Dennis Doleschel

Chemokine Cxcl9 attenuates liver fibrosis‐associated angiogenesis in mice

Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl4)‐induced liver fibrosis in Cxcr3−/− and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet‐inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3−/− mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF‐stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal‐regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter‐regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. Conclusion: The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis. (HEPATOLOGY 2012)

Evolution of contrast agents for ultrasound imaging and ultrasound-mediated drug delivery.

Ultrasound (US) is one of the most frequently used diagnostic methods. It is a non-invasive, comparably inexpensive imaging method with a broad spectrum of applications, which can be increased even more by using bubbles as contrast agents (CAs). There are various different types of bubbles: filled with different gases, composed of soft- or hard-shell materials, and ranging in size from nano- to micrometers. These intravascular CAs enable functional analyses, e.g., to acquire organ perfusion in real-time. Molecular analyses are achieved by coupling specific ligands to the bubbles’ shell, which bind to marker molecules in the area of interest. Bubbles can also be loaded with or attached to drugs, peptides or genes and can be destroyed by US pulses to locally release the entrapped agent. Recent studies show that US CAs are also valuable tools in hyperthermia-induced ablation therapy of tumors, or can increase cellular uptake of locally released drugs by enhancing membrane permeability. This review summarizes important steps in the development of US CAs and introduces the current clinical applications of contrast-enhanced US. Additionally, an overview of the recent developments in US probe design for functional and molecular diagnosis as well as for drug delivery is given.

Failure of annexin-based apoptosis imaging in the assessment of antiangiogenic therapy effects

BackgroundMolecular apoptosis imaging is frequently discussed to be useful for monitoring cancer therapy. We demonstrate that the sole assessment of therapy effects by apoptosis imaging can be misleading, depending on the therapy effect on the tumor vasculature.MethodsApoptosis was investigated by determining the uptake of Annexin Vivo by optical imaging (study part I) and of 99 mTc-6-hydrazinonicotinic [HYNIC]-radiolabeled Annexin V by gamma counting (study part II) in subcutaneous epidermoid carcinoma xenografts (A431) in nude mice after antiangiogenic treatment (SU11248). Optical imaging was performed by optical tomography (3D) and 2D reflectance imaging (control, n = 7; therapy, n = 6). Accumulation of the radioactive tracer was determined ex vivo (control, n = 5; therapy, n = 6). Tumor vascularization was investigated with an optical blood pool marker (study part I) and contrast-enhanced ultrasound (both studies). Data were validated by immunohistology.ResultsA significantly higher apoptosis rate was detected in treated tumors by immunohistological terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining (area fraction: control, 0.023 ± 0.015%; therapy, 0.387 ± 0.105%; P < 0.001). However, both 2D reflectance imaging using Annexin Vivo (control, 13 ± 15 FI/cm2; therapy, 11 ± 7 FI/cm2) and gamma counting using 99 mTc-HYNIC-Annexin V (tumor-to-muscle ratio control, 5.66 ± 1.46; therapy, 6.09 ± 1.40) failed in showing higher accumulation in treated tumors. Optical tomography even indicated higher probe accumulation in controls (control, 81.3 ± 73.7 pmol/cm3; therapy, 27.5 ± 34.7 pmol/cm3). Vascularization was strongly reduced after therapy, demonstrated by contrast-enhanced ultrasound, optical imaging, and immunohistology.ConclusionsThe failure of annexin-based apoptosis assessment in vivo can be explained by the significant breakdown of the vasculature after therapy, resulting in reduced probe/tracer delivery. This favors annexin-based apoptosis imaging only in therapies that do not severely interfere with the vasculature.

Targeted Near-Infrared Imaging of the Erythropoietin Receptor in Human Lung Cancer Xenografts

The putative presence of the erythropoietin receptor (EpoR) on human cancer cells has given rise to controversial discussion about the use of recombinant human erythropoietin (rhuEpo) for treatment of patients with chemotherapy-induced anemia. In vivo analysis of the EpoR status in tumors could help in elucidating the role of erythropoietin in cancer. Thus, the aim of this study was to develop a targeted EpoR probe for the investigation of EpoR expression in human lung cancer xenografts by fluorescence-mediated tomography. Methods: Epo-Cy5.5 was generated by coupling Cy5.5 to rhuEpo. In vitro binding assays were performed using the EpoR-positive non–small cell lung cancer (NSCLC) cell lines A549 (lower EpoR expression) and H838 (higher EpoR expression), the EpoR-negative cell line H2030, and EpoR/EGFP-overexpressing HeLa cells. In vivo specificity of Epo-Cy5.5 was confirmed by competition analyses using micro-CT/fluorescence-mediated tomography fusion imaging. Biodistribution was analyzed over 50 h after injection. Binding of Epo-Cy5.5 was validated on tumor cryosections. Results: After intravenous injection, the probe was rapidly cleared from the circulation. An accumulation was observed in liver and kidneys, with a maximum at 7 h after injection followed by a decline, indicating renal excretion. Almost constant accumulation of Epo-Cy5.5 was found in bone marrow and tumors, indicating specific receptor binding. The probe allowed the discrimination between H838 with higher EpoR expression (89.54 ± 15.91 nM at 25 h) and A549 tumors with lower EpoR expression (60.45 ± 14.59 nM at 25 h, P < 0.05). Tumor accumulation of Epo-Cy5.5 could be significantly reduced by adding unlabeled rhuEpo (P < 0.05 at 4, 7, and 24 h). In vitro validation confirmed specific binding of Epo-Cy5.5 to the tumor cells, and this binding correlated with the EpoR expression level. Binding was also observed on endothelial cells. Vessel density and Epo-Cy5.5 binding on endothelial cells were comparable. Conclusion: Epo-Cy5.5 allows the longitudinal analysis of EpoR expression in tumors and thereby can investigate the influence of erythropoietin on EpoR expression, tumor growth, and angiogenesis.

Hybrid µCT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Erythropoietin Improves the Accumulation and Therapeutic Effects of Carboplatin by Enhancing Tumor Vascularization and Perfusion

Recombinant human erythropoietin (rhuEpo) is currently under debate for the treatment of chemotherapy-induced anemia due to clinical trials showing adverse effects in Epo-treated patients and the discovery of the erythropoietin-receptor (EpoR) in tumor and endothelial cells. Here, using Epo-Cy5.5 as theranostic near-infrared fluorescent probe we analyzed the effects of rhuEpo as co-medication to carboplatin in non-small-cell-lung-cancer (NSCLC)-xenografts with different tumor cell EpoR-expression (H838 ~8-fold higher than A549). Nude mice bearing subcutaneous A549 and H838 NSCLC-xenografts received either only carboplatin or carboplatin and co-medication of rhuEpo in two different doses. Tumor sizes and relative blood volumes (rBV) were longitudinally measured by 3D-contrast-enhanced ultrasound (3D-US). Tumoral EpoR-levels were determined by combined fluorescence molecular tomography (FMT)/ micro computed tomography (µCT) hybrid imaging. We found that rhuEpo predominantly acted on the tumor endothelium. In both xenografts, rhuEpo co-medication significantly increased vessel densities, diameters and the amount of perfused vessels. Accordingly, rhuEpo induced EpoR-phoshorylation and stimulated proliferation of endothelial cells. However, compared with solely carboplatin-treated tumors, tumor growth was significantly slower in the groups co-medicated with rhuEpo. This is explained by the Epo-mediated vascular remodeling leading to improved drug delivery as obvious by a more than 2-fold higher carboplatin accumulation and significantly enhanced tumor apoptosis. In addition, co-medication of rhuEpo reduced tumor hypoxia and diminished intratumoral EpoR-levels which continuously increased during carboplatin (Cp) -treatment. These findings suggest that co-medication of rhuEpo in well balanced doses can be used to improve the accumulation of anticancer drugs. Doses and indications may be personalized and refined using theranostic EpoR-probes.

High accuracy of mesoscopic epi-fluorescence tomography for non-invasive quantitative volume determination of fluorescent protein-expressing tumours in mice

AbstractObjectivesTo compare mesoscopic epi-fluorescence tomography (MEFT) and EPRI-illumination reflectance imaging (EPRI) for quantitative tumour size assessment in mice.MethodsTumour xenografts of green/red fluorescent protein (GFP/RFP)-expressing colon cancer cells were measured using MEFT, EPRI, ultrasound (US) and micro computed tomography (μCT) at day 14 post-injection (n = 6). Results from MEFT and EPRI were correlated with each other and with US and μCT (reference methods). Tumour volumes were measured ex vivo by GFP and RFP fluorescence imaging on cryoslices and compared with the in vivo measurements.ResultsHigh correlation and congruency were observed between MEFT, US and μCT (MEFT/US: GFP: r2 = 0.96; RFP: r2 = 0.97, both P < 0.05; MEFT/μCT: GFP: r2 = 0.93; RFP: r2 = 0.90; both P < 0.05). Additionally, in vivo MEFT data were highly correlated and congruent with ex vivo cryoslice imaging results (GFP: r2 = 0.96; RFP: r2 = 0.99; both P < 0.05). In comparison, EPRI significantly overestimated tumour volumes (P < 0.05), although there was a significant correlation with US and μCT (EPRI/US: GFP: r2 = 0.95; RFP: r2 = 0.94; both P < 0.05; EPRI/μCT GFP: r2 = 0.86; RFP: r2 = 0.86; both P < 0.05).ConclusionsFluorescence distribution reconstruction using MEFT affords highly accurate three-dimensional (3D) tumour volume data showing superior accuracy compared to EPRI. Thus, MEFT is a very suitable technique for quantitatively assessing fluorescence distribution in superficial tumours at high spatial resolution.Key Points• Mesoscopic epi-fluorescence tomography (MEFT) is an important new molecular imaging technique. • MEFT allows accurate size determination of superficial tumours with high resolution.• MEFT is a suitable technique for longitudinal assessment of tumour growth.• MEFT allows 3D reconstruction and quantification of fluorescence distributions.

Nilotinib Enhances Tumor Angiogenesis and Counteracts VEGFR2 Blockade in an Orthotopic Breast Cancer Xenograft Model with Desmoplastic Response

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-targeted therapies predominantly affect nascent, immature tumor vessels. Since platelet-derived growth factor receptor (PDGFR) blockade inhibits vessel maturation and thus increases the amount of immature tumor vessels, we evaluated whether the combined PDGFR inhibition by nilotinib and VEGFR2 blockade by DC101 has synergistic therapy effects in a desmoplastic breast cancer xenograft model. In this context, besides immunohistological evaluation, molecular ultrasound imaging with BR55, the clinically used VEGFR2-targeted microbubbles, was applied to monitor VEGFR2-positive vessels noninvasively and to assess the therapy effects on tumor angiogenesis. DC101 treatment alone inhibited tumor angiogenesis, resulting in lower tumor growth and in significantly lower vessel density than in the control group after 14 days of therapy. In contrast, nilotinib inhibited vessel maturation but enhanced VEGFR2 expression, leading to markedly increased tumor volumes and a significantly higher vessel density. The combination of both drugs led to an almost similar tumor growth as in the DC101 treatment group, but VEGFR2 expression and microvessel density were higher and comparable to the controls. Further analyses revealed significantly higher levels of tumor cell–derived VEGF in nilotinib-treated tumors. In line with this, nilotinib, especially in low doses, induced an upregulation of VEGF and IL-6 mRNA in the tumor cells in vitro, thus providing an explanation for the enhanced angiogenesis observed in nilotinib-treated tumors in vivo. These findings suggest that nilotinib inhibits vessel maturation but counteracts the effects of antiangiogenic co-therapy by enhancing VEGF expression by the tumor cells and stimulating tumor angiogenesis.