ABSTRACT
Hepatic steatosis is characterized by intrahepatic lipid accumulation and may lead to irreversible liver damage if untreated. Here, we investigate whether multispectral optoacoustic tomography (MSOT) can offer label-free detection of liver lipid content to enable non-invasive characterization of hepatic steatosis by analyzing the spectral region around 930 nm, where lipids characteristically absorb. In a pilot study, we apply MSOT to measure liver and surrounding tissues in five patients with liver steatosis and five healthy volunteers, revealing significantly higher absorptions at 930 nm in the patients, while no significant difference was observed in the subcutaneous adipose tissue of the two groups. We further corroborated the human observations with corresponding MSOT measurements in high fat diet (HFD) - and regular chow diet (CD)-fed mice. This study introduces MSOT as a potential non-invasive and portable technique for detecting/monitoring hepatic steatosis in clinical settings, providing justification for larger studies.
ABSTRACT
Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic ß-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic ß-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.
Subject(s)
Extracellular Vesicles , Insulin-Secreting Cells , Islets of Langerhans , Mice , Animals , Insulin Secretion , Insulin/metabolism , Glucose/metabolism , Insulin-Secreting Cells/metabolism , Obesity/metabolism , Adipocytes/metabolism , Extracellular Vesicles/metabolism , Islets of Langerhans/metabolismABSTRACT
Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.
ABSTRACT
Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacoustic method to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO2) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. An initial sharp drop in tumor sO2 and THb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today. SIGNIFICANCE: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.
Subject(s)
Bevacizumab/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents, Immunological/pharmacology , Breast Neoplasms/blood supply , Breast Neoplasms/diagnostic imaging , Cell Line, Tumor , Female , Humans , Mice, SCID , Neovascularization, Pathologic/diagnostic imaging , Oxygen/metabolism , Photoacoustic Techniques/methods , Triple Negative Breast Neoplasms/blood supply , Triple Negative Breast Neoplasms/diagnostic imaging , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Perfusion and oxygenation are critical parameters of muscle metabolism in health and disease. They have been both the target of many studies, in particular using near-infrared spectroscopy (NIRS). However, difficulties with quantifying NIRS signals have limited a wide dissemination of the method to the clinics. Our aim was to investigate whether clinical multispectral optoacoustic tomography (MSOT) could enable the label-free imaging of muscle perfusion and oxygenation under clinically relevant challenges: the arterial and venous occlusion. We employed a hybrid clinical MSOT/ultrasound system equipped with a hand-held scanning probe to visualize hemodynamic and oxygenation changes in skeletal muscle under arterial and venous occlusions. Four (N = 4) healthy volunteers were scanned over the forearm for both 3-minute occlusion challenges. MSOT-recorded pathophysiologically expected results during tests of disturbed blood flow with high resolution and without the need for contrast agents. During arterial occlusion, MSOT-extracted Hb-values showed an increase, while HbO2 - and total blood volume (TBV)-values remained roughly steady, followed by a discrete increase during the hyperemic period after cuff deflation. During venous occlusion, results showed a clear increase in intramuscular HbO2 , Hb and TBV within the segmented muscle area. MSOT was found to be capable of label-free non-invasive imaging of muscle hemodynamics and oxygenation under arterial and venous occlusion. We introduce herein MSOT as a novel modality for the assessment of vascular disorders characterized by disturbed blood flow, such as acute limb ischemia and venous thrombosis.
Subject(s)
Spectroscopy, Near-Infrared , Tomography, X-Ray Computed , Humans , Muscle, Skeletal/diagnostic imaging , Perfusion , Pilot ProjectsABSTRACT
We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.
Subject(s)
Image Enhancement/methods , Lipid Droplets/metabolism , Photoacoustic Techniques/methods , 3T3-L1 Cells , Adipogenesis , Animals , Carbohydrate Metabolism , HeLa Cells , Humans , Mice , Microscopy , Software , Spectroscopy, Fourier Transform InfraredABSTRACT
Imaging has become an indispensable tool in the research and clinical management of cardiovascular disease (CVD). An array of imaging technologies is considered for CVD diagnostics and therapeutic assessment, ranging from ultrasonography, X-ray computed tomography and magnetic resonance imaging to nuclear and optical imaging methods. Each method has different operational characteristics and assesses different aspects of CVD pathophysiology; nevertheless, more information is desirable for achieving a comprehensive view of the disease. Optoacoustic (photoacoustic) imaging is an emerging modality promising to offer novel information on CVD parameters by allowing high-resolution imaging of optical contrast several centimeters deep inside tissue. Implemented with illumination at several wavelengths, multi-spectral optoacoustic tomography (MSOT) in particular, is sensitive to oxygenated and deoxygenated hemoglobin, water and lipids allowing imaging of the vasculature, tissue oxygen saturation and metabolic or inflammatory parameters. Progress with fast-tuning lasers, parallel detection and advanced image reconstruction and data-processing algorithms have recently transformed optoacoustics from a laboratory tool to a promising modality for small animal and clinical imaging. We review progress with optoacoustic CVD imaging, highlight the research and diagnostic potential and current applications and discuss the advantages, limitations and possibilities for integration into clinical routine.
ABSTRACT
OBJECTIVES: Brown adipose tissue (BAT) dissipates nutritional energy as heat through uncoupling protein 1 (UCP1). The discovery of functional BAT in healthy adult humans has promoted the search for pharmacological interventions to recruit and activate brown fat as a treatment of obesity and diabetes type II. These efforts require in vivo models to compare the efficacy of novel compounds in a relevant physiological context. METHODS: We generated a knock-in mouse line expressing firefly luciferase and near-infrared red florescent protein (iRFP713) driven by the regulatory elements of the endogenous Ucp1 gene. RESULTS: Our detailed characterization revealed that firefly luciferase activity faithfully reports endogenous Ucp1 gene expression in response to physiological and pharmacological stimuli. The iRFP713 fluorescence signal was detected in the interscapular BAT region of cold-exposed reporter mice in an allele-dosage dependent manner. Using this reporter mouse model, we detected a higher browning capacity in female peri-ovarian white adipose tissue compared to male epididymal WAT, which we further corroborated by molecular and morphological features. In situ imaging detected a strong luciferase activity signal in a previously unappreciated adipose tissue depot adjunct to the femoral muscle, now adopted as femoral brown adipose tissue. In addition, screening cultured adipocytes by bioluminescence imaging identified the selective Salt-Inducible Kinase inhibitor, HG-9-91-01, to increase Ucp1 gene expression and mitochondrial respiration in brown and brite adipocytes. CONCLUSIONS: In our mouse model, firefly luciferase activity serves as a bona fide reporter for dynamic regulation of Ucp1. In addition, by means of iRFP713 we are able to monitor Ucp1 expression in a non-invasive fashion.
Subject(s)
Adipose Tissue, Beige/diagnostic imaging , Adipose Tissue, Brown/diagnostic imaging , Genes, Reporter , Luciferases, Firefly/genetics , Thermogenesis , Uncoupling Protein 1/genetics , Adipose Tissue, Beige/metabolism , Adipose Tissue, Brown/metabolism , Animals , Cell Line , Cells, Cultured , Luciferases, Firefly/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Uncoupling Protein 1/metabolism , Red Fluorescent ProteinABSTRACT
MSOT has revolutionized biomedical imaging because it allows anatomical, functional, and molecular imaging of deep tissues in vivo in an entirely noninvasive, label-free, and real-time manner. This imaging modality works by pulsing light onto tissue, triggering the production of acoustic waves, which can be collected and reconstructed to provide high-resolution images of features as deep as several centimeters below the body surface. Advances in hardware and software continue to bring MSOT closer to clinical translation. Most recently, a clinical handheld MSOT system has been used to image brown fat tissue (BAT) and its metabolic activity by directly resolving the spectral signatures of hemoglobin and lipids. This opens up new possibilities for studying BAT physiology and its role in metabolic disease without the need to inject animals or humans with contrast agents. In this chapter, we overview how MSOT works and how it has been implemented in preclinical and clinical contexts. We focus on our recent work using MSOT to image BAT in resting and activated states both in mice and humans.
Subject(s)
Adipose Tissue, Brown/metabolism , Photoacoustic Techniques , Animals , Humans , Mice , TomographyABSTRACT
In the original PDF version of this article, affiliation 1, 'Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany', was incorrectly given as 'Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany '. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.
ABSTRACT
The molecular mediator and functional significance of meal-associated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-sympathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, which is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.
Subject(s)
Adipocytes, Brown/metabolism , Adipose Tissue, Brown/metabolism , Eating , Secretin/metabolism , Thermogenesis , Adipocytes, Brown/cytology , Adipose Tissue, Brown/cytology , Animals , HEK293 Cells , Humans , Lipolysis , Mice , Mice, Knockout , Mice, Obese , Secretin/geneticsABSTRACT
Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3ß4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3ß4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Obesity/metabolism , Receptors, Nicotinic/metabolism , TRPM Cation Channels/antagonists & inhibitors , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Animals , Body Weight/drug effects , Cold Temperature , Diabetes Mellitus, Type 2/drug therapy , Diet , Dimethylphenylpiperazinium Iodide/pharmacology , Dimethylphenylpiperazinium Iodide/therapeutic use , Energy Metabolism/drug effects , Fatty Liver/pathology , Glucose Intolerance/pathology , Insulin Resistance , Male , Melanocortins/metabolism , Mice, Inbred C57BL , Mice, Obese , Obesity/drug therapy , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , Receptor, Melanocortin, Type 4/metabolism , TRPM Cation Channels/metabolism , Thermogenesis/drug effectsABSTRACT
Metabolism is a fundamental process of life. However, non-invasive measurement of local tissue metabolism is limited today by a deficiency in adequate tools for in vivo observations. We designed a multi-modular platform that explored the relation between local tissue oxygen consumption, determined by label-free optoacoustic measurements of hemoglobin, and concurrent indirect calorimetry obtained during metabolic activation of brown adipose tissue (BAT). By studying mice and humans, we show how video-rate handheld multi-spectral optoacoustic tomography (MSOT) in the 700-970 nm spectral range enables non-invasive imaging of BAT activation, consistent with positron emission tomography findings. Moreover, we observe BAT composition differences between healthy and diabetic tissues. The study consolidates hemoglobin as a principal label-free biomarker for longitudinal non-invasive imaging of BAT morphology and bioenergetics in situ. We also resolve water and fat components in volunteers, and contrast MSOT readouts with magnetic resonance imaging data.
Subject(s)
Adipose Tissue, Brown/metabolism , Hemoglobins/metabolism , Oxygen Consumption , Photoacoustic Techniques , Adult , Animals , Energy Metabolism , Female , Humans , Lipid Metabolism , Male , Mice, Inbred BALB CABSTRACT
Optoacoustic (photoacoustic) imaging enables high-resolution optical imaging at depths well beyond optical microscopy, revolutionizing optical interrogation of tissues. Operation in the near-infrared (NIR) is nevertheless necessary to capitalize on the technology potential and reach depths of several centimeters. Using Flash NanoPrecipitation for highly-scalable single-step encapsulation of hydrophobic hexacene at self-quenching concentrations, we propose quenched fluorescence-dye nanoparticles as a potent alternative to NIR metal nanoparticles for strong optoacoustic signal generation. Comprehensive hexacene-based nanoparticle characterization was based on a 5-step approach that examined the physicochemical features (Step 1), optoacoustic signal generation (Step 2), stability (Step 3), biocompatibility (Step 4) and spectral sensitivity (Step 5). Using this characterization framework we showcase the discovery of two nanoparticle formulations, QH2-50 nm and QH2-100 nm that attain superior stability characteristics and optimal optoacoustic properties compared to gold standards commonly employed for near-infrared optoacoustics. We discuss encapsulation and self-quenching (ESQ) of organic dyes as a promising strategy to generate optimal optoacoustic particles.
ABSTRACT
Label-free multispectral optoacoustic tomography (MSOT) has recently shown superior performance in visualizing the morphology of human vasculature, especially of smaller vessels, compared to ultrasonography. Herein, we extend these observations towards MSOT interrogation of macrovascular endothelial function. We employed a real-time handheld MSOT scanner to assess flow-mediated dilatation (FMD), a technique used to characterize endothelial function. A data processing scheme was developed to quantify the dimensions and diameter changes of arteries in humans and determine wall distensibility parameters. By enabling high-resolution delineation of the blood-vessel wall in a cross-sectional fashion, the findings suggest MSOT as a capable alternative to ultrasonography for clinical FMD measurements.
ABSTRACT
The folate receptor (FR) is upregulated in various epithelial cancer types (FR α-isoform), while healthy tissues show only restricted expression. FR-targeted imaging using folate radiopharmaceuticals is therefore a promising approach for the detection of FR-positive cancer tissue. Almost all folate-based radiopharmaceuticals have been prepared by conjugation at the γ-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated α- and γ-conjugated folate derivatives were synthesized and their in vitro and in vivo properties compared. The syntheses of all six regioisomers were obtained in good chemical yields using a multistep synthetic approach including the highly selective Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radiosyntheses of the α- and γ-conjugated (18)F-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/µmol. In vitro, all folate derivatives showed high binding affinity to the FR-α (IC50 = 1.4-2.2 nM). In vivo PET imaging and biodistribution studies in FR-positive KB tumor-bearing mice demonstrated similar FR-specific tumor uptake for both regioisomers of each pair of compounds. However, FR-unspecific liver uptake was significantly lower for the α-regioisomers compared to the corresponding γ-regioisomers. In contrast, kidney uptake was up to 50% lower for the γ-regioisomers than for the α-regioisomers. These results show that the site of conjugation in the glutamyl moiety of folic acid has a significant impact on the in vivo behavior of (18)F-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel (18)F-labeled folate-based radiotracers.
Subject(s)
Fluorine Radioisotopes/chemistry , Folic Acid/chemistry , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemistry , Animals , Catalysis , Copper , Female , Fluorine Radioisotopes/pharmacokinetics , Folate Receptors, GPI-Anchored/metabolism , Humans , Isomerism , Isotope Labeling , KB Cells , Mice, Nude , Molecular Structure , Radiochemistry/methods , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacokinetics , Tissue DistributionABSTRACT
INTRODUCTION: Application of therapeutic folate radioconjugates is a promising option for the treatment of folate receptor (FR)-positive tumors, although high uptake of radiofolates in the kidneys remains a critical issue. Recently, it was shown that enhancing the blood circulation of radiofolates results in increased tumor uptake and reduced retention of radioactivity in the kidneys. In this study, we investigated and compared the anti-tumor effects and potential long-term damage to the kidneys after application of an albumin-binding ((177)Lu-cm09), and a conventional ((177)Lu-EC0800) folate radioconjugate. METHODS: In vivo studies were performed with KB tumor-bearing nude mice. (177)Lu-EC0800 and (177)Lu-cm09 were applied at variable quantities (10-30 MBq/mouse), and the tumor growth was monitored over time. Mice without tumors were injected with the same radiofolates and investigated over eight months by determination of creatinine and blood urea nitrogen plasma levels and by measuring renal uptake of (99m)Tc-DMSA using SPECT. At the study end, the morphological changes were examined on renal tissue sections using variable staining methods. RESULTS: Compared to untreated controls, dose-dependent tumor growth inhibition and prolonged survival was observed in all treated mice. In line with the resulting absorbed dose, the treatment was more effective with (177)Lu-cm09 than with (177)Lu-EC0800, enabling complete tumor remission after application of ≥20MBq (≥28Gy). Application of radiofolates with an absorbed renal dose ≥23 Gy showed increased levels of renal plasma parameters and reduced renal uptake of (99m)Tc-DSMA. Morphological changes observed on tissue sections confirmed radionephropathy of variable stages. CONCLUSIONS: (177)Lu-cm09 showed more favorable anti-tumor effects and significantly less damage to the kidneys compared to (177)Lu-EC0800 as was expected based on improved tumor-to-kidney ratios. It was demonstrated that enhancing the blood circulation time of radiofolates was favorable regarding the risk-benefit profile of a therapeutic application. These results hold promise for future translation of the albumin-binder concept to the clinics, potentially enabling FR-targeted radionuclide therapy in patients.
Subject(s)
Coordination Complexes/adverse effects , Folic Acid Transporters/metabolism , Folic Acid/adverse effects , Kidney Diseases/etiology , Neoplasms/radiotherapy , Radiation Injuries/etiology , Radiopharmaceuticals/adverse effects , Radiopharmaceuticals/therapeutic use , Animals , Biological Transport/radiation effects , Body Weight/radiation effects , Cell Survival/radiation effects , Coordination Complexes/pharmacokinetics , Coordination Complexes/therapeutic use , Female , Folic Acid/analogs & derivatives , Folic Acid/pharmacokinetics , Folic Acid/therapeutic use , Humans , KB Cells , Kidney/metabolism , Kidney/pathology , Kidney/radiation effects , Leukocytes/cytology , Leukocytes/radiation effects , Mice , Mice, Nude , Neoplasms/blood , Neoplasms/metabolism , Radiometry , Radiopharmaceuticals/pharmacokinetics , Survival Analysis , Technetium Tc 99m Dimercaptosuccinic Acid/metabolismABSTRACT
There is growing interest in genetically expressed reporters for in vivo studies of bacterial colonization in the context of infectious disease research, studies of the bacterial microbiome or cancer imaging and treatment. To empower non-invasive high-resolution bacterial tracking with deep tissue penetration, we herein use the genetically controlled biosynthesis of the deep-purple pigment Violacein as a photobleaching-resistant chromophore label for in vivo optoacoustic (photoacoustic) imaging in the near-infrared range. We demonstrate that Violacein-producing bacteria can be imaged with high contrast-to-noise in strongly vascularized xenografted murine tumors and further observe that Violacein shows anti-tumoral activity. Our experiments thus identify Violacein as a robust bacterial label for non-invasive optoacoustic imaging with high potential for basic research and future theranostic applications in bacterial tumor targeting.
Subject(s)
Bacterial Proteins , Escherichia coli , Indoles/metabolism , Molecular Imaging/methods , Photoacoustic Techniques/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromobacterium/genetics , Escherichia coli/cytology , Escherichia coli/enzymology , Escherichia coli/geneticsABSTRACT
Terbium-149 is among the most interesting therapeutic nuclides for medical applications. It decays by emission of short-range α-particles (Eα = 3.967 MeV) with a half-life of 4.12 h. The goal of this study was to investigate the anticancer efficacy of a 149Tb-labeled DOTA-folate conjugate (cm09) using folate receptor (FR)-positive cancer cells in vitro and in tumor-bearing mice. 149Tb was produced at the ISOLDE facility at CERN. Radiolabeling of cm09 with purified 149Tb resulted in a specific activity of ~1.2 MBq/nmol. In vitro assays performed with 149Tb-cm09 revealed a reduced KB cell viability in a FR-specific and activity concentration-dependent manner. Tumor-bearing mice were injected with saline only (group A) or with 149Tb-cm09 (group B: 2.2 MBq; group C: 3.0 MBq). A significant tumor growth delay was found in treated animals resulting in an increased average survival time of mice which received 149Tb-cm09 (B: 30.5 d; C: 43 d) compared to untreated controls (A: 21 d). Analysis of blood parameters revealed no signs of acute toxicity to the kidneys or liver in treated mice over the time of investigation. These results demonstrated the potential of folate-based α-radionuclide therapy in tumor-bearing mice.
ABSTRACT
INTRODUCTION: We assessed the suitability of the radiolanthanide (155)Tb (t1/2=5.32 days, Eγ=87 keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. METHODS: (155)Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~1.4 GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. (155)Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules - a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) - were radiolabeled with (155)Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. RESULTS: The total yield of the two-step separation process of (155)Tb was 86%. (155)Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The (155)Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (>95%). (155)Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of (155)Tb-DOTATATE and (155)Tb-MD, respectively. The relatively long physical half-life of (155)Tb matched in particular the biological half-lives of (155)Tb-cm09 and (155)Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. CONCLUSIONS: The radiolanthanide (155)Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the ß(-)-emitting radiolanthanides (177)Lu, (161)Tb, (166)Ho, and the pseudo-radiolanthanide (90)Y.