Carsten H. Nielsen

Matrix-insensitive protein assays push the limits of biosensors in medicine

Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.

Targeted microbubbles for imaging tumor angiogenesis: assessment of whole-body biodistribution with dynamic micro-PET in mice.

PURPOSE To evaluate in vivo whole-body biodistribution of microbubbles (MBs) targeted to tumor angiogenesis-related vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by using dynamic micro-positron emission tomography (PET) in living mice. MATERIALS AND METHODS Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Lipid-shell perfluorocarbon-filled MBs, targeted to VEGFR2 via anti-VEGFR2 antibodies, were radiolabeled by conjugating the radiofluorination agent N-succinimidyl-4-[(18)F]fluorobenzoate (SFB) to the anti-VEGFR2 antibodies. These MBs were then injected intravenously into nude mice (n = 4) bearing angiosarcomas, and the whole-body biodistribution of these probes was assessed for 60 minutes by using dynamic micro-PET. Results were compared with ex vivo gamma counting (n = 6) and immunofluorescence staining (n = 6). Control studies in angiosarcoma-bearing mice were performed with injection of the radiolabeled antibodies alone (n = 3) or free SFB (n = 3). A mixed-effects regression of MB accumulation on fixed effects of time and tissue type (tumor or muscle) and random effect of animal was performed. RESULTS VEGFR2-targeted MBs rapidly cleared from the blood circulation (50% blood clearance after approximately 3.5 minutes) and accumulated in the liver (mean, 33.4% injected dose [ID]/g +/- 13.7 [standard deviation] at 60 minutes) and spleen (mean, 9.3% ID/g +/- 6.5 at 60 minutes) on the basis of micro-PET imaging. These findings were confirmed with ex vivo gamma counting. Uptake of targeted MBs was significantly higher (P < .0001) in tumor than in adjacent skeletal muscle tissue. Immunofluorescence staining demonstrated accumulation of the targeted MBs within hepatic Kupffer cells and splenic macrophages. Biodistribution of the radiolabeled antibodies and free SFB differed from the distribution of the targeted MBs. CONCLUSION Dynamic micro-PET allows assessment of in vivo biodistribution of VEGFR2-targeted MBs.

Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

Preclinical Evaluation of Raman Nanoparticle Biodistribution for their Potential Use in Clinical Endoscopy Imaging

Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Ramans limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with (64) Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (μCi) (3.7 megabecquerel (MBq)) of (64) Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g(-1)) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g(-1); 24 h = 8.27% ID g(-1)) than IR-injected mice (5 h = 0.09% ID g(-1); 24 h = 0.08% ID g(-1)). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g(-1); 24 h = 0.42% ID g(-1)) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool.

New positron emission tomography (PET) radioligand for imaging σ-1 receptors in living subjects.

σ-1 receptor (S1R) radioligands have the potential to detect and monitor various neurological diseases. Herein we report the synthesis, radiofluorination, and evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([(18)F]FTC-146, [(18)F]13). [(18)F]13 was synthesized by nucleophilic fluorination, affording a product with >99% radiochemical purity (RCP) and specific activity (SA) of 2.6 ± 1.2 Ci/μmol (n = 13) at end of synthesis (EOS). Positron emission tomography (PET) and ex vivo autoradiography studies of [(18)F]13 in mice showed high uptake of the radioligand in S1R rich regions of the brain. Pretreatment with 1 mg/kg haloperidol (2), nonradioactive 13, or BD1047 (18) reduced the binding of [(18)F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [(18)F]13 accumulation in mouse brain represents specific binding to S1Rs. These results indicate that [(18)F]13 is a promising candidate radiotracer for further evaluation as a tool for studying S1Rs in living subjects.

Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vectors ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis

Objective—Atherosclerotic lesions contain hypoxic areas, but the pathophysiological importance of hypoxia is unknown. Hypoxia-inducible factor-1&agr; (HIF-1&agr;) is a key transcription factor in cellular responses to hypoxia. We investigated the hypothesis that HIF-1&agr; has effects on macrophage biology that promotes atherogenesis in mice. Approach and Results—Studies with molecular probes, immunostaining, and laser microdissection of aortas revealed abundant hypoxic, HIF-1&agr;–expressing macrophages in murine atherosclerotic lesions. To investigate the significance of macrophage HIF-1&agr;, Ldlr−/− mice were transplanted with bone marrow from mice with HIF-1&agr; deficiency in the myeloid cells or control bone marrow. The HIF-1&agr; deficiency in myeloid cells reduced atherosclerosis in aorta of the Ldlr−/− recipient mice by ≈72% (P=0.006).In vitro, HIF-1&agr;–deficient macrophages displayed decreased differentiation to proinflammatory M1 macrophages and reduced expression of inflammatory genes. HIF-1&agr; deficiency also affected glucose uptake, apoptosis, and migratory abilities of the macrophages. Conclusions—HIF-1&agr; expression in macrophages affects their intrinsic inflammatory profile and promotes development of atherosclerosis.

First-in-human uPAR PET: Imaging of Cancer Aggressiveness

A first-in-human clinical trial with Positron Emission Tomography (PET) imaging of the urokinase-type plasminogen activator receptor (uPAR) in patients with breast, prostate and bladder cancer, is described. uPAR is expressed in many types of human cancers and the expression is predictive of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with 64Cu for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of 64Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment with laboratory blood screening tests was performed before and after PET ligand injection. In a subgroup of the patients, the in vivo stability of our targeted PET ligand was determined in collected blood and urine. No adverse or clinically detectable side effects in any of the 10 patients were found. The ligand exhibited good in vivo stability and fast clearance from plasma and tissue compartments by renal excretion. In addition, high uptake in both primary tumor lesions and lymph node metastases was seen and paralleled high uPAR expression in excised tumor tissue. Overall, this first-in-human study therefore provides promising evidence for safe use of 64Cu-DOTA-AE105 for uPAR PET imaging in cancer patients.

Evaluation of σ-1 Receptor Radioligand 18F-FTC-146 in Rats and Squirrel Monkeys Using PET

The noninvasive imaging of σ-1 receptors (S1Rs) could provide insight into their role in different diseases and lead to novel diagnostic/treatment strategies. The main objective of this study was to assess the S1R radiotracer 18F-FTC-146 in rats. Preliminary squirrel monkey imaging and human serum/liver microsome studies were performed to gain information about the potential of 18F-FTC-146 for eventual clinical translation. Methods: The distribution and stability of 18F-FTC-146 in rats were assessed via PET/CT, autoradiography, γ counting, and high-performance liquid chromatography (HPLC). Preliminary PET/MRI of squirrel monkey brain was conducted along with HPLC assessment of 18F-FTC-146 stability in monkey plasma and human serum. Results: Biodistribution studies showed that 18F-FTC-146 accumulated in S1R-rich rat organs, including the lungs, pancreas, spleen, and brain. Pretreatment with known S1R compounds, haloperidol, or BD1047, before radioligand administration, significantly attenuated 18F-FTC-146 accumulation in all rat brain regions by approximately 85% (P < 0.001), suggesting radiotracer specificity for S1Rs. Similarly, PET/CT and autoradiography results demonstrated accumulation of 18F-FTC-146 in rat brain regions known to contain S1Rs and that this uptake could be blocked by BD1047 pretreatment. Ex vivo analysis of 18F-FTC-146 in the brain showed that only intact radiotracer was present at 15, 30, and 60 min, whereas rapid metabolism of residual 18F-FTC-146 was observed in rat plasma. Preliminary monkey PET/MRI studies demonstrated specific accumulation of 18F-FTC-146 in the brain (mainly in cortical structures, cerebellum, and vermis) that could be attenuated by pretreatment with haloperidol. HPLC of monkey plasma suggested radioligand metabolism, whereas 18F-FTC-146 appeared to be stable in human serum. Finally, liver microsome studies revealed that 18F-FTC-146 has a longer half-life in human microsomes, compared with rodents. Conclusion: Together, these results indicate that 18F-FTC-146 is a promising tool for visualizing S1Rs in preclinical studies and that it has potential for mapping these sites in the human brain.

18F-FDG and 18F-FLT-PET Imaging for Monitoring Everolimus Effect on Tumor-Growth in Neuroendocrine Tumors: Studies in Human Tumor Xenografts in Mice

Introduction The mTOR inhibitor everolimus has shown promising results in some but not all neuroendocrine tumors. Therefore, early assessment of treatment response would be beneficial. In this study, we investigated the in vivo and in vitro treatment effect of everolimus in neuroendocrine tumors and evaluated the performance of 18F-FDG and the proliferation tracer 18F-FLT for treatment response assessment by PET imaging. Methods The effect of everolimus on the human carcinoid cell line H727 was examined in vitro with the MTT assay and in vivo on H727 xenograft tumors. The mice were scanned at baseline with 18F-FDG or 18F-FLT and then treated with either placebo or everolimus (5 mg/kg daily) for 10 days. PET/CT scans were repeated at day 1,3 and 10. Results Everolimus showed significant inhibition of H727 cell proliferation in vitro at concentrations above 1 nM. In vivo tumor volumes measured relative to baseline were significantly lower in the everolimus group compared to the control group at day 3 (126±6% vs. 152±6%; p = 0.016), day 7 (164±7% vs. 226±13%; p<0.001) and at day 10 (194±10% vs. 281±18%; p<0.001). Uptake of 18F-FDG and 18F-FLT showed little differences between control and treatment groups, but individual mean uptake of 18F-FDG at day 3 correlated with tumor growth day 10 (r2 = 0.45; P = 0.034), 18F-FLT mean uptake at day 1 correlated with tumor growth day 7 (r2 = 0.63; P = 0.019) and at day 3 18F-FLT correlated with tumor growth day 7 (r2 = 0.87; P<0.001) and day 10 (r2 = 0.58; P = 0.027). Conclusion Everolimus was effective in vitro and in vivo in human xenografts lung carcinoid NETs and especially early 18F-FLT uptake predicted subsequent tumor growth. We suggest that 18F-FLT PET can be used for tailoring therapy for neuroendocrine tumor patients through early identification of responders and non-responders.