Proteome-wide profiling and mapping of post translational modifications in human hearts.

Post translational modifications (PTMs) are covalent modifications of proteins that can range from small chemical modifications to addition of entire proteins. PTMs contribute to regulation of protein function and thereby greatly increase the functional diversity of the proteome. In the heart, a few well-studied PTMs, such as phosphorylation and glycosylation, are known to play essential roles for cardiac function. Yet, only a fraction of the ~ 300 known PTMs have been studied in a cardiac context. Here we investigated the proteome-wide map of PTMs present in human hearts by utilizing high-resolution mass spectrometry measurements and a suite of PTM identification algorithms. Our approach led to identification of more than 150 different PTMs across three of the chambers in human hearts. This finding underscores that decoration of cardiac proteins by PTMs is much more diverse than hitherto appreciated and provides insights in cardiac protein PTMs not yet studied. The results presented serve as a catalogue of which PTMs are present in human hearts and outlines the particular protein and the specific amino acid modified, and thereby provides a detail-rich resource for exploring protein modifications in human hearts beyond the most studied PTMs.

Diffusion weighted magnetic resonance imaging (DW-MRI) as a non-invasive, tissue cellularity marker to monitor cancer treatment response.

BACKGROUND: Diffusion weighted magnetic resonance imaging (DW-MRI) holds great potential for monitoring treatment response in cancer patients shortly after initiation of radiotherapy. It is hypothesized that a decrease in cellular density of irradiated cancerous tissue will lead to an increase in quantitative apparent diffusion coefficient (ADC) values. DW-MRI can therefore serve as a non-invasive marker of cell death and apoptosis in response to treatment. In the present study, we aimed to investigate the applicability of DW-MRI in preclinical models to monitor radiation-induced treatment response. In addition, we compared DW-MRI with ex vivo measures of cell density, cell death and apoptosis. METHODS: DW-MRI was tested in two different syngeneic mouse models, a colorectal cancer (CT26) and a breast cancer (4 T1). ADC values were compared with quantitative determinations of apoptosis and cell death by flow cytometry. Furthermore, ADC-values were also compared to histological measurement of cell density on tumor sections. RESULTS: We found a significant correlation between ADC-values and apoptotic state in the CT26 model (P = 0.0031). A strong correlation between the two measurements of ADC-value and apoptotic state was found in both models, which were also present when comparing ADC-values to cell densities. CONCLUSIONS: Our findings demonstrate that DW-MRI can be used for non-invasive monitoring of radiation-induced changes in cell state during cancer therapy. ADC values reflect ex vivo cell density and correlates well with apoptotic state, and can hereby be described as a marker for the cell state after therapy and used as a non-invasive response marker.

Injectable iodine-125 labeled tissue marker for radioactive localization of non-palpable breast lesions.

We have developed a 125I-radiolabeled injectable fiducial tissue marker with the potential to replace current methods used for surgical guidance of non-palpable breast tumors. Methods in routine clinical use today such as radioactive seed localization, radio-guided occult lesion localization and wire-guided localization suffers from limitations that this injectable fiducial tissue marker offers solutions to. The developed 125I-radiolabeled injectable fiducial tissue marker is based on highly viscous sucrose acetate isobutyrate. The marker was readily inserted in NMRI mice and proved to be spatially well-defined and stable over a seven day period with excellent CT contrast (>1500 HU), enabling fluoroscopic visualization of the marker during placement. The radioactivity remains strongly associated with the marker during the implantation period, which limits exposure to healthy tissue. Biodistribution studies show that there is negligible radioactivity in all non-tumor tissues sampled, with the exception of the thyroid gland, where limited accumulation was observed (0.06% of injected dose after 7 days). Based on the excellent performance of the marker and the fact that it can be delivered through thin hypodermic needles (≥27G), the marker holds great promise for clinical application, since patient discomfort is reduced significantly compared to current methods. STATEMENT OF SIGNIFICANCE: A new type of tissue marker for local administration to non-palpable breast tumors has been developed. The surgical guidance marker is based on derivatives of the biomaterial sucrose acetate isobutyrate and unlike currently used markers it is injectable in the tissue using thin needles, reducing the discomfort to the patients significantly. The marker confers CT contrast and has radioactive properties, meaning it also could find use in brachytherapy. The design of the iodine-125 labeled fiducial tissue marker enables control of dosimetry as well as a choice of iodine isotope used. The marker is anticipated to be clinical applicable due to its contrast performance in mice and its potential for enhanced flexibility in surgical procedures, compared to current methods.

Uremia does not affect neointima formation in mice.

Atherosclerotic cardiovascular disease is a major complication of chronic kidney disease (CKD). CKD leads to uremia, which modulates the phenotype of aortic smooth muscle cells (SMCs). Phenotypic modulation of SMCs plays a key role in accelerating atherosclerosis. We investigated the hypothesis that uremia potentiates neointima formation in response to vascular injury in mice. Carotid wire injury was performed on C57BL/6 wt and apolipoprotein E knockout (Apoe -/-) mice two weeks after induction of uremia by 5/6 nephrectomy. Wire injury led to neointima formation and downregulation of genes encoding classical SMC markers (i.e., myocardin, α-smooth muscle actin, SM22-alpha, and smooth muscle myosin heavy chain) in both wt and Apoe -/- mice. Contrary to our expectations, uremia did not potentiate neointima formation, nor did it affect intimal lesion composition as judged from magnetic resonance imaging and histological analyses. Also, there was no effect of uremia on SMC marker gene expression in the injured carotid arteries, suggesting that there may be different effects of uremia on SMCs in different vascular beds. In conclusion, uremia does not accelerate neointima formation in response to wire injury of the carotid artery in mice.

T-cells and macrophages peak weeks after experimental stroke: Spatial and temporal characteristics.

The activities of the central and peripheral immune systems impact neurological outcome after ischemic stroke. However, studies investigating the temporal profile of leukocyte infiltration, especially T-cell recruitment, are sparse. Our aim was to investigate leukocyte infiltration at different time points after experimental stroke in mice. Permanent middle cerebral artery occlusion was performed on 11 weeks old C57BL/6J mice, allowed to survive for 1, 3, 8, 14 or 28 days. In addition to infarct size measurements, detailed immunohistochemical analyses of T-cell and macrophage influx were performed. A recently introduced F-19 MR probe (V-sense), designed to track macrophages, was furthermore tested. Fourteen and 28 days after permanent middle cerebral artery occlusion a significant increase in CD3+ T-cells was found within the ipsilateral hemisphere compared to controls, especially within the infarct core and the corpus callosum. The number of CD68+ cells within the infarct core was significantly increased at days 8, 14 and 28. This temporal pattern was also seen in MRI. After experimental stroke within the infarcted cortex we found a delayed (day 14) infiltration of T-cells and macrophages. Furthermore, our data show that T-cells are present in higher numbers in the corpus callosum compared to the rest of the brain (except from the infarct core where they were highest).

PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-Labeled Active Site-Inhibited Factor VII.

UNLABELLED: Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site-inhibited FVII (FVIIai) labeled with (64)Cu for PET imaging of TF expression. METHODS: FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu ((64)Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of (64)Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with (64)Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using (64)Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. RESULTS: Longitudinal PET imaging with (64)Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to-normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of (64)Cu-NOTA-FVIIai was colocalized with viable tumor tissue. CONCLUSION: (64)Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.

Micro Regional Heterogeneity of 64Cu-ATSM and 18F-FDG Uptake in Canine Soft Tissue Sarcomas: Relation to Cell Proliferation, Hypoxia and Glycolysis.

OBJECTIVES: Tumour microenvironment heterogeneity is believed to play a key role in cancer progression and therapy resistance. However, little is known about micro regional distribution of hypoxia, glycolysis and proliferation in spontaneous solid tumours. The overall aim was simultaneous investigation of micro regional heterogeneity of 64Cu-ATSM (hypoxia) and 18F-FDG (glycolysis) uptake and correlation to endogenous markers of hypoxia, glycolysis, proliferation and angiogenesis to better therapeutically target aggressive tumour regions and prognosticate outcome. METHODS: Exploiting the different half-lives of 64Cu-ATSM (13 h) and 18F-FDG (2 h) enabled simultaneous investigation of micro regional distribution of hypoxia and glycolysis in 145 tumour pieces from four spontaneous canine soft tissue sarcomas. Pairwise measurements of radioactivity and gene expression of endogenous markers of hypoxia (HIF-1α, CAIX), glycolysis (HK2, GLUT1 and GLUT3), proliferation (Ki-67) and angiogenesis (VEGFA and TF) were performed. Dual tracer autoradiography was compared with Ki-67 immunohistochemistry. RESULTS: Micro regional heterogeneity in hypoxia and glycolysis within and between tumour sections of each tumour piece was observed. The spatial distribution of 64Cu-ATSM and 18F-FDG was rather similar within each tumour section as reflected in moderate positive significant correlations between the two tracers (ρ = 0.3920-0.7807; p = 0.0180 -<0.0001) based on pixel-to-pixel comparisons of autoradiographies and gamma counting of tumour pieces. 64Cu-ATSM and 18F-FDG correlated positively with gene expression of GLUT1 and GLUT3, but negatively with HIF-1α and CAIX. Significant positive correlations were seen between Ki-67 gene expression and 64Cu-ATSM (ρ = 0.5578, p = 0.0004) and 18F-FDG (ρ = 0.4629-0.7001, p = 0.0001-0.0151). Ki-67 gene expression more consistently correlated with 18F-FDG than with 64Cu-ATSM. CONCLUSIONS: Micro regional heterogeneity of hypoxia and glycolysis was documented in spontaneous canine soft tissue sarcomas. 64Cu-ATSM and 18F-FDG uptakes and distributions showed significant moderate correlations at the micro regional level indicating overlapping, yet different information from the tracers.18F-FDG better reflected cell proliferation as measured by Ki-67 gene expression than 64Cu-ATSM.

Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging.

UNLABELLED: (64)Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of (64)Cu-DOTA-AE105. METHODS: Five mice received iv tail injection of (64)Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22 h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another (64)Cu-DOTA peptide-based tracer, (64)Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using (64)Cu-DOTA-TATE. RESULTS: Human estimates of (64)Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02-0.04 mSv/MBq. The mean effective whole-body dose of (64)Cu-DOTA-AE105 was estimated to be 0.0317 mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for (64)Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high precision (predicted value: 0.0252 mSv/Mbq, Observed value: 0.0315 mSv/MBq) thus validating our approach for human dosimetry estimation. CONCLUSION: Favorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of (64)Cu-DOTA-AE105.

Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and (64)Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors.

INTRODUCTION: The aim of this study was to synthesize and perform a side-by-side comparison of two new tumor-angiogenesis PET tracers (68)Ga-NODAGA-E[c(RGDyK)](2) and (64)Cu-NODAGA-E[c(RGDyK)](2) in vivo using human xenograft tumors in mice. Human radiation burden was estimated to evaluate potential for future use as clinical PET tracers for imaging of neo-angiogenesis. METHODS: A (68)Ge/(68)Ga generator was used for the synthesis of (68)Ga-NODAGA-E[c(RGDyK)](2). (68)Ga and (64)Cu labeled NODAGA-E[c(RGDyK)](2) tracers were administrated in nude mice bearing either human glioblastoma (U87MG) or human neuroendocrine (H727) xenograft tumors. PET/CT scans at 3 time points were used for calculating the tracer uptake in tumors (%ID/g), integrin αVß3 target specificity was shown by blocking with cold NODAGA-E[c(RGDyK)](2), and biodistribution in normal organs were also examined. From biodistribution data in mice human radiation-absorbed doses were estimated using OLINDA/EXM software. RESULTS: (68)Ga-NODAGA-E[c(RGDyK)](2) was synthesized with a radiochemical purity of 89%-99% and a specific activity (SA) of 16-153 MBq/nmol. (64)Cu-NODAGA-E[c(RGDyK)](2) had a purity of 92%-99% and an SA of 64-78 MBq/nmol. Both tracers showed similar uptake in xenograft tumors 1h after injection (U87MG: 2.23 vs. 2.31%ID/g; H727: 1.53 vs. 1.48%ID/g). Both RGD dimers showed similar tracer uptake in non-tumoral tissues and a human radiation burden of less than 10 mSv with an administered dose of 200 MBq was estimated. CONCLUSION: (68)Ga-NODAGA-E[c(RGDyK)](2) and (64)Cu-NODAGA-E[c(RGDyK)](2) can be easily synthesized and are both promising candidates for PET imaging of integrin αVß3 positive tumor cells. (68)Ga-NODAGA-E[c(RGDyK)](2) showed slightly more stable tumor retention. With the advantage of in-house commercially (68)Ge/(68)Ga generators, (68)Ga-NODAGA-E[c(RGDyK)](2) may be the best choice for future clinical PET imaging in humans.

(64)Cu-NODAGA-c(RGDyK) Is a Promising New Angiogenesis PET Tracer: Correlation between Tumor Uptake and Integrin α(V)ß(3) Expression in Human Neuroendocrine Tumor Xenografts.

Purpose. The purpose of this paper is to evaluate a new PET tracer (64)Cu-NODAGA-c(RGDyK) for imaging of tumor angiogenesis using gene expression of angiogenesis markers as reference and to estimate radiation dosimetry for humans. Procedures. Nude mice with human neuroendocrine tumor xenografts (H727) were administered (64)Cu-NODAGA-c(RGDyK) i.v. for study of biodistribution as well as for dynamic PET. Gene expression of angiogenesis markers integrin α(V), integrin ß(3), and VEGF-A were analyzed using QPCR and correlated to the tracer uptake in the tumors (%ID/g). From biodistribution data human radiation-absorbed doses were estimated using OLINDA/EXM. Results. Tumor uptake was 1.2%ID/g with strong correlations between gene expression and tracer uptake, for integrin α(V) R = 0.76, integrin ß(3) R = 0.75 and VEGF-A R = 0.81 (all P < 0.05). The whole body effective dose for humans was estimated to be 0.038 and 0.029 mSv/MBq for females and males, respectively, with highest absorbed dose in bladder wall. Conclusion. (64)Cu-NODAGA-c(RGDyK) is a promising new angiogenesis PET tracer with potential for human use.

The Combination of In vivo (124)I-PET and CT Small Animal Imaging for Evaluation of Thyroid Physiology and Dosimetry.

OBJECTIVE: A thyroid rat model combining functional and anatomical information would be of great benefit for better modeling of thyroid physiology and for absorbed dose calculations. Our aim was to show that (124)I-PET and CT small animal imaging are useful as a combined model for studying thyroid physiology and dose calculation. METHODS: Seven rats were subjects for multiple thyroid (124)I-imaging and CT-scans. S-values [mGy/MBqs] for different thyroid sizes were simulated. A phantom with spheres was designed for validation of performances of the small animal PET and CT imaging systems. RESULTS: Small animal image-based measurements of the activity amount and the volumes of the spheres with a priori known volumes showed a good agreement with their corresponding actual volumes. The CT scans of the rats showed thyroid volumes from 34-70 mL. CONCLUSIONS: The wide span in volumes of thyroid glands indicates the importance of using an accurate volume-measuring technique such as the small animal CT. The small animal PET system was on the other hand able to accurately estimate the activity concentration in the thyroid volumes. We conclude that the combination of the PET and CT image information is essential for quantitative thyroid imaging and accurate thyroid absorbed dose calculation.

Importance of Attenuation Correction (AC) for Small Animal PET Imaging.

UNLABELLED: The purpose of this study was to investigate whether a correction for annihilation photon attenuation in small objects such as mice is necessary. The attenuation recovery for specific organs and subcutaneous tumors was investigated. A comparison between different attenuation correction methods was performed. METHODS: Ten NMRI nude mice with subcutaneous implantation of human breast cancer cells (MCF-7) were scanned consecutively in small animal PET and CT scanners (MicroPET(TM) Focus 120 and ImTek's MicroCAT(TM) II). CT-based AC, PET-based AC and uniform AC methods were compared. RESULTS: The activity concentration in the same organ with and without AC revealed an overall attenuation recovery of 9-21% for MAP reconstructed images, i.e., SUV without AC could underestimate the true activity at this level. For subcutaneous tumors, the attenuation was 13 ± 4% (9-17%), for kidneys 20 ± 1% (19-21%), and for bladder 18 ± 3% (15-21%). The FBP reconstructed images showed almost the same attenuation levels as the MAP reconstructed images for all organs. CONCLUSIONS: The annihilation photons are suffering attenuation even in small subjects. Both PET-based and CT-based are adequate as AC methods. The amplitude of the AC recovery could be overestimated using the uniform map. Therefore, application of a global attenuation factor on PET data might not be accurate for attenuation correction.