetation carbon sinks in coal mining areas. The results can provide technical reference for the scientific research and engineering construction of ecological carbon sinks in coal mining areas.

Longitudinal Molecular Imaging of Progesterone Receptor Reveals Early Differential Response to Endocrine Therapy in Breast Cancer with an Activating ESR1 Mutation.

Activating mutations in the estrogen receptor (ER) α-gene (ESR1) result in constitutive transcriptional activity in the absence of estrogen and are associated with endocrine resistance in metastatic ER-positive (+) breast cancer. It is not known how activating ESR1 mutations may alter the predictive values of molecular imaging agents for endocrine therapy response. This study investigated the effect of an activating ESR1 mutation on pretreatment 18F-fluoroestradiol (18F-FES) uptake and early assessment of endocrine therapy response using 18F-FDG and 18F-fluorofuranylnorprogesterone (18F-FFNP) PET/CT imaging of tumor glucose metabolism and progesterone receptor (PR) expression, respectively. Methods: ER+, PR+ T47D breast cancer cells expressing wild-type (WT) ER or an activating ESR1 mutation, Y537S-ER, were used to generate tumor xenografts in ovariectomized female immunodeficient mice supplemented with 17ß-estradiol. Tumor growth curves were determined in the presence or absence of estrogen and for ethanol vehicle control or fulvestrant treatment, a selective ER degrader. Pretreatment 18F-FES uptake was compared between Y537S-ER and WT-ER tumors. Longitudinal PET/CT imaging with 18F-FFNP and 18F-FDG was performed before and 7-9 d after the start of endocrine therapy with fulvestrant. Radiopharmaceutical uptake in Y537S-ER and WT-ER tumors was compared between baseline and follow-up scans. Statistical significance was determined using paired t testing for longitudinal imaging and 2-way ANOVA for the 18F-FFNP tissue biodistribution assay. Results: Y537S-ER xenografts showed estrogen-independent growth, whereas WT-ER tumors grew only with estrogen. Fulvestrant treatment for 28 d significantly reduced tumor volumes for WT-ER but only stabilized volumes for Y537S-ER. Baseline 18F-FES uptake did not significantly differ between WT-ER and Y537S-ER tumors. Fulvestrant treatment induced a similar early metabolic response for both WT-ER and Y537S-ER tumors. 18F-FFNP uptake in WT-ER tumors was significantly reduced after 7 d of fulvestrant treatment; however, this reduction did not occur in Y537S-ER tumors, which showed no significant change between baseline and follow-up PET/CT. Conclusion: Molecular imaging of PR expression dynamics could be a noninvasive approach for early identification of reduced effectiveness of endocrine therapy resulting from activating ESR1 mutations.

Low interferon-gamma release in response to phytohemagglutinin predicts the high severity of diseases.

A clinically useful immune biomarker could potentially assist clinicians in their decision making. We stimulated T-cell proliferation to secret interferon gamma (IFN-γ) by phytohemagglutinin, and then measured the production of IFN-γ (mitogen value [M value]). We aimed to determine the relationship between the M value, clinical severity, and outcomes of diseases.In all, 484 patients admitted to intensive care units were enrolled in this retrospective study. The Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were collected within the first 24 hours. M value, C-reaction protein (CRP), procalcitonin (PCT), erythrocyte sedimentation rate (ESR), and routine blood tests were analyzed and collected during the study.When APACHE II scores were greater than 15 and M values were less than 6, the hospital mortality rose in a straight line. There was an inverse correlation between APACHE II score and M value (rs = -0.212, P < .001). There was a positive correlation between M value and lymphocyte numbers (b' = 0.249, P < .001); however, there was an inverse correlation between M value and WBC (b' = -0.230, P < .001), and ESR (b' = -0.100, P = .029). Neurological diseases had the greatest influence on APACHE II scores (b' = 10.356, P < .001), whereas respiratory diseases had the greatest influence on M value (b' = 1.933, P < .001). Furthermore, in the respiratory system, severe pneumonia had a greater influence on M value. Taking the APACHE II score as the gold standard, the area under the curve of M was 0.632 (95% confidence interval [CI] 0.575-0.690, P < .001), PCT was 0.647 (95% CI 0.589-0.705, P < .001), CRP was 0.570 (95% CI 0.511-0.629, P = .022), and ESR was 0.553 (95% CI 0.494-0.612, P = .078). Divided by M value = 5, the positive predictive value of the M value is 37.22% (115/309) and negative predictive value is 75.43% (132/175).The results show that the M values, PCT, and CRP were better than ESR to predict the severity of diseases. The number and proportion of lymphocytes also affected the result of the M value. To a certain extent, the M value may be a clinically useful immune biomarker, which may help clinicians objectively evaluate the severity of diseases, especially in the respiratory system.

18F-Fluoroestradiol PET Imaging of Activating Estrogen Receptor-α Mutations in Breast Cancer.

The purpose of this study was to determine the effect of estrogen receptor-α gene (ESR1) mutations at the tyrosine (Y) 537 amino acid residue within the ligand binding domain on 18F-fluoroestradiol (18F-FES) binding and in vivo tumor uptake compared with wild-type (WT)-estrogen receptor α (ER). Methods: ER-negative MDA-MB-231 breast cancer cells were used to generate stable cell lines that express WT-ER, Y537S, or Y537C mutant ER. Receptor expression and localization were confirmed by Western blot and immunofluorescence, respectively. ER transcriptional function was measured using an estrogen response element-luciferase reporter gene assay and quantitative polymerase chain reaction analysis of ER-regulated endogenous target genes. Saturation binding and competition assays were performed to determine equilibrium dissociation constant (Kd) and half maximal inhibitory concentration (IC50) values. 18F-FES uptake was measured in tumor xenografts grown in female athymic nude mice by small-animal PET/CT imaging and tissue biodistribution using 5.55 MBq (150 µCi) of 18F-FES. A 10-fold-lower injected dose of 0.555 MBq (15 µCi) of 18F-FES was also used for tissue biodistribution. Statistical significance was determined using ANOVA. Results: Y537S and Y537C mutations resulted in increased ER transcriptional activity in the absence of estrogen compared with WT-ER (11.48 ± 2.42 fold; P = 0.0002, and 5.89 ± 0.94 fold; P = 0.04, respectively). Constitutive ER activation of two target genes (PGR and TFF1) in the absence of estrogen was also observed in Y537S- and Y537C-ER cells compared with WT-ER. Kd values for 18F-FES were 0.98 ± 0.54 nM for Y537S-ER (P = 0.27) and 0.24 ± 0.03 nM for Y537C-ER (P = 0.95) compared with 0.07 ± 0.03 nM for WT-ER. IC50 values were 0.22 ± 0.09 nM for Y537S-ER (P = 0.97), 0.18 ± 0.09 nM for Y537C-ER (P = 0.99), and 0.19 ± 0.11 nM for WT-ER. Tumor xenografts expressing Y537S-ER (mean percentage injected dose per gram, 1.45 ± 0.06; P = 0.77) and Y537C-ER (2.09 ± 0.20; P = 0.21) had similar 18F-FES uptake compared with WT-ER (1.68 ± 0.12). Comparable 18F-FES uptake between Y537S-, Y537C-, and WT-ER xenografts was also observed using a 10-fold-lower injected dose with the tissue biodistribution assay. Conclusion: Since tumoral uptake of 18F-FES is not significantly impacted by Y537S-ER or Y537C-ER mutations, the potential diagnostic utility of 18F-FES PET imaging is expected to be equally valid for patients with or without these activating ESR1 mutations.

Sensitivity and Isoform Specificity of 18F-Fluorofuranylnorprogesterone for Measuring Progesterone Receptor Protein Response to Estradiol Challenge in Breast Cancer.

The purpose of this study was to evaluate the ability of 21-18F-fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione (18F-FFNP) to measure alterations in progesterone receptor (PR) protein level and isoform expression in response to estradiol challenge. Methods: T47D human breast cancer cells and female mice-bearing T47D tumor xenografts were treated with 17ß-estradiol (E2) to increase PR expression. 18F-FFNP uptake was measured using cell uptake and tissue biodistribution assays. MDA-MB-231 breast cancer clonal cell lines were generated that express the A or B isoforms of human PR. PR protein levels, transcriptional function, and subcellular localization were determined. In vitro 18F-FFNP binding was measured via saturation and competitive binding curves. In vivo 18F-FFNP uptake was measured using tumor xenografts and positron emission tomography. Statistical significance was determined using analysis of variance and t-tests. Results: After 48 and 72 h of E2, 18F-FFNP uptake in T47D cells was maximally increased compared to both vehicle and 24 h E2 treatment (p<0.0001 vs ethanol; P = 0.02 and P = 0.0002 vs 24 h for 48 and 72 h, respectively). T47D tumor xenografts in mice treated with 72 h E2 had maximal 18F-FFNP uptake compared to ethanol-treated mice (11.3±1.4 vs 5.2±0.81 %ID/g; P = 0.002). Corresponding tumor-to-muscle uptake ratios were 4.1±0.6, 3.9±0.5, and 2.3±0.4 for 48 h E2, 72 h E2, and ethanol-treated mice, respectively. There was no significant preferential 18F-FFNP binding or uptake by PR-A versus PR-B in the PR isoform-specific cell lines and tumor xenografts. Conclusion:18F-FFNP is capable of measuring estrogen-induced shifts in total PR expression in human breast cancer cells and tumor xenografts with equivalent isoform binding.

DNA origami nanostructures can exhibit preferential renal uptake and alleviate acute kidney injury.

Patients with acute kidney injury (AKI) frequently require kidney transplantation and supportive therapies, such as rehydration and dialysis. Here, we show that radiolabelled DNA origami nanostructures (DONs) with rectangular, triangular and tubular shapes accumulate preferentially in the kidneys of healthy mice and mice with rhabdomyolysis-induced AKI, and that rectangular DONs have renal-protective properties, with efficacy similar to the antioxidant N-acetylcysteine-a clinically used drug that ameliorates contrast-induced AKI and protects kidney function from nephrotoxic agents. We evaluated the biodistribution of DONs non-invasively via positron emission tomography, and the efficacy of rectangular DONs in the treatment of AKI via dynamic positron emission tomography imaging with 68Ga-EDTA, blood tests and kidney tissue staining. DNA-based nanostructures could become a source of therapeutic agents for the treatment of AKI and other renal diseases.

Molybdenum-based nanoclusters act as antioxidants and ameliorate acute kidney injury in mice.

Acute kidney injury (AKI) is a common reactive oxygen species (ROS)-related renal disease that causes numerous deaths annually, yet only supportive treatment is currently available in the clinics. Development of antioxidants with high accumulation rates in kidneys is highly desired to help prevent AKI. Here we report molybdenum-based polyoxometalate (POM) nanoclusters with preferential renal uptake as novel nano-antioxidants for kidney protection. These POM nanoclusters, with a readily variable valence state of molybdenum ions, possess the capability to scavenge detrimental ROS. Our results demonstrate that POM nanoclusters can efficiently alleviate clinical symptoms in mice subjected to AKI, as verified by dynamic PET imaging with 68Ga-EDTA, serum tests, kidney tissue staining, and biomarkers detection in the kidneys. The protective effect of POM nanoclusters against AKI in living animals suggests exploring their use for the treatment of AKI patients, as well as patients with other ROS-related diseases.

Determination of binding affinity of molecular imaging agents for steroid hormone receptors in breast cancer.

16α-[18F]Fluoro-17ß-estradiol ([18F]FES) and 21-[18F]-Fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxyl]-19-norpregn-4-ene-3,20-dione ([18F]FFNP) are being investigated as imaging biomarkers for breast cancer patients. Quantitative positron emission tomography (PET) reflects both total receptor content and binding affinity. To study factors that may alter radiopharmaceutical binding and impact PET accuracy, assays that can separate receptor amount from binding affinity are needed. The study purpose was to quantify the binding parameters of [18F]FES and [18F]FFNP in breast cancer. Estrogen receptor-alpha (ER) and progesterone receptor (PR) positive breast cancer cell lines (MCF-7 and T47D) were used to measure [18F]FES and [18F]FFNP binding parameters via saturation and competitive binding curves. The equilibrium dissociation constant (Kd) and total receptor density (Bmax) were determined using nonlinear regression of the saturation binding curves. Half-maximal inhibitory concentration (IC50) was determined using nonlinear regression of the competitive binding curves. Linear correlation between increasing cell number and tracer uptake was observed for both [18F]FES and [18F]FFNP (R2=0.99 and 0.91, respectively). Using [18F]FES, the Kd for ER in MCF-7 cells was 0.13±0.02 nM with a Bmax of 1901±89.3 fmol/mg protein and IC50 of 0.085 nM (95% CI: 0.069-0.104 nM). Using [18F]FFNP, the Kd for PR in T47D cells was 0.41±0.05 nM with a Bmax of 1984±75.6 fmol/mg protein and IC50 of 2.6 nM (95% CI: 2.0-3.4 nM). The ligand binding function of ER and PR can be quantified using [18F]FES and [18F]FFNP and are comparable to previous studies using tritiated radioligands. [18F]FES and [18F]FFNP can be used in cell-based assays to quantify receptor-radioligand binding affinity, which cannot be obtained from a single PET examination.

18F-16α-17ß-Fluoroestradiol Binding Specificity in Estrogen Receptor-Positive Breast Cancer.

Purpose To determine the binding specificity of 18F-16α-17ß-fluoroestradiol (FES) in estrogen receptor (ER) α-positive breast cancer cells and tumor xenografts. Materials and Methods Protocols were approved by the office of biologic safety and institutional animal care and use committee. By using ER-negative MDA-MB-231 breast cancer cells, clonal lines were created that expressed either wild-type (WT; 231 WT ER) or G521R mutant ERα (231 G521R ER), which is defective in estradiol binding. ERα protein levels, subcellular localization, and transcriptional function were confirmed. FES binding was measured by using an in vitro cell uptake assay. In vivo FES uptake was measured in tumor xenografts by using small-animal positron emission tomographic/computed tomographic imaging of 24 mice (17 WT ER tumors, nine mutant G521R ER tumors, eight MDA-MB-231 tumors, and four MCF-7 ER-positive tumors). Statistical significance was determined by using Mann-Whitney (Wilcoxon rank sum) test. Results ERα transcriptional function was abolished in the mutated 231 G521R ER cells despite appropriate receptor protein expression and nuclear localization. In vitro FES binding in the 231 G521R ER cells was reduced to that observed in the parental cells. Similarly, there was no significant FES uptake in the 231 G521R ER xenografts (percent injected dose [ID] per gram, 0.49 ± 0.042), which was similar to the negative control MDA-MB-231 xenografts (percent ID per gram, 0.42 ± 0.051; P = .20) and nonspecific muscle uptake (percent ID per gram, 0.41 ± 0.0095; P = .06). Conclusion This study showed that FES retention in ER-positive breast cancer is strictly dependent on an intact receptor ligand-binding pocket and that FES binds to ERα with high specificity. These results support the utility of FES imaging for assessing tumor heterogeneity by localizing immunohistochemically ER-positive metastases that lack receptor-binding functionality. © RSNA, 2017 Online supplemental material is available for this article.

Sex as a Biologic Variable in Preclinical Imaging Research: Initial Observations with 18F-FLT.

The study objective was to investigate whether sex influences 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) uptake and tissue distribution in mouse models of cancer. Methods:18F-FLT biodistribution was measured in 3 strains of male and female mice (129S6/SvEv, athymic nude, and BALB/c). 18F-FDG biodistribution was measured for comparison. 18F-FLT uptake was also measured in female 129S6/SvEv mice bearing estrogen-dependent SSM3 mouse mammary tumors, male athymic nude mice bearing androgen-dependent CWR22 prostate cancer xenografts, and male and female athymic nude mice bearing estrogen-independent MDA-MB-231 human breast cancer xenografts. Ki-67 expression was assayed by immunohistochemistry. PET/CT imaging was performed to visualize 18F-FLT biodistribution and to determine pharmacokinetics. Results: Greater 18F-FLT activity was observed in blood, liver, muscle, heart, kidney, and bone in female than male mice. Pharmacokinetic analysis demonstrated higher early renal 18F-FLT activity and greater accumulation of 18F-FLT in the urinary bladder in male than female mice. The differential pattern of 18F-FLT biodistribution between the sexes seen with 18F-FLT was not observed with 18F-FDG. Increased tumoral 18F-FLT uptake compared with muscle was observed in both the SSM3 mammary tumors (2.4 ± 0.17 vs. 1.6 ± 0.14 percentage injected dose [%ID]/g at 2 h after injection, P = 0.006) and the CWR22 prostate cancer xenografts (0.34 ± 0.08 vs. 0.098 ± 0.033 %ID/g at 2 h after injection, P = 0.03). However, because of higher nonspecific muscle uptake in female mice, tumor-to-muscle uptake ratios were greater for CWR22 tumors than for SSM3 tumors (4.2 ± 0.78 vs. 1.5 ± 0.049 at 2 h after injection, P = 0.008). Sex-dependent differences in 18F-FLT uptake were also observed for MDA-MB-231 xenografts (tumor-to-muscle ratio, 7.2 ± 0.9 for female vs. 16.9 ± 8.6 for male, P = 0.039). Conversely, greater tumoral Ki-67 staining was observed in female mice (71% ± 3% for female vs. 54% ± 2% for male, P = 0.009), and this finding more closely matched the relative differences in absolute 18F-FLT tumor uptake values (4.5 ± 0.99 %ID/g for female vs. 1.9 ± 0.30 %ID/g for male, P = 0.03). Conclusion: Depending on whether female or male mice are used, differences in biodistribution and nonspecific tissue uptake can adversely affect quantitative measures of 18F-FLT uptake. Thus, sex is a potential variable to consider in defining quantitative imaging metrics using 18F-FLT to assess tumor proliferation.

Hydrogen sulfide ameliorates chronic renal failure in rats by inhibiting apoptosis and inflammation through ROS/MAPK and NF-κB signaling pathways.

Chronic renal failure (CRF) is a major public health problem worldwide. Hydrogen sulfide (H2S) plays important roles in renal physiological and pathophysiological processes. However, whether H2S could protect against CRF in rats remains unclear. In this study, we found that H2S alleviated gentamicin-induced nephrotoxicity by reducing reactive oxygen species (ROS)-mediated apoptosis in normal rat kidney-52E cells. We demonstrated that H2S significantly improved the kidney structure and function of CRF rats. We found that H2S decreased the protein levels of Bax, Caspase-3, and Cleaved-caspase-3, but increased the expression of Bcl-2. Treatment with H2S reduced the levels of malondialdehyde and ROS and increased the activities of superoxide dismutase and glutathione peroxidase. H2S significantly abolished the phosphorylation of extracellular signal-regulated protein kinase 1/2, c-Jun N-terminal kinase, and p38 in the kidney of CRF rats. Furthermore, H2S decreased the expression levels of tumor necrosis factor-α, interleukin (IL)-6, IL-10, and monocyte chemoattractant protein-1, as well as the protein levels of p50, p65, and p-p65 in the kidney of CRF rats. In conclusion, H2S could ameliorate adenine-induced CRF in rats by inhibiting apoptosis and inflammation through ROS/mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways.

Epidermal growth factor promotes mesenchymal stem cell-mediated wound healing and hair follicle regeneration.

BACKGROUND AND AIM: Bone marrow mesenchymal stem cells (MSC) are receiving increasing attention for skin wound repair. However, the specific mechanisms underlying MSC-mediated improvement in wound healing have not been fully elucidated. This study aims at testing whether epidermal growth factor (EGF) can promote MSC-mediated wound healing and hair follicle regeneration. METHODS: Excisional wounds in rats were transplanted with different collagen-chitosan scaffolds: control, MSC, and MSC + EGF. Regenerated tissues were harvested 1, 3, or 5 weeks following transplantation, stained with hematoxylin and eosin and evaluated microscopically. The formation of sebaceous glands was examined by Oil red staining and the regeneration of hair follicles by immunohistochemical staining and Western blot to test the expression of hair follicle-specific factors. RESULTS: Gross observations showed that the wounds were much smaller and the hairs grew faster in the MSC + EGF group. Histological analysis demonstrated that there were more hair follicles, sebaceous glands, and newly formed blood vessels in the MSC + EGF group compared with that in the MSC group. In addition, oil red staining showed that MSCs + EGF induced sebaceous gland regeneration. Finally, immunohistochemistry and western blot revealed that MSCs + EGF increased the expression of hair follicle-specific factors. CONCLUSION: MSCs alone cannot achieve the regeneration of hair follicles and EGF can promote MSC-mediated wound healing and hair follicle regeneration.

An Investigation on a Novel Anti-tumor Fusion Peptide of FSH33-53-IIKK.

A novel fusion peptide FSH33-53-IIKK was designed and expected to combine the follicle stimulating hormone receptor (FSHR) targeting and tumor toxicity. In vitro and in vivo study showed the anti-tumor activity of FSH33-53-IIKK was enhanced compared to that of IIKK only. FSH33-53-IIKK could inhibit the growth of tumor via apoptosis and autophagy pathways. In summary, combining the tumor marker-target peptide and anti-tumor peptide together may be an efficient way to search for better anti-tumor candidates.

PET of follicle-stimulating hormone receptor: broad applicability to cancer imaging.

Selective overexpression of follicle-stimulating hormone receptor (FSHR) inside the vascular endothelium of tumors has been confirmed to play critical roles in angiogenesis, tumor invasion, and metastases. The expression level of FSHR correlates strongly with the response of tumors to antiangiogenic therapies. In this study, an immunoPET tracer was developed for imaging of FSHR in different cancer types. A monoclonal antibody (FSHR-mAb) against FSHR was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and used for subsequent (64)Cu-labeling. NOTA-FSHR-mAb preserved FSHR specificity/affinity, confirmed by flow cytometry measurements. (64)Cu-labeling was successfully conducted with decent yields (∼25%) and high specific activity (0.93 GBq/mg). The uptake of (64)Cu-NOTA-FSHR-mAb was 3.6 ± 0.8, 13.2 ± 0.7, and 14.6 ± 0.4 %ID/g in FSHR-positive CAOV-3 tumors at 4, 24, and 48 h postinjection, respectively (n = 3), significantly higher (p < 0.05) than that in FSHR-negative SKOV-3 tumors (2.3 ± 1.2, 8.0 ± 0.9, and 9.1 ± 1.3 %ID/g at 4, 24, and 48 h postinjection, respectively (n = 3)) except at 4 h p.i. FSHR-relevant uptake of (64)Cu-NOTA-FSHR-mAb was also readily observed in other tumor types (e.g., triple-negative breast tumor MDA-MB-231 or prostate tumor PC-3). Histology studies showed universal FSHR expression in microvasculature of these four tumor types and also prominent expression in tumor cells of CAOV-3, PC-3, and MDA-MB-231. Correlations between tumor FSHR level and uptake of (64)Cu-NOTA-FSHR-mAb were witnessed in this study. FSHR-specific uptake of (64)Cu-NOTA-FSHR mAb in different tumors enables its applicability for future cancer theranostic applications and simultaneously establishes FSHR as a promising clinical target for cancer.

PET imaging of prostate tumors with 18F-Al-NOTA-MATBBN.

Overexpression of the gastrin-releasing peptide receptor (GRPR) in prostate cancer provides a promising target for detection the disease. MATBBN is a new bombesin analog originating from the GRPR antagonists with a hydrophilic linker. In this study NOTA-conjugated MATBBN was labeled by the Al(18)F method and the potential of (18)F-Al-NOTA-MATBBN for prostate tumor PET imaging was also evaluated. NOTA-MATBBN was radiolabeled with (18) F using Al(18)F complexes. Partition coefficient, in vitro stability and GRPR binding affinity were also determined. PET studies were performed with (18)F-Al-NOTA-MATBBN in PC-3 tumor-bearing mice. (18)F-Al-NOTA-MATBBN can be produced within 30 min with a decay-corrected yield of 62.5 ± 2.1% and a radiochemical purity of >98%. The logP octanol-water value for the Al(18)F-labeled BBN analog was -2.40 ± 0.07 and the radiotracer was stable in phosphate-buffered saline and human serum for 2 h. The IC50 values of displacement for the (18)F-Al-NOTA-MATBBN with MATBBN was 126.9 ± 2.75 nm. The PC-3 tumors were clearly visible with high contrast after injection of the labeled peptide. At 60 min post-injection, the tumor uptakes for (18)F-Al-NOTA-MATBBN and (18)F-FDG were 4.59 ± 0.43 and 1.98 ± 0.35% injected dose/g, and tumor to muscle uptake radios for two tracers were 6.77 ± 1.10 and 1.78 ± 0.32, respectively. Dynamic PET revealed that (18) F-Al-NOTA-MATBBN was excreted mainly through the kidneys. GRPR-binding specificity was also demonstrated by reduced tumor uptake of (18)F-Al-NOTA-MATBBN after coinjection with excess unlabeled MATBBN peptide at 1 h post-injection. NOTA- MATBBN could be labeled rapidly with (18)F using one step method. (18)F-Al-NOTA-MATBBN may be a promising PET imaging agent for prostate cancer.

A new (68)Ga-labeled BBN peptide with a hydrophilic linker for GRPR-targeted tumor imaging.

Bombesin (BBN) is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPR), which is overexpressed on several types of cancers. Various GRPR antagonists and agonists have been labeled with radiometals for positron emission tomography (PET) imaging of GRPR-positive tumors. However, unfavorable hepatobiliary excretion such as high intestinal activity may prohibit their clinical utility for imaging abdominal cancer. In this study, the modified BBN peptide with a new hydrophilic linker was labeled with (68)Ga for PET imaging of GRPR-expressing PC-3 prostate cancer xenograft model. GRPR antagonists, MATBBN (Gly-Gly-Gly-Arg-Asp-Asn-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH2CH3) and ATBBN (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH2CH3), were conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with (68)Ga. Partition coefficient and in vitro stability were also determined. GRPR binding affinity of both tracers was investigated by competitive radioligand binding assay. The in vivo receptor targeting potential and pharmacokinetic of (68)Ga-NOTA-MATBBN were also evaluated in PC-3 prostate tumor model and compared with those of (68)Ga-NOTA-ATBBN. NOTA-conjugated BBN analogs were labeled with (68)Ga within 20 min with a decay-corrected yield ranging from 90 to 95 % and a radiochemical purity of more than 98 %. The specific activity of (68)Ga-NOTA-MATBBN and (68)Ga-NOTA-ATBBN was at least 16.5 and 11.9 GBq/µmol, respectively. The radiotracers were stable in phosphate-buffered saline and human serum. (68)Ga-NOTA-MATBBN was more hydrophilic than (68)Ga-NOTA-ATBBN, as indicated by their log P values (-2.73 ± 0.02 vs. -1.20 ± 0.03). The IC50 values of NOTA-ATBBN and NOTA-MATBBN were similar (102.7 ± 1.18 and 124.6 ± 1.21 nM). The accumulation of (68)Ga-labeled GRPR antagonists in the subcutaneous PC-3 tumors could be visualized via small animal PET. The tumors were clearly visible, and the tumor uptakes of (68)Ga-NOTA-MATBBN and (68)Ga-NOTA-ATBBN were determined to be 4.19 ± 0.32, 4.00 ± 0.41, 2.93 ± 0.35 and 4.70 ± 0.40, 4.10 ± 0.30, 3.14 ± 0.30 %ID/g at 30, 60, and 120 min, respectively. There was considerable accumulation and retention of (68)Ga-NOTA-ATBBN in the liver and intestines. In contrast, the abdominal area does not have much retention of (68)Ga-NOTA-MATBBN. Biodistribution data were in accordance with the PET results, showing that (68)Ga-NOTA-MATBBN had more favorable pharmacokinetics and higher tumor to background ratios than those of (68)Ga-NOTA-ATBBN. At 1 h postinjection, the tumor to liver and intestine of (68)Ga-NOTA-MATBBN were 8.05 ± 0.56 and 21.72 ± 3.47 and the corresponding values of unmodified counterpart were 0.85 ± 0.23 and 3.45 ± 0.43, respectively. GRPR binding specificity was demonstrated by reduced tumor uptake of radiolabeled tracers after coinjection of an excess of unlabeled BBN peptides. (68)Ga-NOTA-MATBBN exhibited GRPR-targeting properties both in vitro and in vivo. The favorable characterizations of (68)Ga-NOTA-MATBBN such as convenient synthesis, specific GRPR targeting, high tumor uptake, and satisfactory pharmacokinetics warrant its further investigation for clinical cancer imaging.

Pilot study of a novel (18)F-labeled FSHR probe for tumor imaging.

PURPOSE: Follicle-stimulating hormone receptor (FSHR) is overexpressed in primary and metastatic tumor. Molecular imaging of FSHR is beneficial for prognosis and therapy of cancer. FSHß(33-53) (YTRDLVYKDPARPKIQKTCTF), denoted as FSH1, is a FSHR antagonist. In the present study, maleimide-NOTA conjugate of FSH1 (NOTA-MAL-FSH1) was designed and labeled with [(18)F] aluminum fluoride. The resulting tracer, (18)F-Al-NOTA-MAL-FSH1, was preliminarily evaluated in PET imaging of FSHR-positive tumor. PROCEDURES: NOTA-MAL-FSH1 was synthesized and radiolabeled with Al(18)F complex. The tumor-targeting potential and pharmacokinetic profile of the (18)F-labeled compound were evaluated in vitro and in vivo using a PC3 human prostate tumor model. RESULTS: (18)F-Al-NOTA-MAL-FSH1 can be efficiently produced within 30 min with a non-decay-corrected yield of 48.6 ± 2.1 % and a radiochemical purity of more than 95 %. The specific activity was at least 30 GBq/µmol. The radiotracer was stable in phosphate-buffered saline and human serum for at least 2 h. The IC50 values of displacement (18)F-Al-NOTA-MAL-FSH1 with FSH1 were 252 ± 1.12 nM. The PC3 human prostate tumor xenografts were clearly visible with high contrast after injection of (18)F-Al-NOTA-MAL-FSH1 via microPET. At 30, 60 and 120 min postinjection, the tumor uptakes were 2.98 ± 0.29 % injected dose (ID)/g, 2.53 ± 0.20 %ID/g and 1.36 ± 0.12 %ID/g, respectively. Dynamic PET scanning showed that tumor uptake reached a plateau by about 6 min. Heart peaked earlier and then cleared quickly. Biodistribution studies confirmed that the normal organs except kidney uptakes were all below 1 %ID/g at 1 h p.i. The tumor-to-blood and tumor-to-muscle ratio at 10 min, 0.5, 1, and 2 h after injection were 1.64 ± 0.36, 2.97 ± 0.40, 9.31 ± 1.06, and 13.59 ± 2.33 and 7.05 ± 1.10, 10.10 ± 1.48, 16.17 ± 3.29, and 30.88 ± 4.67, respectively. The tracer was excreted mainly through the renal system, as evidenced by high levels of radioactivity in the kidneys. FSHR-binding specificity was also demonstrated by reduced tumor uptake of (18)F-Al-NOTA-MAL-FSH1 after coinjection with an excess of unlabeled FSH1 peptide. CONCLUSION: NOTA-MAL-FSH1 could be labeled rapidly and efficiently with (18)F using one step method. Favorable preclinical data suggest that (18)F-Al-NOTA-MAL-FSH1 may be a suitable radiotracer for the non-invasive visualization of FSHR positive tumor in vivo.

Application of (99m)Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7-14) SPECT/CT in prostate cancer patients: a first-in-man study.

RATIONALE: The peptide bombesin (BBN) and its derivatives exhibit high binding affinity for the gastrin-releasing peptide receptor (GRPR), which is highly expressed in prostate cancer. We used the BBN-based radiopharmaceutical (99m)Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7-14) ((99m)Tc-HABBN) to perform a first-in-man clinical pilot study to evaluate the feasibility of (99m)Tc-HABBN SPECT/CT for detection of prostate cancer in patients. METHODS: Eight patients with biopsy-proven prostate cancer who were scheduled for either radical prostatectomy or external beam radiotherapy underwent (99m)Tc-HABBN scintigraphy and SPECT/CT prior to treatment. Serial blood samples were taken to assess blood radioactivity and to determine in vivo metabolic stability. Clinical parameters were measured and reported side effects, if present, were recorded. Prostate cancer specimens of all patients were immunohistochemically stained for GRPR. RESULTS: (99m)Tc-HABBN was synthesized with high radiochemical yield, purity and specific activity. There were no significant changes in clinical parameters, and there were no adverse or subjective side effects. Low metabolic stability was observed, as less than 20% of (99m)Tc-HABBN was intact after 30 min. Immunohistochemical staining for GRPR was observed in the prostate cancer specimens in all patients. (99m)Tc-HABBN scintigraphy and SPECT/CT did not detect prostate cancer in patients with proven disease. CONCLUSIONS: (99m)Tc-HABBN SPECT/CT for visualization of prostate cancer is safe but hampered by an unexpected low in vivo metabolic stability in man. The difference between the excellent in vitro stability of (99m)Tc-HABBN in human serum samples determined in our previous study regarding (99m)Tc-HABBN and the low in vivo metabolic stability determined in this study, is striking. This issue warrants further study of peptide-based radiopharmaceuticals.