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Microbubbles (MB) are used as ultrasound (US) contrast agents in clinical settings because of their ability to oscillate upon exposure to acoustic pulses and generate nonlinear responses with a stable cavitation profile. Polymeric MB have recently attracted increasing attention as molecular imaging probes and drug delivery agents based on their tailorable acoustic responses, high drug loading capacity, and surface functionalization capabilities. While many of these applications require MB to be functionalized with biological ligands, the impact of bioconjugation on polymeric MB cavitation and acoustic properties remains poorly understood. Hence, we here evaluated the effects of MB shell hydrolysis and subsequent streptavidin conjugation on the acoustic behavior of poly(butyl cyanoacrylate) (PBCA) MB. We show that upon biofunctionaliza-tion, MB display higher acoustic stability, stronger stable cavitation, and enhanced second harmonic generation. Furthermore, functionalized MB preserve the binding capabilities of streptavidin conjugated on their surface. These findings provide insights into the effects of bioconjugation chemistry on polymeric MB acoustic properties, and they contribute to improving the performance of polymer-based US imaging and theranostic agents.

PURPOSE: Evaluation of [68Ga]NODAGA-duramycin as a positron emission tomography (PET) tracer of cell death for whole-body detection of chemotherapy-induced organ toxicity.PROCEDURES: Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2 h after intravenous injection of [68Ga]NODAGA-duramycin. In vivo data were validated by immunohistology and blood parameters.RESULTS: In vitro experiments confirmed specific binding of [68Ga]NODAGA-duramycin. Organ toxicities were detected successfully using [68Ga]NODAGA-duramycin PET/X-ray computed tomography (CT) and confirmed by immunohistochemistry and blood parameter analysis. Organ toxicities in livers and kidneys showed similar trends in PET/CT and immunohistology. Busulfan and cisplatin-related organ toxicities in heart, liver, and lungs were detected earlier by PET/CT than by blood parameters and immunohistology.CONCLUSION: [68Ga]NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in mice. It, therefore, represents a promising alternative to standard toxicological analyses with a high translational potential.

Acetylsalicylic acid (ASA) is a well-established drug for heart attack and stroke prophylaxis. Furthermore, numerous studies have reported an anti-carcinogenic effect, but its exact mechanism is still unknown. Here, we applied VEGFR-2-targeted molecular ultrasound to explore a potential inhibitory effect of ASA on tumor angiogenesis in vivo. Daily ASA or placebo therapy was performed in a 4T1 tumor mouse model. During therapy, ultrasound scans were performed using nonspecific microbubbles (CEUS) to determine the relative intratumoral blood volume (rBV) and VEGFR-2-targeted microbubbles to assess angiogenesis. Finally, vessel density and VEGFR-2 expression were assessed histologically. CEUS indicated a decreasing rBV in both groups over time. VEGFR-2 expression increased in both groups up to Day 7. Towards Day 11, the binding of VEGFR-2-specific microbubbles further increased in controls, but significantly (p = 0.0015) decreased under ASA therapy (2.24 ± 0.46 au vs. 0.54 ± 0.55 au). Immunofluorescence showed a tendency towards lower vessel density under ASA and confirmed the result of molecular ultrasound. Molecular US demonstrated an inhibitory effect of ASA on VEGFR-2 expression accompanied by a tendency towards lower vessel density. Thus, this study suggests the inhibition of angiogenesis via VEGFR-2 downregulation as one of the anti-tumor effects of ASA.

Molecular imaging & biology : MIB 24(2), 288-297 (2022). doi:10.1007/s11307-021-01686-x, Published by Springer Nature Switzerland, Cham

Journal of experimental & clinical cancer research 40(1), 288 (2021). doi:10.1186/s13046-021-02043-0, Published by Springer, Berlin ; Heidelberg

Introduction: Ultrasound (US) imaging enables tissue visualization in high spatial resolution with short examination times. Thus, it is often applied in preclinical research. Diagnostic US, including contrast-enhanced US (CEUS), is considered to be well-tolerated by laboratory animals although no systematic study has been performed to confirm this claim. Therefore, the aim of this study was to screen for possible effects of US and CEUS examinations on welfare of healthy mice. Additionally, the potential influence of CEUS and molecular CEUS on well-being and therapy response to regorafenib was investigated in breast cancer-bearing mice. Material and Methods: Forty healthy Balb/c mice were randomly assigned for examination with US or CEUS (3×/week) for 4 weeks. Untreated healthy mice and mice receiving only isoflurane anesthesia served as controls (n = 10/group). Ninety-four 4T1 tumor-bearing Balb/c mice were allocated randomly to the following groups: no imaging, isoflurane anesthesia, CEUS, and molecular CEUS. They either received 10 mg/kg regorafenib or vehicle solution daily by oral gavage. Animals were examined three times within 2 weeks. CEUS measurements were performed using phospholipid microbubbles, and phospholipid microbubbles targeting the vascular endothelial growth factor receptor-2 were applied for molecular CEUS. Welfare evaluation was performed by daily observational score sheets, measuring the heart rate, Rotarod performance, and fecal corticosterone metabolites twice a week. On the last day, pathological changes in serum corticosterone concentrations, hemograms, and organ weights were obtained. Moreover, a potential influence of isoflurane anesthesia, CEUS, and molecular CEUS on regorafenib response in tumor-bearing mice was examined. Analysis of variance and Dunnett’s post hoc test were performed as statistical analyses. Results: Severity parameters were not altered after repeated US and CEUS examinations of healthy mice, but spleen sizes were significantly lower after isoflurane anesthesia. In tumor-bearing mice, no effect on animal welfare after repeated CEUS and molecular CEUS could be observed. However, leukocyte counts and spleen weights of tumor-bearing mice were significantly lower in animals examined with CEUS and molecular CEUS compared to the control groups. This effect was not visible in regorafenib-treated animals. Conclusions: Repeated US and (molecular) CEUS have no detectable impact on animal welfare in healthy and tumor-bearing mice. However, CEUS and molecular CEUS in combination with isoflurane anesthesia might attenuate immunological processes in tumor-bearing animals and may consequently affect responses to antitumor therapy.

Background and Aims Chronic hypokalemia causes kidney fibrosis with cystic lesions and arterial hypertension. In contrast, potassium-rich diet lowers blood pressure. The acute effects of hypo- and hyperkalemia on heart and kidney are not well understood. Method Wild-type mice were fed with low (LK), normal (NK) and high (HK) potassium diet for 4 and 20 days. Kidneys were examined for site of acute injury, inflammation and fibrosis. Blood analysis of electrolytes and kidney parameters were analyzed. Echocardiography and ECG were used to assess heart function. Further, KCNJ10 knockout mice were used to investigate kidney damage in a genetically induced hypokalemia model. Results Proximal tubule injury as detected by KIM-1+ staining and yH2AX+ DNA-damage was observed after 4 and 20 days of LK diet. Injury was associated with strong Ki-67+ proliferation of proximal tubule cells. No injury was detected in mice on NK and HK diet. After 20 days of LK diet, F4/80+ inflammation and aSMA+ extracellular matrix accumulation, typical for fibrosis, were observed. LK mice developed polyurie, volume depletion, loss of body weight and high BUN. Lower cardiac output and signs of myocardial stress was seen in echocardiography and ECG. Consistent with WT mice on LK diet, KCNJ10 knockout mice developed same pattern of kidney injury. Nine months after deletion of KCNJ10, cysts were observed in the proximal tubule in outer medzulla. Conclusion Acute hypokalemia causes kidney injury and myocardial stress. Cystic lesions originate from late proximal tubule. Hypokalemia should be corrected rapidly to stop progression into kidney fibrosis.

Contrast media & molecular imaging 2021, (2021). doi:10.1155/2021/6676337, Published by Hindawi, London

The authors regret that a statement on author contributions was not correctly formulated. With this corrigendum we provide the corrected version. Author contributions FW and SK prepared the tissue-engineered vascular grafts and were responsible of the study's coordination. FW, SK, AR and PM performed the tissue analysis and interpretation. SJ, TL, FM and FK were responsible for the study design. VF, MM and NG-W performed the in vitro and in vivo MR monitoring of grafts and the MRI data analysis together with TL, FK, PM and FW. OW and AM performed the PET- CT monitoring of grafts and the data analysis together with FM, PM and FW. HS and KC were responsible for the surgical implantation of the grafts. FW drafted the original manuscript; PM, TL and FK critically revised it. All authors discussed the results and approved the final version of the manuscript. The authors would like to apologise for any inconvenience caused.

(1) Background: Radiogenomics is motivated by the concept that biomedical images contain information that reflects underlying pathophysiology. This review focused on papers that used genetics to validate their radiomics models and outcomes and assess their contribution to this emerging field. (2) Methods: All original research with the words radiomics and genomics in English and performed in humans up to 31 January 2022, were identified on Medline and Embase. The quality of the studies was assessed with Radiomic Quality Score (RQS) and the Cochrane recommendation for diagnostic accuracy study Quality Assessment 2. (3) Results: 45 studies were included in our systematic review, and more than 50% were published in the last two years. The studies had a mean RQS of 12, and the studied tumors were very diverse. Up to 83% investigated the prognosis as the main outcome, with the rest focusing on response to treatment and risk assessment. Most applied either transcriptomics (54%) and/or genetics (35%) for genetic validation. (4) Conclusions: There is enough evidence to state that new science has emerged, focusing on establishing an association between radiological features and genomic/molecular expression to explain underlying disease mechanisms and enhance prognostic, risk assessment, and treatment response radiomics models in cancer patients.

Theranostics 11(19), 9557-9570 (2021). doi:10.7150/thno.64767, Published by Ivyspring, Wyoming, NSW

Methods For apoptosis imaging, the near-infrared probe Annexin Vivo750 was used in combination with fluorescence molecular tomography and microcomputed tomography (FMT/µCT). Glucose metabolism was assessed using 18F-FDG-PET/CT. Five groups of nude mice bearing lung cancer xenografts (A549) were investigated: (i) untreated controls and two groups after (ii) cytotoxic (carboplatin) or (iii) anti-angiogenic (sunitinib) treatment for four and nine days, respectively. Imaging data were validated by immunohistochemistry. Results In response to carboplatin treatment, an inverse relation was found between the change in glucose metabolism and apoptosis in A549 tumors. Annexin Vivo showed a continually increasing tumor accumulation, while the tumor-to-muscle ratio of 18F-FDG continuously decreased during therapy. Immunohistochemistry revealed a significantly higher tumor apoptosis (p=0.007) and a minor but not significant reduction in vessel density only at day 9 of carboplatin therapy. Interestingly, during anti-angiogenic treatment there was an early drop in the tumor-to-muscle ratio between days 0 and 4, followed by a subsequent minor decrease (18F-FDG tumor-to-muscle-ratio: 1.9 ± 0.4; day 4: 1.1 ± 0.2; day 9: 1.0 ± 0.2; p=0.021 and p=0.001, respectively). The accumulation of Annexin Vivo continuously increased over time (Annexin Vivo: untreated: 53.7 ± 36.4 nM; day 4: 87.2 ± 53.4 nM; day 9: 115.1 ± 103.7 nM) but failed to display the very prominent early induction of tumor apoptosis that was found by histology already at day 4 (TUNEL: p=0.0036) together with a decline in vessel density (CD31: p=0.004), followed by no significant changes thereafter. Conclusion Both molecular imaging approaches enable visualizing the effects of cytotoxic and anti-angiogenic therapy in A549 tumors. However, the early and strong tumor apoptosis induced by the anti-angiogenic agent sunitinib was more sensitively and reliably captured by monitoring of the glucose metabolism as compared to Annexin V-based apoptosis imaging.

According to European Union directive 2010/63/EU a severity classification of experimental procedures performed on laboratory animals is mandatory. This includes a prospective evaluation of all interventions performed within the experiment, as well as an assessment of the actual burden of each animal during the experiment. In this regard, the evaluation and scoring of defined criteria regarding the health state of animals could help to early identify deteriorations in animal health and facilitate the application of humane endpoints. This article discusses the applicability of an adapted score sheet in BALB/cAnNRj mice receiving either cisplatin, doxorubicin or busulfan, three chemotherapeutic agents with different toxicological profiles and longitudinal non-invasive molecular imaging. The health state was investigated by score sheets documenting general state, body weight, spontaneous behaviour and treatment specific parameters (e.g. anaemia, neurotoxicity, persistent diarrhoea). Although blood and serum analyses clearly indicated various organ damage, most scoring parameters except for body weight did not report on the deceasing animal health state. Thus, there is need for more sensitive observational parameters to judge the animal's health state and welfare.

Contrast-enhanced ultrasound (CEUS) imaging is a valuable tool for preclinical and clinical diagnostics. The most frequently used ultrasound contrast agents are microbubbles. Besides them, novel nano-sized materials are under investigation, which are briefly discussed in this chapter. For molecular CEUS, the ultrasound contrast agents are modified to actively target disease-associated molecular markers with a site-specific ligand. The most common markers for tumor imaging are related to neoangiogenesis, like the vascular endothelial growth factor receptor-2 (VEGFR2) and α

Magnetic resonance imaging (MRI) is considered to be well tolerated by laboratory animals. However, no systematic study has been performed yet, proving this assumption. Therefore, the aim of this study was to investigate the possible effects of longitudinal native and contrast-enhanced (CE) 1-T and 7-T MRI examinations on mouse welfare as well as 4T1 breast cancers progression and therapy response.Forty-seven healthy and 72 breast cancer-bearing mice (4T1) were investigated. One-Tesla (ICON) and 7-T (Biospec) MRI measurements were performed thrice per week under isoflurane anesthesia in healthy BALB/c mice for 4 weeks and 3 times within 2 weeks in tumor-bearing animals. Animal welfare was examined by an observational score sheet, rotarod performance, heart rate measurements, and assessment of fecal corticosterone metabolites. Furthermore, we investigated whether CE-MRI influences the study outcome. Therefore, hemograms and organ weights were obtained, and 4T1 tumor growth, perfusion, immune cell infiltration, as well as response to the multikinase inhibitor regorafenib were investigated. Statistical comparisons between groups were performed using analysis of variance and Tukey or Bonferroni post hoc tests.Mice showed no alterations in the observational score sheet rating, rotarod performance, heart rate, and fecal corticosterone metabolites (P0.05) after repeated MRI at both field strengths. However, spleen weights were reduced in all healthy mouse groups that received isoflurane anesthesia (P0.001) including the groups investigated by 1-T and 7-T MRI (P = 0.02). Neither tumor progression nor response to the regorafenib treatment was affected by isoflurane anesthesia or CE-MRI monitoring. Furthermore, immunohistological tumor analysis did not indicate an effect of isoflurane and MRI on macrophage infiltration of tumors, perfusion of tumor vessels, and apoptotic cell rate (P0.05).Repeated MRI did not influence the welfare of mice and did not affect tumor growth and therapy response of 4T1 tumors. However, systemic immunological effects of isoflurane anesthesia need to be considered to prevent potential bias.

Objective— The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. Approach and Results— Flow-dependent endothelial dysfunction was induced in apolipoprotein E–deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A–targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [ P P Conclusions— Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A–targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.

Microbubbles (MB) are used as ultrasound (US) contrast agents and can be efficiently targeted against markers of angiogenesis and inflammation. Due to their gas core, MB locally alter susceptibilities in magnetic resonance imaging (MRI), but unfortunately, the resulting contrast is low and not sufficient to generate powerful molecular MRI probes. Therefore, we investigated whether a potent molecular MR agent can be generated by encapsulating superparamagnetic iron oxide nanoparticles (SPION) in the polymeric shell of poly (n-butylcyanoacrylate) (PBCA) MB and targeted them against α vβ3 integrins on the angiogenic vasculature of 4T1 murine breast carcinomas. SPION-MB consist of an air core and a multi-layered polymeric shell enabling efficient entrapment of SPION. The mean size of SPION-MB was 1.61 ± 0.32 μm. Biotin-streptavidin coupling was employed to functionalize the SPION-MB with cyclic RGDfK (Arg-Gly-Asp) and RADfK (Arg-Ala-Asp) peptides. Cells incubated with RGD-SPION-MB showed enhanced transverse relaxation rates compared with SPION-MB and blocking α vβ3 integrin receptors with excess free cRGDfK significantly reduced RGD-SPION-MB binding. Due to the fast binding of RGD-SPION-MB in vivo , dynamic susceptibility contrast MRI was employed to track their retention in tumors in real-time. Higher retention of RGD-SPION-MB was observed compared with SPION-MB and RAD-SPION-MB. To corroborate our MRI results, molecular US was performed the following day using the destruction-replenishment method. Both imaging modalities consistently indicated higher retention of RGD-SPION-MB in angiogenic vessels compared with SPION-MB and RAD-SPION-MB. Competitive blocking experiments in mice further confirmed that the binding of RGD-SPION-MB to α vβ3 integrin receptors is specific. Overall, this study demonstrates that RGD-SPION-MB can be employed as molecular MR/US contrast agents and are capable of assessing the αvβ3 integrin expression in the neovasculature of malignant tumors.

Contrast-enhanced ultrasound (CEUS) imaging is a valuable tool for preclinical and clinical diagnostics. The most frequently used ultrasound contrast agents are microbubbles. Besides them, novel nano-sized materials are under investigation, which are briefly discussed in this chapter. For molecular CEUS, the ultrasound contrast agents are modified to actively target disease-associated molecular markers with a site-specific ligand. The most common markers for tumor imaging are related to neoangiogenesis, like the vascular endothelial growth factor receptor-2 (VEGFR2) and αvβ3 integrin. In this chapter, applications of molecular ultrasound to longitudinally monitor receptor expression during tumor growth, to detect neovascularization, and to evaluate therapy responses are described. Furthermore, we report on first clinical trials of molecular CEUS with VEGFR2-targeted phospholipid microbubbles showing promising results regarding patient safety and its ability to detect tumors of prostate, breast, and ovary. The chapter closes with an outlook on ultrasound theranostics, where (targeted) ultrasound contrast agents are used to increase the permeability of tumor tissues and to support drug delivery.

The modification of biomaterials to comply with clinically employed monitoring techniques is a promising strategy to support clinical translation in regenerative medicine. Here, multimodal imaging of tissue-engineered vascular grafts (TEVG) was enabled by functionalizing the textile scaffold with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The resulting MR-imageable grafts (iTEVG) were monitored non-invasively throughout their whole life-cycle, from initial quality control to longitudinal functional evaluation in an ovine model for up to 8 weeks. Crucial features such as the complete embedding of the textile mesh in the developing tissue and the grafts' structural stability were assessed in vitro using 1T-, 3T- and 7T-MRI scanners. In vivo, the grafts were imaged by 3T-MRI and PET-CT. Contrary to unlabeled constructs, iTEVG could be delineated from native arteries and precisely localized by MRI. USPIO labeling neither induced calcifications, nor negatively affected their remodeling with respect to tissue-specific extracellular matrix composition and endothelialization. Functionality was confirmed by MR-angiography. 18F-FDG uptake (assessed via PET-CT) indicated only transient post-surgical inflammation. In conclusion, USPIO-labeling enables accurate localization of TEVG and opens up opportunities for multimodal imaging approaches to assess transplant acceptance and function. Thereby, it can support clinical decision-making on the need for further pharmacological or surgical interventions.

Evaluation of [Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2 h after intravenous injection of [In vitro experiments confirmed specific binding of [[

Compared to standard toxicological techniques in preclinical toxicity studies, non-invasive imaging of organ toxicity enables fast and longitudinal investigation of the whole animal. Therefore, we set out to evaluate [68Ga]Ga-NODAGA-duramycin as a positron emission tomography (PET)-tracer of cell death for detecting chemotherapy-induced organ toxicity.MethodsNODAGA-duramycin was radiolabeled with 68Ga, and quality control was done by thin layer chromatography and high performance liquid chromatography. Tracer specificity was determined in vitro by performing competitive binding experiments on ethanol treated cells. To optimize the timing of the PET/CT-based tracer evaluation, kinetic studies were performed in untreated and cisplatin-treated (20 mg/kg BW, intraperitoneal (i.p.)) BALB/cAnNRj mice. Organ uptake was analyzed in doxorubicin (4 mg/kg BW, i.p.)-, busulfan (18.8 mg/kg KG, i.p.)-, and cisplatin-treated (20 mg/kg BW, i.p.) mice, and in untreated control mice 2 hours after intravenous injection of 5–10 MBq [68Ga]Ga-NODAGA-duramycin. For immunofluorescence validation, tissue sections were stained with anti-active caspase-3 antibody. Blood and serum samples were collected to determine platelet count, aspartate transaminase, alanine transaminase, urea, creatinine, and creatine kinase values.ResultsIn vitro experiments confirmed specific binding of [68Ga]Ga-NODAGA-duramycin to dying cells. The biodistribution analysis revealed a blood half-life of 10–17 minutes and a predominantly urinary excretion of the radiotracer. Doxorubicin-, busulfan-, and cisplatin-induced organ toxicities were detected successfully using [68Ga]Ga-NODAGA-duramycin PET/CT and confirmed by immunohistochemistry as well as blood parameter analysis. Busulfan-related spleno-, cardio-, and pneumotoxicity as well as cisplatin-induced cardio- and pneumotoxicity were detected even earlier by [68Ga]Ga-NODAGA-duramycin PET/CT than by blood parameters and histological stainings. In livers and kidneys, differences between treated and untreated animals tended to occur in PET/CT at later time points than in histology due to the relatively high background in these organs. However, trends over time were comparable.Conclusion[68Ga]Ga-NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in preclinical studies. It even depicted some toxic effects prior to immunohistochemistry and blood parameter analysis and represents a promising alternative or complementary method to standard toxicological analyses. Furthermore, the tracer has a high translational potential and may provide a valuable link between preclinical and clinical research.

Ultrasound (US) imaging of heart and major arteries and veins is among the most frequently used diagnostic techniques applied in humans. Conventional cardiovascular US sessions include anatomical B-mode and functional M-, pulsed-wave- and Doppler mode, which have their limitations in both precise cardiac chambers' delineation and small vessel imaging. The introduction of contrast-enhanced US, employing microbubble suspensions as contrast agent, has enabled a better delineation of heart chambers, the visualization of myocardial microvasculature, and the atherosclerotic plaque neovascularization. Moreover, specific disease-related molecular tracers have been developed by modifying the microbubbles with targeting ligands directed to biological markers exposed to the luminal side of the blood vessels. Microbubble functionalization has enabled in vivo molecular US imaging of various stages of atherosclerosis, from plaque initiation to plaque vulnerability, and neointima formation following revascularization procedures. Furthermore, oscillating microbubbles have been used to mechanically dissolve thrombus material and may act as carriers of drugs and nucleic acids that are released locally by US pulses. This review article summarizes recent advances in functional and molecular US images and discusses therapeutic applications of microbubbles. The addressed topics include an overview on microbubble formats, microbubble detection methods, molecular targets of cardiovascular diseases, and the use of microbubbles for thrombolysis and drug delivery.

Interventions such as balloon angioplasty can cause vascular injury leading to platelet activation, thrombus formation, and inflammatory response. This induces vascular smooth muscle cell activation and subsequent re-endothelialization with expression of αvβ3-integrin by endothelial cells and vascular smooth muscle cell. Thus, poly-N-butylcyanoacrylate microbubbles (MBs) targeted to αvβ3-integrin were evaluated for monitoring vascular healing after vessel injury in pigs using molecular ultrasound imaging.Approval for animal experiments was obtained. The binding specificity of αvβ3-integrin-targeted MB to human umbilical vein endothelial cells was tested with fluorescence microscopy. In vivo imaging was performed using a clinical ultrasound system and an 8-MHz probe. Six mini pigs were examined after vessel injury in the left carotid artery. The right carotid served as control. Uncoated MB, cDRG-coated MB, and αvβ3-integrin-specific cRGD-coated MB were injected sequentially. Bound MBs were assessed 8 minutes after injection using ultrasound replenishment analysis. Measurements were performed 2 hours, 1 and 5 weeks, and 3 and 6 months after injury. In vivo data were validated by immunohistochemistry.Significantly stronger binding of cRGD-MB than MB and cDRG-MB to human umbilical vein endothelial cells was found (P0.01). As vessel injury leads to upregulation of αvβ3-integrin, cRGD-MBs bound significantly stronger (P0.05) in injured carotid arteries than at the counter side 1 week after vessel injury and significant differences could also be observed after 5 weeks. After 3 months, αvβ3-integrin expression decreased to baseline and binding of cRGD-MB was comparable in both vessels. Values remained at baseline also after 6 months.Ultrasound imaging with RGD-MB is promising for monitoring vascular healing after vessel injury. This may open new perspectives to assess vascular damage after radiological interventions.

Immune checkpoint inhibitors have shown clinical benefit in several cancer entities including metastatic microsatellite instable colorectal carcinomas. However, for the majority of metastatic colorectal carcinomas the potential and limitations of immune checkpoint inhibition is not fully understood. In this study, the effects of sole and dual CTLA-4 and PD-L1 blockade were investigated in a microsatellite stable highly aggressive orthotopic mouse model of colon cancer. Dual CTLA-4 and PD-L1 inhibition resulted in tumor growth stagnation and completely blocked liver metastasis. Sole CTLA-4 and PD-L1 inhibition only moderately reduced metastatic spread of the colon cancer cells, though CTLA-4 blockade being superior to PD-L1 inhibition. Dual immune checkpoint blockade and sole CTLA-4 inhibition significantly increased intratumoral CD8+ and CD4+ T cells and reduced FOXP3+/CD4+ Treg cells. This was associated with increased expression levels of the pro-inflammatory Th1/M1-related cytokines IFN-γ, IL-1α, IL-2, and IL-12. Moreover, tumors treated with combined immune checkpoint blockade showed the strongest increase in intratumoral iNOS+ macrophages, reduction of PD-L1+ and Tie2+ macrophages and the lowest expression of M2/Th2-related IL-4, TARC and COX-2. The assessment of further microenvironmental changes by DCE-MRI and immunohistology revealed no alterations in functional tumor vascularization upon combined immune checkpoint blockade, but a significant increase in intratumoral fibroblasts and collagen I deposition. Thus, the synergistic inhibitory effects of dual immune checkpoint inhibition can be explained by anti-tumorigenic T cell responses mediated by CTLA-4 inhibition and M1 macrophage polarization predominantly induced by PD-L1 blockade. This was accompanied by pronounced fibroblast activation highlighting the interconnection between immunogenicity and desmoplasia.

Photoacoustic imaging presents an innocuous imaging modality with good penetration depth and resolution. To use this modality for detection and imaging of pathological sites, new imaging probes need to be developed to enhance the contrast over endogenous sonophores. These contrast agents should specifically bind to the site of interest, be non-toxic and be cleared renally if applied intravenously. Small organic dyes with absorption in the near infrared spectrum often exhibit good photoacoustic response. However, such dyes are often not water soluble or they are cytotoxic. Here, we present a novel PEGylated sonophore based on diketopyrrolopyrrole (DPP), which overcomes these limitations and can be functionalized with desired biological recognition motifs using thiol–yne click chemistry. Proof of concept is demonstrated by functionalizing the DPP-based probe with an RGD peptide, resulting in specific binding to endothelial (HUVEC) cells and an efficient photoacoustic response.

Ultrasound is among the most rapidly advancing imaging techniques. Functional methods such as elastography have been clinically introduced, and tissue characterization is improved by contrast-enhanced scans. Here, novel super-resolution techniques provide unique morphological and functional insights into tissue vascularisation. Functional analyses are complemented with molecular ultrasound imaging, to visualize markers of inflammation and angiogenesis. The full potential of diagnostic ultrasound may become apparent by integrating these multiple imaging features in radiomics approaches. Emerging interest in ultrasound also results from its therapeutic potential. Various applications on tumor ablation with high intensity focused ultrasound (HIFU) are clinically evaluated and its performance strongly benefits from the integration into Magnetic Resonance Imaging (MRI). Additionally, oscillating microbubbles mediate sonoporation to open biological barriers, thus improving the delivery of drugs or nucleic acids that are co-administered or co-formulated with microbubbles. This article provides an overview of recent developments in diagnostic and therapeutic ultrasound, highlighting multiple innovation tracks and their translational potential.

Super-resolution imaging methods promote tissue characterization beyond the spatial resolution limits of the devices and bridge the gap between histopathological analysis and non-invasive imaging. Here, we introduce Ultrasound Bubble Tracking (UBT) as an easily applicable and robust new tool to morphologically and functionally characterize fine vascular networks in tumors at super-resolution. In tumor-bearing mice and for the first time in patients, we demonstrate that within less than one minute scan time UBT can be realized using conventional preclinical and clinical ultrasound devices. In this context, next to highly detailed images of tumor microvascularization and the reliable quantification of relative blood volume and perfusion, UBT provides access to multiple new functional and morphological parameters that showed superior performance in discriminating tumors with different vascular phenotypes. Furthermore, our initial clinical results indicate that UBT is a highly translational technology with strong potential for the multiparametric characterization of tumors and the assessment of therapy response.

Super-resolution imaging methods promote tissue characterization beyond the spatial resolution limits of the devices and bridge the gap between histopathological analysis and non-invasive imaging. Here, we introduce motion model ultrasound localization microscopy (mULM) as an easily applicable and robust new tool to morphologically and functionally characterize fine vascular networks in tumors at super-resolution. In tumor-bearing mice and for the first time in patients, we demonstrate that within less than 1 min scan time mULM can be realized using conventional preclinical and clinical ultrasound devices. In this context, next to highly detailed images of tumor microvascularization and the reliable quantification of relative blood volume and perfusion, mULM provides multiple new functional and morphological parameters that discriminate tumors with different vascular phenotypes. Furthermore, our initial patient data indicate that mULM can be applied in a clinical ultrasound setting opening avenues for the multiparametric characterization of tumors and the assessment of therapy response., The vascular structure of tumors impacts diagnosis, prognosis and drug response; however, imaging methods to analyse this important feature have been hindered by spatial resolution limitations. Here the authors present a tool called motion model ultrasound localization microscopy to morphologically and functionally characterize fine vascular networks in tumors at super-resolution.

Recombinant human erythropoietin (rhuEpo) is used clinically to treat anaemia. However, rhuEpo-treated cancer patients show decreased survival rates and erythropoietin receptor (EpoR) expression has been found in patient tumour tissue. Thus, rhuEpo application might promote EpoR(+) tumour progression. We therefore developed the positron emission tomography (PET)-probe (68)Ga-DOTA-rhuEpo and evaluated its performance in EpoR(+) A549 non-small-cell lung cancer (NSCLC) xenografts.(68)Ga-DOTA-rhuEpo was generated by coupling DOTA-hydrazide to carbohydrate side-chains of rhuEpo. Biodistribution was determined in tumour-bearing mice 0.5, 3, 6, and 9 h after probe injection. Competition experiments were performed by co-injecting (68)Ga-DOTA-rhuEpo and rhuEpo in five-fold excess. Probe specificity was further evaluated histologically using Epo-Cy5.5 stainings.The blood half-life of (68)Ga-DOTA-rhuEpo was 2.6 h and the unbound fraction was cleared by the liver and kidney. After 6 h, the highest tumour to muscle ratio was reached. The highest (68)Ga-DOTA-rhuEpo accumulation was found in liver (10.06 ± 6.26%ID/ml), followed by bone marrow (1.87 ± 0.53%ID/ml), kidney (1.58 ± 0.39%ID/ml), and tumour (0.99 ± 0.16%ID/ml). EpoR presence in these organs was histologically confirmed. Competition experiments showed significantly (p 0.05) lower PET-signals in tumour and bone marrow at 3 and 6 h.(68)Ga-DOTA-rhuEpo shows favourable pharmacokinetic properties and detects EpoR specifically. Therefore, it might become a valuable radiotracer to monitor EpoR status in tumours and support decision-making in anaemia therapy.• PET-probe (68) Ga-DOTA-rhuEpo was administered to assess the EpoR status in vivo • (68) Ga-DOTA-rhuEpo binds specifically to EpoR positive organs in vivo • Tumour EpoR status determination might enable decision-making in anaemia therapy with rhuEpo.

To investigate the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted ultrasonographic (US) microbubbles for the assessment of liver dysplasia in transgenic mice.Animal experiments were approved by the governmental review committee. Nuclear factor-κB essential modulator knock-out mice with liver dysplasia and wild-type mice underwent liver imaging by using a clinical US system. Two types of contrast agents were investigated: nontargeted, commercially available, second-generation microbubbles (SonoVue) and clinically translatable PEGylated VEGFR2-targeted microbubbles (BR55). Microbubble kinetics was investigated over the course of 4 minutes. Targeted contrast material-enhanced US signal was quantified 5 minutes after injection. Competitive in vivo binding experiments with BR55 were performed in knock-out mice. Immunohistochemical and hematoxylin-eosin staining of liver sections was performed to validate the in vivo US results. Groups were compared by using the Mann-Whitney test.Peak enhancement after injection of SonoVue and BR55 did not differ in healthy and dysplastic livers (SonoVue, P = .46; BR55, P = .43). Accordingly, immunohistochemical findings revealed comparable vessel densities in both groups. The specificity of BR55 to VEGFR2 was proved by in vivo competition (P = .0262). While the SonoVue signal decreased similarly in healthy and dysplastic livers during the 4 minutes, there was an accumulation of BR55 in dysplastic livers compared with healthy ones. Furthermore, targeted contrast-enhanced US signal indicated a significantly higher site-specific binding of BR55 in dysplastic than healthy livers (P = .005). Quantitative immunohistologic findings confirmed significantly higher VEGFR2 levels in dysplastic livers (P = .02).BR55 enables the distinction of early stages of liver dysplasia from normal liver.

To compare non-enhanced and contrast-enhanced high-frequency 3D Doppler ultrasound with contrast-enhanced 2D and 3D B-mode imaging for assessing tumor vascularity during antiangiogenic treatment using soft-shell and hard-shell microbubbles.Antiangiogenic therapy effects (SU11248) on vascularity of subcutaneous epidermoid-carcinoma xenografts (A431) in female CD1 nude mice were investigated longitudinally using non-enhanced and contrast-enhanced 3D Doppler at 25 MHz. Additionally, contrast-enhanced 2D and 3D B-mode scans were performed by injecting hard-shell (poly-butyl-cyanoacrylate-based) and soft-shell (phospholipid-based) microbubbles. Suitability of both contrast agents for high frequency imaging and the sensitivity of the different ultrasound methods to assess early antiangiogenic therapy effects were investigated. Ultrasound data were validated by immunohistology.Hard-shell microbubbles induced higher signal intensity changes in tumors than soft-shell microbubbles in 2D B-mode measurements (424 ± 7 vs. 169 ± 8 A.U.; p0.01). In 3D measurements, signals of soft-shell microbubbles were hardly above the background (5.48 ± 4.57 vs. 3.86 ± 2.92 A.U.), while signals from hard-shell microbubbles were sufficiently high (30.5 ± 8.06 A.U). Using hard-shell microbubbles 2D and 3D B-mode imaging depicted a significant decrease in tumor vascularity during antiangiogenic therapy from day 1 on. Using soft-shell microbubbles significant therapy effects were observed at day 4 after therapy in 2D B-mode imaging but could not be detected in the 3D mode. With non-enhanced and contrast-enhanced Doppler imaging significant differences between treated and untreated tumors were found from day 2 on.Hard-shell microbubble-enhanced 2D and 3D B-mode ultrasound achieved highest sensitivity for assessing therapy effects on tumor vascularisation and were superior to B-mode ultrasound with soft-shell microbubbles and to Doppler imaging.

Our objective was to determine the lowest diagnostically effective dose for E-selectin-targeted poly n-butyl cyanoacrylate (PBCA)-shelled microbubbles and to apply it to monitor antiangiogenic therapy effects. PBCA-shelled microbubbles (MBs) coupled to an E-selectin-specific peptide were applied in mice carrying MLS or A431 carcinoma xenografts scaling down the MB dosage to the lowest level where binding could be examined with a 18-MHz small animal ultrasound transducer. Differences in E-selectin expression in the two carcinoma xenografts were confirmed by enzyme-linked immunosorbent assay (ELISA). In addition, MLS tumor-bearing mice under antiangiogenic therapy were monitored using E-selectin-targeted MBs at the lowest applicable dose. Therapy effects on tumor vascularization were verified by immunohistological analyses. The minimally required dosage was 7 × 107 MBs/kg body weight. This dosage was sufficient to enable E-selectin detection in high E-selectin-expressing MLS tumors, while low E-selectin-expressing A431 tumors required almost 2.5-fold higher doses. At the dose of 7 × 107 MBs/kg body weight, a decrease in E-selectin MB binding under antiangiogenic therapy could be assessed (being significant after 3 days of treatment; p

Purpose: To assess the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)–targeted and nontargeted ultrasonography (US) to depict antiangiogenic therapy effects and to investigate whether first-pass kinetics obtained with VEGFR2-targeted microbubbles provide independent data about tumor vascularization. Materials and Methods: Governmental approval was obtained for animal experiments. Vascularization in response to anti–vascular endothelial growth factor receptor or vehicle-control treatment (10 per group) in HaCaT-ras A-5RT3 xenografts was longitudinally assessed in mice by means of first-pass kinetics of nontargeted microbubbles (BR1, BR38; Bracco, Geneva, Switzerland) and VEGFR2-targeted microbubbles (BR55, Bracco) before and 4, 7, and 14 days after therapy. VEGFR2 expression was determined 8 minutes after BR55 injection with destruction-replenishment analysis. US data were validated with immunohistochemistry. Significant differences were evaluated with the Mann-Whitney test. Results: First-pass analysis with BR1, BR38, and BR55 showed similar tendencies toward decreasing vascularization, with a stronger decrease in tumors treated with anti-VEGF antibody. The median signal intensity (in arbitrary units [au]) of anti-VEGF antibody–treated versus control tumors at day 14 was as follows: BR1, 5.2 au (interquartile range [IQR], 3.2 au) vs 11.3 au (IQR, 10.0 au), respectively; BR38, 6.2 au (IQR, 3.5) vs 10.0 au (IQR, 7.8); and BR55, 9.5 au (IQR, 6.0 au) vs 13.8 au (IQR, 9.8) (P = .0230). VEGFR2 assessment with BR55 demonstrated significant differences between both groups throughout the therapy period (median signal intensity of anti-VEGF antibody–treated vs control tumors: 0.04 au [IQR, 0.1 au] vs 0.14 au [IQR, 0.08 au], respectively, at day 4, P = .0058; 0.04 au [IQR, 0.06 au] vs 0.13 au [IQR, 0.09 au] at day 7, P = .0058; and 0.06 au [IQR, 0.11 au] vs 0.16 au [IQR, 0.15 au] at day 14, P = .0247). Immunohistochemistry confirmed the lower microvessel density and VEGFR2-positive area fraction in tumors treated with anti-VEGF antibody. Conclusion: Antiangiogenic therapy effects were detected earlier and more distinctly with VEGFR2-targeted US than with functional US. First-pass analyses with BR55, BR38, and BR1 revealed similar results, with a decrease in vascularization during therapy. Functional data showed that BR55 is not strongly affected by early binding of the microbubbles to VEGFR2. Thus, functional and molecular imaging of angiogenesis can be performed with BR55 within one examination.

Theranostics 5(8), 905-918 (2015). doi:10.7150/thno.11304, Published by Ivyspring, Wyoming, NSW

Chemical communications 51(45), 9358-9361(2015). doi:10.1039/C4CC10092A, Published by Royal Society of Chemistry, Cambridge

OBJECTIVES The purposes of this study were the development and preclinical evaluation of clinically translatable E-selectin-specific ultrasound contrast agents based on a peptide ligand with the recognition sequence IELLQAR. MATERIALS AND METHODS The E-selectin-specific peptide was synthesized through solid phase peptide synthesis and covalently attached to poly n-butylcyanoacrylate-stabilized microbubbles with an air core. Quantification of the microbubble surface coverage with peptides was performed through flow cytometry. Targeted adhesion of peptide-coated microbubbles was investigated in vitro using parallel plate flow chamber assays on tumor necrosis factor-α-stimulated human umbilical vein endothelial cells. In vivo imaging was performed in nude mice bearing human ovarian carcinoma xenografts (MLS), followed by ex vivo immunohistochemistry validation of E-selectin expression. RESULTS Success of peptide synthesis was validated through preparative reverse phase high-pressure liquid chromatography and electronspray ionization-mass spectrometry. Results of the flow cytometry revealed approximately 4000 E-selectin-specific peptides/microbubble surface. Results of the in vitro experiments demonstrated the specificity of peptide-coated microbubbles to E-selectin (1.10 ± 0.48 vs 0.19 ± 0.09 bound microbubbles per cell, before and after competition respectively; P < 0.01). The in vivo imaging enabled specific assessment of E-selectin expression in MLS carcinoma xenografts (5.21 ± 3.41 vs 1.37 ± 0.67 contrast intensity before and after competition, respectively; P < 0.05). CONCLUSIONS Clinically translatable microbubbles that were covalently coupled to the short E-selectin-specific peptide (IELLQAR) enabled specific imaging of the E-selectin expression in tumor vessels in vivo.

Quantitative contrast-enhanced ultrasound plays an important role in tumor characterization and treatment assessment. Besides established functional ultrasound techniques, ultrasound molecular imaging using microbubbles targeted to disease-associated markers is increasingly being applied in pre-clinical studies. Often, repeated injections of non-targeted or targeted microbubbles during the same imaging session are administered. However, the influence of repeated injections on the accuracy of the quantitative data is unclear. Therefore, in tumor-bearing mice, we investigated the influence of multiple injections of non-targeted microbubbles (SonoVue) on time to peak and peak enhancement in liver and tumor tissue and of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agents (MicroMarker) on specific tumor accumulation. We found significantly decreasing values for time to peak and a tendency for increased values for peak enhancement after multiple injections. Repeated injections of VEGFR2-targeted microbubbles led to significantly increased tumor accumulation, which may result from the exposure of additional binding sites at endothelial surfaces caused by mechanical forces from destroyed microbubbles.