Carsten Höltke

Purpose: A hypoxic tumor microenvironment promotes cancer progression, with endothelin-A receptor (ET _A R) signaling playing a key role in tumor neoangiogenesis and macrophage infiltration. We hypothesize that multispectral optoacoustic tomography (MSOT) using an ET _A R-specific probe could provide improved insights into the hypoxic characteristics of the tumor microenvironment (TME), either alone or in combination with endogenous markers, and that alterations in ET _A R expression may correlate with increased tumor oxygenation serving as an early indicator of response to anti-angiogenic or immune-modulating therapy. Methods: A fluorescent ET _A R probe was applied for in vivo MSOT evaluation of ET _A R expression in hypoxic murine breast cancer. Optoacoustic signal intensity (SI) of deoxygenated and oxygenated hemoglobin served as additive intrinsic readouts. Furthermore, therapeutic interventions utilizing Bevacizumab , Clodronate and Sorafenib were evaluated with regard to effects on ET _A R expression and hemoglobin oxygen saturation. Imaging results were validated ex vivo via immunohistochemistry. Results: Exposure of 4T1 murine breast cancer cells to hypoxic conditions led to upregulation of ET _A R in vitro. In vivo, tumor growth correlated with increased ET _A R probe signal intensity in 4T1 tumors. All therapeutic interventions significantly reduced ET _A R SI following treatment. Anti-angiogenic therapies also increased tumor oxygen saturation, indicating therapy-induced re-oxygenation. Conclusion: ET _A R expression in hypoxic tumor regions can be visualized non-invasively by MSOT using an exogenously administered targeted probe. Combining ET _A R-targeted imaging with intrinsic hemoglobin readouts enables assessment of reoxygenation and immune cell modulation in response to therapy. Thus, ET _A R has potential as an in vivo imaging biomarker for early therapy response in experimental breast cancer studies.

Endothelin-1 (ET-1) levels are altered in atherosclerosis, while the roles of the endothelin receptors ET_AR and ET_BR during the pathogenesis of atherosclerosis remain unclear. Therefore, the focus of this study was to clarify how endothelin receptors are expressed in advanced human atherosclerotic plaques and how this is related to atherosclerotic risk factors. Ex vivo expression analysis was performed by quantitative real-time PCR (qRT-PCR) of 98 atherosclerotic plaques and controls that were obtained from adult patients undergoing vascular surgery. Correlation analyses of atherosclerosis-promoting factors were accomplished using a linear regression model. We found an overall reduced expression of ET receptors and smooth muscle actin (SMA), a marker of healthy vascular smooth muscle cells, in atherosclerotic plaques, whereas the levels of ET-1 and matrix metalloproteinase 2 (MMP-2), a marker of atherosclerosis progression, remained unchanged. Reduced expression was predominantly correlated with hypertension, which affects both receptors as well as SMA. Age, body mass index (BMI) and gender also correlated with either ET_AR, ET_BR or SMA expression in advanced plaques. In contrast, no effect of diabetes mellitus or smoking was found, indicating an ancillary effect of those risk factors. The results of our study indicate that endothelin receptor expression during the pathogenesis of atherosclerosis is predominantly correlated with hypertension.

Endothelin-1/endothelin A receptor (ET-1/ETAR) pathway plays an important role in the progression of liver fibrosis by activating hepatic stellate cells (HSCs) - a key cell type involved in the pathogenesis of liver fibrosis. Inactivating HSCs by blocking the ET-1/ETAR pathway using a selective ETAR antagonist (ERA) represents a promising therapeutic approach for liver fibrosis. Unfortunately, small-molecule ERAs possess limited clinical potential due to poor bioavailability, short half-life, and rapid renal clearance. To improve the clinical applicability, we conjugated ERA to superparamagnetic iron-oxide nanoparticles (SPIONs) and investigated the therapeutic efficacy of ERA and ERA-SPIONs in vitro and in vivo and analyzed liver uptake by in vivo and ex vivo magnetic resonance imaging (MRI), HSCs-specific localization, and ET-1/ETAR-pathway antagonism in vivo. In murine and human liver fibrosis/cirrhosis, we observed overexpression of ET-1 and ETAR that correlated with HSC activation, and HSC-specific localization of ETAR. ERA and successfully synthesized ERA-SPIONs demonstrated significant attenuation in TGFβ-induced HSC activation, ECM production, migration, and contractility. In an acute CCl₄-induced liver fibrosis mouse model, ERA-SPIONs exhibited higher liver uptake, HSC-specific localization, and ET-1/ETAR pathway antagonism. This resulted in significantly reduced liver-to-body weight ratio, plasma ALT levels, and α-SMA and collagen-I expression, indicating attenuation of liver fibrosis. In conclusion, our study demonstrates that the delivery of ERA using SPIONs enhances the therapeutic efficacy of ERA in vivo. This approach holds promise as a theranostic strategy for the MRI-based diagnosis and treatment of liver fibrosis.

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 α_vβ₃ 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 α_v-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 α_vβ₃ was detected within vascular structures.

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 α_vβ₃ 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.

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 (ET_AR) 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 ET_AR-positive HT-1080 fibrosarcoma and ET_AR-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.

(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.

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 (ET_AR) 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 ET_AR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ET_AR 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 ET_AR is upregulated during the progression of early atherosclerosis in the ApoE^−/− mouse model, we found that ET_AR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ET_AR-Cy 5.5 probe confirming its specificity and potential use in future studies.

The endothelin-(ET-)axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ET_AR) expression over time in the course of myocardial injury and healing may offer valuable information towards the understanding of ET-axis involvement in MI. We developed an approach to track the expression of ET_AR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ET_AR selective antagonist 3-(1,3-Benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-tri-methoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labelled 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 i.v. injection of the ET_AR 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. 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 localization of probe and ET_AR 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 ET_AR expression over time in affected murine myocardium after MI in vivo and ex vivo.