Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy.

BACKGROUND: With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy. METHODS: Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry. RESULTS: During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models. CONCLUSION: Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

Development, synthesis and evaluation of novel fluorescent Endothelin-B receptor probes.

The endothelin (ET) signaling system is comprised of three endothelin peptides (ET-1, -2 and -3) and two corresponding endothelin-A and -B receptors (ETAR and ETBR), which belong to the G-protein coupled receptor (GPCR) superfamily. The endothelin axis, as this system is also referred to, contributes to the maintenance of vascular tone, functions as regulator of inflammation and proliferation and helps in balancing water homeostasis. In pathological settings, the ET axis is known to contribute to endothelial activation in cardiovascular diseases, to cell proliferation, chemoresistance and metastasis in cancer and to inflammation and fibrosis in renal disease. Antagonists of ETAR and ETBR, either subtype-specific compounds or substances with high affinity to both receptors, have been developed for more than 30 years. In the preclinical context, in vivo imaging of endothelin receptor expression has been utilized to assess ET-axis contribution to e.g. cancer or myocardial infarction. In this work, we present the development and synthesis of two novel ETBR-specific fluorescent probes, based on the available antagonists BQ788 and IRL2500 and their preliminary evaluation in a breast cancer context.

Detection of Early Endothelial Dysfunction by Optoacoustic Tomography.

Variations in vascular wall shear stress are often presumed to result in the formation of atherosclerotic lesions at specific arterial regions, where continuous laminar flow is disturbed. The influences of altered blood flow dynamics and oscillations on the integrity of endothelial cells and the endothelial layer have been extensively studied in vitro and in vivo. Under pathological conditions, the Arg-Gly-Asp (RGD) motif binding integrin αvß3 has been identified as a relevant target, as it induces endothelial cell activation. Animal models for in vivo imaging of endothelial dysfunction (ED) mainly rely on genetically modified knockout models that develop endothelial damage and atherosclerotic plaques upon hypercholesterolemia (ApoE-/- and LDLR-/-), thereby depicting late-stage pathophysiology. The visualization of early ED, however, remains a challenge. Therefore, a carotid artery cuff model of low and oscillating shear stress was applied in CD-1 wild-type mice, which should be able to show the effects of altered shear stress on a healthy endothelium, thus revealing alterations in early ED. Multispectral optoacoustic tomography (MSOT) was assessed as a non-invasive and highly sensitive imaging technique for the detection of an intravenously injected RGD-mimetic fluorescent probe in a longitudinal (2-12 weeks) study after surgical cuff intervention of the right common carotid artery (RCCA). Images were analyzed concerning the signal distribution upstream and downstream of the implanted cuff, as well as on the contralateral side as a control. Subsequent histological analysis was applied to delineate the distribution of relevant factors within the carotid vessel walls. Analysis revealed a significantly enhanced fluorescent signal intensity in the RCCA upstream of the cuff compared to the contralateral healthy side and the downstream region at all time points post-surgery. The most obvious differences were recorded at 6 and 8 weeks after implantation. Immunohistochemistry revealed a high degree of αv-positivity in this region of the RCCA, but not in the left common carotid artery (LCCA) or downstream of the cuff. In addition, macrophages could be detected by CD68 immunohistochemistry in the RCCA, showing ongoing inflammatory processes. In conclusion, MSOT is capable of delineating alterations in endothelial cell integrity in vivo in the applied model of early ED, where an elevated expression of integrin αvß3 was detected within vascular structures.

Vascular response patterns to targeted therapies in murine breast cancer models with divergent degrees of malignancy.

BACKGROUND: Response assessment of targeted cancer therapies is becoming increasingly challenging, as it is not adequately assessable with conventional morphological and volumetric analyses of tumor lesions. The tumor microenvironment is particularly constituted by tumor vasculature which is altered by various targeted therapies. The aim of this study was to noninvasively assess changes in tumor perfusion and vessel permeability after targeted therapy in murine models of breast cancer with divergent degrees of malignancy. METHODS: Low malignant 67NR or highly malignant 4T1 tumor-bearing mice were treated with either the multi-kinase inhibitor sorafenib or immune checkpoint inhibitors (ICI, combination of anti-PD1 and anti-CTLA4). Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with i.v. injection of albumin-binding gadofosveset was conducted on a 9.4 T small animal MRI. Ex vivo validation of MRI results was achieved by transmission electron microscopy, immunohistochemistry and laser ablation-inductively coupled plasma-mass spectrometry. RESULTS: Therapy-induced changes in tumor vasculature differed between low and highly malignant tumors. Sorafenib treatment led to decreased tumor perfusion and endothelial permeability in low malignant 67NR tumors. In contrast, highly malignant 4T1 tumors demonstrated characteristics of a transient window of vascular normalization with an increase in tumor perfusion and permeability early after therapy initiation, followed by decreased perfusion and permeability parameters. In the low malignant 67NR model, ICI treatment also mediated vessel-stabilizing effects with decreased tumor perfusion and permeability, while ICI-treated 4T1 tumors exhibited increasing tumor perfusion with excessive vascular leakage. CONCLUSION: DCE-MRI enables noninvasive assessment of early changes in tumor vasculature after targeted therapies, revealing different response patterns between tumors with divergent degrees of malignancy. DCE-derived tumor perfusion and permeability parameters may serve as vascular biomarkers that allow for repetitive examination of response to antiangiogenic treatment or immunotherapy.

Profiling specific cell populations within the inflammatory tumor microenvironment by oscillating-gradient diffusion-weighted MRI.

BACKGROUND: The inflammatory tumor microenvironment (TME) is formed by various immune cells, being closely associated with tumorigenesis. Especially, the interaction between tumor-infiltrating T-cells and macrophages has a crucial impact on tumor progression and metastatic spread. The purpose of this study was to investigate whether oscillating-gradient diffusion-weighted MRI (OGSE-DWI) enables a cell size-based discrimination between different cell populations of the TME. METHODS: Sine-shaped OGSE-DWI was combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) approach to measure microscale diffusion distances, here relating to cell sizes. The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages, or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1, 67NR) during tumor progression, clodronate liposome-mediated depletion of macrophages, and immune checkpoint inhibitor (ICI) treatment. Ex vivo analysis of IMPULSED-derived cell radii was conducted by immunohistochemical wheat germ agglutinin staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining. RESULTS: OGSE-DWI detected mean cell radii of 8.8±1.3 µm for 4T1, 8.2±1.4 µm for 67NR, 13.0±1.7 for macrophage, and 3.8±1.8 µm for T-cell spheroids. While T-cell infiltration during progression of 4T1 tumors was observed by decreasing mean cell radii from 9.7±1.0 to 5.0±1.5 µm, increasing amount of intratumoral macrophages during progression of 67NR tumors resulted in increasing mean cell radii from 8.9±1.2 to 12.5±1.1 µm. After macrophage depletion, mean cell radii decreased from 6.3±1.7 to 4.4±0.5 µm. T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models, with more pronounced effects in the 67NR tumor model. CONCLUSIONS: OGSE-DWI provides a versatile tool for non-invasive profiling of the inflammatory TME by assessing the dominating cell type T-cells or macrophages.

Multiparametric MRI enables for differentiation of different degrees of malignancy in two murine models of breast cancer.

Objective: The objective of this study was to non-invasively differentiate the degree of malignancy in two murine breast cancer models based on identification of distinct tissue characteristics in a metastatic and non-metastatic tumor model using a multiparametric Magnetic Resonance Imaging (MRI) approach. Methods: The highly metastatic 4T1 breast cancer model was compared to the non-metastatic 67NR model. Imaging was conducted on a 9.4 T small animal MRI. The protocol was used to characterize tumors regarding their structural composition, including heterogeneity, intratumoral edema and hemorrhage, as well as endothelial permeability using apparent diffusion coefficient (ADC), T1/T2 mapping and dynamic contrast-enhanced (DCE) imaging. Mice were assessed on either day three, six or nine, with an i.v. injection of the albumin-binding contrast agent gadofosveset. Ex vivo validation of the results was performed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), histology, immunhistochemistry and electron microscopy. Results: Significant differences in tumor composition were observed over time and between 4T1 and 67NR tumors. 4T1 tumors showed distorted blood vessels with a thin endothelial layer, resulting in a slower increase in signal intensity after injection of the contrast agent. Higher permeability was further reflected in higher Ktrans values, with consecutive retention of gadolinium in the tumor interstitium visible in MRI. 67NR tumors exhibited blood vessels with a thicker and more intact endothelial layer, resulting in higher peak enhancement, as well as higher maximum slope and area under the curve, but also a visible wash-out of the contrast agent and thus lower Ktrans values. A decreasing accumulation of gadolinium during tumor progression was also visible in both models in LA-ICP-MS. Tissue composition of 4T1 tumors was more heterogeneous, with intratumoral hemorrhage and necrosis and corresponding higher T1 and T2 relaxation times, while 67NR tumors mainly consisted of densely packed tumor cells. Histogram analysis of ADC showed higher values of mean ADC, histogram kurtosis, range and the 90th percentile (p90), as markers for the heterogenous structural composition of 4T1 tumors. Principal component analysis (PCA) discriminated well between the two tumor models. Conclusions: Multiparametric MRI as presented in this study enables for the estimation of malignant potential in the two studied tumor models via the assessment of certain tumor features over time.

In vivo imaging of microenvironmental and anti-PD-L1-mediated dynamics in cancer using S100A8/S100A9 as an imaging biomarker.

PURPOSE: As a promotor of tumor invasion and tumor microenvironment (TME) formation, the protein complex S100A8/S100A9 is associated with poor prognosis. Our aim was to further evaluate its origin and regulatory effects, and to establish an imaging biomarker for TME activity. METHODS: S100A9-/-cells (ko) were created from syngeneic murine breast cancer 4T1 (high malignancy) and 67NR (low malignancy) wildtype (wt) cell lines and implanted into either female BALB/c wildtype or S100A9-/- mice (n = 10 each). Anti-S100A9-Cy5.5-targeted fluorescence reflectance imaging was performed at 0 h and 24 h after injection. Potential early changes of S100A9-presence under immune checkpoint inhibition (anti-PD-L1, n = 7 vs. rat IgG2b as isotype control, n = 3) were evaluated. RESULTS: In S100A9-/-mice contrast-to-noise-ratios were significantly reduced for wt and S100A9-/-tumors. No significant differences were detected for 4T1 ko and 67NR ko cells as compared to wildtype cells. Under anti-PD-L1 treatment S100A9 presence significantly decreased compared with the control group. CONCLUSION: Our results confirm a secretion of S100A8/S100A9 by the TME, while tumor cells do not apparently release the protein. Under immune checkpoint inhibition S100A9-imaging reports an early decrease of TME activity. Therefore, S100A9-specific imaging may serve as an imaging biomarker for TME formation and activity.

How Different Albumin-Binders Drive Probe Distribution of Fluorescent RGD Mimetics.

The biodistribution of medical imaging probes depends on the chemical nature of the probe and the preferred metabolization and excretion routes. Especially targeted probes, which have to reach a certain (sub)cellular destination, have to be guided to the tissue of interest. Therefore, small molecular probes need to exhibit a well-balanced polarity and lipophilicity to maintain an advantageous bioavailability. Labelled antibodies circulate for several days due to their size. To alter the biodistribution behavior of probes, different strategies have been pursued, including utilizing serum albumin as an inherent transport mechanism for small molecules. We describe here the modification of an existing fluorescent RGD mimetic probe targeted to integrin αvß3 with three different albumin binding moieties (ABMs): a diphenylcyclohexyl (DPCH) group, a p-iodophenyl butyric acid (IPBA) and a fatty acid (FA) group with the purpose to identify an optimal ABM for molecular imaging applications. All three modifications result in transient albumin binding and a preservation of the target binding capability. Spectrophotometric measurements applying variable amounts of bovine serum albumin (BSA) reveal considerable differences between the compounds concerning their absorption and emission characteristics and hence their BSA binding mode. In vivo the modified probes were investigated in a murine U87MG glioblastoma xenograft model over the course of 1 wk by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT). While the unmodified probe was excreted rapidly, the albumin-binding probes were accumulating in tumor tissue for at least 5 days. Considerable differences between the three probes in biodistribution and excretion characteristics were proved, with the DPCH-modified probe showing the highest overall signal intensities, while the FA-modified probe exhibits a low but more specific fluorescent signal. In conclusion, the modification of small molecular RGD mimetics with ABMs can precisely fine-tune probe distribution and offers potential for future clinical applications.

Exploring the Influence of Different Albumin Binders on Molecular Imaging Probe Distribution.

The biodistribution of molecular imaging probes or tracers mainly depends on the chemical nature of the probe and the preferred metabolization and excretion routes. Small molecules have rather short half-lives while antibodies reside inside the organism for a longer period of time. An excretion via kidneys and bladder is faster than a mainly hepatobiliary elimination. To manipulate the biodistribution behavior of probes, different strategies have been pursued, including utilizing serum albumin as an inherent transport mechanism for small molecules. Here, we modified an existing small molecular fluorescent probe targeted to the endothelin-A receptor (ETAR) with three different albumin-binding moieties to search for an optimal modification strategy. A diphenylcyclohexyl (DPCH) group, a p-iodophenyl butyric acid (IPBA), and a fatty acid (FA) group were attached via amino acid linkers. All three modifications result in transient albumin binding of the developed compounds, as concluded from gel electrophoresis investigations. Spectrophotometric measurements applying variable amounts of bovine, murine, and human serum albumin (BSA, MSA, and HSA) reveal distinct variations of absorption and emission intensities and shifts of their maximum wavelengths. Binding to MSA results in the weakest effects, while binding to HSA leads to the strongest. Cell-based in vitro investigations utilizing ETAR-positive HT-1080 fibrosarcoma and ETAR-negative BT-20 breast adenocarcinoma cells support a retained specific target-binding capacity of the modified compounds and different degrees of unspecific binding. In vivo analysis of a HT-1080 xenograft model in nude mice over the course of 1 week by fluorescence reflectance imaging illustrates noticeable differences between the four examined probes. While the IPBA-modified probe shows the highest absolute signal intensity values, the FA-modified probe exhibits the most favorable tumor-to-organ ratios. In summary, reversible binding to albumin enhances the biological half-life of the designed probes substantially and enables near infrared optical imaging of subcutaneous tumors for several days in vivo. Because the unmodified probe already exhibits reasonable results, the attachment of albumin-binding moieties does not lead to a substantially improved imaging outcome in terms of target-to-background ratios. On the other hand, because the implemented transient albumin binding results in an overall higher amount of probe inside tumor lesions, this strategy might be adaptable for theranostic or therapeutic approaches in a future clinical routine.

S100A9-Imaging Enables Estimation of Early Therapy-Mediated Changes in the Inflammatory Tumor Microenvironment.

(1) Background: The prognosis of cancer is dependent on immune cells in the tumor microenvironment (TME). The protein S100A9 is an essential regulator of the TME, associated with poor prognosis. In this study, we evaluated early therapy effects on the TME in syngeneic murine breast cancer via S100A9-specific in vivo imaging. (2) Methods: Murine 4T1 cells were implanted orthotopically in female BALB/c mice (n = 59). Tumor size-adapted fluorescence imaging was performed before and 5 days after chemo- (Doxorubicin, n = 20), anti-angiogenic therapy (Bevacizumab, n = 20), or placebo (NaCl, n = 19). Imaging results were validated ex vivo (immunohistochemistry, flow cytometry). (3) Results: While tumor growth revealed no differences (p = 0.48), fluorescence intensities (FI) for S100A9 in Bevacizumab-treated tumors were significantly lower as compared to Doxorubicin (2.60 vs. 15.65 AU, p < 0.0001). FI for Doxorubicin were significantly higher compared to placebo (8.95 AU, p = 0.01). Flow cytometry revealed shifts in monocytic and T-cell cell infiltrates under therapy, correlating with imaging. (4) Conclusions: S100A9-specific imaging enables early detection of therapy effects visualizing immune cell activity in the TME, even before clinically detectable changes in tumor size. Therefore, it may serve as a non-invasive imaging biomarker for early therapy effects.

Monitoring Endothelin-A Receptor Expression during the Progression of Atherosclerosis.

Cardiovascular disease remains the most frequent cause of death worldwide. Atherosclerosis, an underlying cause of cardiovascular disease, is an inflammatory disorder associated with endothelial dysfunction. The endothelin system plays a crucial role in the pathogenesis of endothelial dysfunction and is involved in the development of atherosclerosis. We aimed to reveal the expression levels of the endothelin-A receptor (ETAR) in the course of atherogenesis to reveal possible time frames for targeted imaging and interventions. We used the ApoE-/- mice model and human specimens and evaluated ETAR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ETAR after 22 weeks of high-fat diet in the aortae of ApoE-/- mice. With regard to translation to human disease, we applied the fluorescent probe to fresh explants of human carotid and femoral artery specimens. The findings were correlated with qPCR and histology. While ETAR is upregulated during the progression of early atherosclerosis in the ApoE-/- mouse model, we found that ETAR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ETAR-Cy 5.5 probe confirming its specificity and potential use in future studies.

Mass Spectrometry Imaging of atherosclerosis-affine Gadofluorine following Magnetic Resonance Imaging.

Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr-/- mice were investigated by high-field MRI (7 T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis.

Target-Specific Imaging of Cathepsin and S100A8/A9 Reflects Specific Features of Malignancy and Enables Estimation of Tumor Malignancy.

PURPOSE: Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential. PROCEDURES: Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry. RESULTS: At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p < 0.0001 each), reflecting local vessel invasion and tumor cell shedding. Immunohistology supported the in vivo imaging results. CONCLUSIONS: Non-invasive in vivo imaging of S100A8 and monocytic proteases allows for differentiation of the tumors' local invasive and systemic metastatic potential in reflecting the TME formation. While proteases augment local tumor cell invasion, solid metastases seem to be dependent on a pro-tumoral microenvironment.

Targeting Endothelin Receptors in a Murine Model of Myocardial Infarction Using a Small Molecular Fluorescent Probe.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

Introducing Specificity to Iron Oxide Nanoparticle Imaging by Combining 57Fe-Based MRI and Mass Spectrometry.

Iron oxide nanoparticles (ION) are highly sensitive probes for magnetic resonance imaging (MRI) that have previously been used for in vivo cell tracking and have enabled implementation of several diagnostic tools to detect and monitor disease. However, the in vivo MRI signal of ION can overlap with the signal from endogenous iron, resulting in a lack of detection specificity. Therefore, the long-term fate of administered ION remains largely unknown, and possible tissue deposition of iron cannot be assessed with established methods. Herein, we combine nonradioactive 57Fe-ION MRI with ex vivo laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging, enabling unambiguous differentiation between endogenous iron (56Fe) and iron originating from applied ION in mice. We establish 57Fe-ION as an in vivo MRI sensor for cell tracking in a mouse model of subcutaneous inflammation and for assessing the long-term fate of 57Fe-ION. Our approach resolves the lack of detection specificity in ION imaging by unambiguously recording a 57Fe signature.

Optoacoustic properties of Doxorubicin - A pilot study.

Doxorubicin (DOX) is a widely used chemotherapeutic anticancer drug. Its intrinsic fluorescence properties enable investigation of tumor response, drug distribution and metabolism. First phantom studies in vitro showed optoacoustic property of DOX. We therefore aimed to further investigate the optoacoustic properties of DOX in biological tissue in order to explore its potential as theranostic agent. We analysed doxorubicin hydrochloride (Dox·HCl) and liposomal encapsulated doxorubicin hydrochloride (Dox·Lipo), two common drugs for anti-cancer treatment in clinical medicine. Optoacoustic measurements revealed a strong signal of both doxorubicin substrates at 488 nm excitation wavelength. Post mortem analysis of intra-tumoral injections of DOX revealed a detectable optoacoustic signal even at three days after the injection. We thereby demonstrate the general feasibility of doxorubicin detection in biological tissue by means of optoacoustic tomography, which could be applied for high resolution imaging at mesoscopic depths dictated by effective penetration of visible light into the biological tissues.

isoDGR-Peptides for Integrin Targeting: Is the Time Up for RGD?

Integrins are the main cell adhesion mediators on the cell surface. Especially integrin αvß3 has gained a lot of attention as a target in cancer therapy because it mediates diverse angiogenic processes during tumor development. The peptide sequence Arg-Gly-Asp (RGD), which is present in a number of endogenous integrin ligands like fibronectin, vitronectin, and related proteins of the extracellular matrix (ECM), has been extensively used as a targeting vector for therapeutic as well as diagnostic purposes, and cilengitide, a cyclic RGD peptide, has entered clinical trials for the treatment of various cancers. However, recent advancements utilizing isoDGR, a sequence that was found in aged fibronectin, already show that RGD-based targeting is not the end of the line. Novel developments and a closer investigation of the binding mode of these peptides now show promising results for the future use of such compounds.

Molecular imaging of myocardial infarction with Gadofluorine P - A combined magnetic resonance and mass spectrometry imaging approach.

BACKGROUND: Molecular MRI is becoming increasingly important for preclinical research. Validation of targeted gadolinium probes in tissue however has been cumbersome up to now. Novel methodology to assess gadolinium distribution in tissue after in vivo application is therefore needed. PURPOSE: To establish combined Magnetic Resonance Imaging (MRI) and Mass Spectrometry Imaging (MSI) for improved detection and quantification of Gadofluorine P deposition in scar formation and myocardial remodeling. MATERIALS AND METHODS: Animal studies were performed according to institutionally approved protocols. Myocardial infarction was induced by permanent ligation of the left ascending artery (LAD) in C57BL/6J mice. MRI was performed at 7T at 1 week and 6 weeks after myocardial infarction. Gadofluorine P was used for dynamic T1 mapping of extracellular matrix synthesis during myocardial healing and compared to Gd-DTPA. After in vivo imaging contrast agent concentration as well as distribution in tissue were validated and quantified by spatially resolved Matrix-Assisted Laser Desorption Ionization (MALDI) MSI and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. RESULTS: Both Gadofluorine P enhancement as well as local tissue content in the myocardial scar were highest at 15 minutes post injection. R1 values increased from 1 to 6 weeks after MI (1.62 s-1 vs 2.68 s-1, p = 0.059) paralleled by an increase in Gadofluorine P concentration in the infarct from 0.019 mM at 1 week to 0.028 mM at 6 weeks (p = 0.048), whereas Gd-DTPA enhancement showed no differences (3.95 s-1 vs 3.47 s-1, p = 0.701). MALDI-MSI results were corroborated by elemental LA-ICP-MS of Gadolinium in healthy and infarcted myocardium. Histology confirmed increased extracellular matrix synthesis at 6 weeks compared to 1 week. CONCLUSION: Adding quantitative MSI to MR imaging enables a quantitative validation of Gadofluorine P distribution in the heart after MI for molecular imaging.

Targeting Mucosal Endothelin-A-Receptor Expression by Fluorescence Endoscopy is Feasible to Detect and Characterize Colitis-Associated Cancer in Mice.

Background: To facilitate onsite decision-making during endoscopy, both accurate detection and in vivo characterization of preneoplasia are prerequisites. However, no endoscopy technique is available that meets both demands satisfactorily. We evaluated endothelin-receptor A (ETAR)-guided fluorescence endoscopy (FE) in vivo and fluorescence reflectance imaging (FRI) ex vivo for detection and characterization of early dysplastic colitis-associated colonic lesions. Methods: Colorectal cancerogenesis was investigated in the inflammatory driven AOM-DSS model and spontaneous adenoma development in ApcMin mice. A Cy5.5-labeled nonpeptidic ETAR-specific imaging probe was injected intravenously to assess tumor development in vivo by white light endoscopy (WLE) and FE. Ex vivo tumors were evaluated by FRI, histological examination, and western blot analysis. In addition, tissue samples from patients with colitis-associated malignant and nonmalignant mucosal alterations were analyzed. Specificity experiments were performed using an unspecific Cy3.5-glycine tracer. Results: Overall, 62 adenomas were observed. FE was able to detect and quantify ETAR expression targeting the ETAR-specific photoprobe. A significantly higher fluorescent contrast was detected in colonic adenomas compared to adjacent nonmalignant mucosa by FE (64.3 ± 7.9 vs. 56.6. ± 7.0; P < 0.001). These results were confirmed by FRI examination, immunochemistry, and western blot analysis. Additionally, ETAR expression in samples from human patients with colitis-associated cancer was highly elevated compared to nonmalignant alterations. Specificity experiments indicated a high binding-specificity of the applied ETAR photoprobe (1.4 ± 0.3 vs. 2.5 ± 0.7; P < 0.001). Conclusions: We introduced ETAR guided FE in mice for successful in vivo detection and characterization of colorectal neoplasia on a molecular level.

Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction.

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.