In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis.

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.

Assessment of an scFv Antibody Fragment Against ELTD1 in a G55 Glioblastoma Xenograft Model.

Glioblastoma (GBM), the most common primary brain tumor found in adults, is extremely aggressive. These high-grade gliomas, which are very diffuse, highly vascular, and invasive, undergo unregulated vascular angiogenesis. Despite available treatments, the median survival for patients is dismal. ELTD1 (EGF, latrophilin, and 7 transmembrane domain containing protein 1) is an angiogenic biomarker highly expressed in human high-grade gliomas. Recent studies have demonstrated that the blood-brain barrier, as well as the blood-tumor barrier, is not equally disrupted in GBM patients. This study therefore aimed to optimize an antibody treatment against ELTD1 using a smaller scFv fragment of a monoclonal antibody that binds against the external region of ELTD1 in a G55 glioma xenograft glioma preclinical model. Morphological magnetic resonance imaging (MRI) was used to determine tumor volumes and quantify perfusion rates. We also assessed percent survival following tumor postdetection. Tumor tissue was also assessed to confirm and quantify the presence of the ELTD1 scFv molecular targeted MRI probe, as well as microvessel density and Notch1 levels. In addition, we used molecular-targeted MRI to localize our antibodies in vivo. This approach showed that our scFv antibody attached-molecular MRI probe was effective in targeting and localizing diffuse tumor regions. Through this analysis, we determined that our anti-ELTD1 scFv antibody treatments were successful in increasing survival, decreasing tumor volumes, and normalizing vascular perfusion and Notch1 levels within tumor regions. This study demonstrates that our scFv fragment antibody against ELTD1 may be useful and potential antiangiogenic treatments against GBM.

Targeting ELTD1, an angiogenesis marker for glioblastoma (GBM), also affects VEGFR2: molecular-targeted MRI assessment.

Glioblastomas (GBM) are deadly brain tumors that currently do not have long-term patient treatments available. GBM overexpress the angiogenesis factor VEGF and its receptor VEGFR2. ETLD1 (epidermal growth factor, latrophilin and seven transmembrane domain-containing protein 1), a G-protein coupled receptor (GPCR) protein, we discovered as a biomarker for high-grade gliomas, is also a novel regulator of angiogenesis. Since it was established that VEGF regulates ELTD1, we wanted to establish if VEGFR2 is also associated with ELTD1, by targeted antibody inhibition. G55 glioma-bearing mice were treated with either anti-ELTD1 or anti-VEGFR2 antibodies. With the use of MRI molecular imaging probes, we were able to detect in vivo levels of either ELTD1 (anti-ELTD1 probe) or VEGFR2 (anti-VEGFR2 probe). Protein expressions were obtained for ELTD1, VEGF or VEGFR2 via immunohistochemistry (IHC). VEGFR2 levels were significantly decreased (P < 0.05) with anti-ELTD1 antibody treatment, and ELTD1 levels were significantly decreased (P < 0.05) with anti-VEGFR2 antibody treatment, both compared to untreated tumors. IHC from mouse tumor tissues established that VEGFR2 and ELTD1 were co-localized. The mouse anti-ELTD1 antibody treatment study indicated that anti-VEGFR2 antibody treatment does not significantly increase survival, decrease tumor volumes, or alter tumor perfusion (measured as relative cerebral blood flow or rCBF). Conversely, anti-ELTD1 antibody treatment was able to significantly increase animal survival (P < 0.01), decrease tumor volumes (P < 0.05), and reduce change in rCBF (P < 0.001), when compared to untreated or IgG-treated tumor bearing mice. Anti-ELTD1 antibody therapy could be beneficial in targeting ELTD1, as well as simultaneously affecting VEGFR2, as a possible GBM treatment.

ELTD1, an effective anti-angiogenic target for gliomas: preclinical assessment in mouse GL261 and human G55 xenograft glioma models.

Background: Despite current therapies, glioblastoma is a devastating cancer, and validation of effective biomarkers for it will enable better diagnosis and therapeutic intervention for this disease. We recently discovered a new biomarker for high-grade gliomas, ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1) via bioinformatics, and validated that ELTD1 protein levels are significantly higher in human and rodent gliomas. The focus of this study was to assess the effect on tumor growth of an antibody against ELTD1 in orthotopic, GL261, and G55 xenograft glioma models. Methods: The effect of anti-ELTD1 antibody therapy was assessed by animal survival, MRI measured tumor volumes, MR angiography, MR perfusion imaging, and immunohistochemistry (IHC) characterization of microvessel density in mouse glioma models. Comparative treatments included anti-vascular endothelial growth factor (VEGF) and anti-c-Met antibody therapies, compared with untreated controls. Results: Tumor volume and survival data in this study show that antibodies against ELTD1 inhibit glioma growth just as effectively or even more so compared with other therapeutic targets studied, including anti-VEGF antibody therapy. Untreated GL261 or G55 tumors were found to have significantly higher ELTD1 levels (IHC) compared with contralateral normal brain. The anti-angiogenic effect of ELTD1 antibody therapy was observed in assessment of microvessel density, as well as from MR angiography and perfusion measurements, which indicated that anti-ELTD1 antibody therapy significantly decreased vascularization compared with untreated controls. Conclusions: Either as a single therapy or in conjunction with other therapeutic approaches, anti-ELTD1 antibodies could be a valuable new clinical anti-angiogenic therapeutic for high-grade gliomas.

A new anti-glioma therapy, AG119: pre-clinical assessment in a mouse GL261 glioma model.

BACKGROUND: High grade gliomas (HGGs; grades III and IV) are the most common primary brain tumors in adults, and their malignant nature ranks them fourth in incidence of cancer death. Standard treatment for glioblastomas (GBM), involving surgical resection followed by radiation and chemotherapy with temozolomide (TMZ) and the anti-angiogenic therapy bevacizumab, have not substantially improved overall survival. New therapeutic agents are desperately needed for this devastating disease. Here we study the potential therapeutic agent AG119 in a pre-clinical model for gliomas. AG119 possesses both anti-angiogenic (RTK inhibition) and antimicrotubule cytotoxic activity in a single molecule. METHODS: GL261 glioma-bearing mice were either treated with AG119, anti-VEGF (vascular endothelial growth factor) antibody, anti c-Met antibody or TMZ, and compared to untreated tumor-bearing mice. Animal survival was assessed, and tumor volumes and vascular alterations were monitored with morphological magnetic resonance imaging (MRI) and perfusion-weighted imaging, respectively. RESULTS: Percent survival of GL261 HGG-bearing mice treated with AG119 was significantly higher (p < 0.001) compared to untreated tumors. Tumor volumes (21-31 days following intracerebral implantation of GL261 cells) were found to be significantly lower for AG119 (p < 0.001), anti-VEGF (p < 0.05) and anti-c-Met (p < 0.001) antibody treatments, and TMZ-treated (p < 0.05) mice, compared to untreated controls. Perfusion data indicated that both AG119 and TMZ were able to reduce the effect of decreasing perfusion rates significantly (p < 0.05 for both), when compared to untreated tumors. It was also found that IC50 values for AG119 were much lower than those for TMZ in T98G and U251 cells. CONCLUSIONS: These data support further exploration of the anticancer activity AG119 in HGG, as this compound was able to increase animal survival and decrease tumor volumes in a mouse GL261 glioma model, and that AG119 is also not subject to methyl guanine transferase (MGMT) mediated resistance, as is the case with TMZ, indicating that AG119 may be potentially useful in treating resistant gliomas.

Assessment of thermal effects of interstitial laser phototherapy on mammary tumors using proton resonance frequency method.

Laser immunotherapy (LIT) uses a synergistic approach to treat cancer systemically through local laser irradiation and immunological stimulation. Currently, LIT utilizes dye-assisted noninvasive laser irradiation to achieve selective photothermal interaction. However, LIT faces difficulties treating deeper tumors or tumors with heavily pigmented overlying skin. To circumvent these barriers, we use interstitial laser irradiation to induce the desired photothermal effects. The purpose of this study is to analyze the thermal effects of interstitial irradiation using proton resonance frequency (PRF). An 805-nm near-infrared laser with an interstitial cylindrical diffuser was used to treat rat mammary tumors. Different power settings (1.0, 1.25, and 1.5 W) were applied with an irradiation duration of 10 min. The temperature distributions of the treated tumors were measured by a 7 T magnetic resonance imager using PRF. We found that temperature distributions in tissue depended on both laser power and time settings, and that variance in tissue composition has a major influence in temperature elevation. The temperature elevations measured during interstitial laser irradiation by PRF and thermocouple were consistent, with some variations due to tissue composition and the positioning of the thermocouple's needle probes. Our results indicated that, for a tissue irradiation of 10 min, the elevation of rat tumor temperature ranged from 8 to 11°C for 1 W and 8 to 15°C for 1.5 W. This is the first time a 7 T magnetic resonance imager has been used to monitor interstitial laser irradiation via PRF. Our work provides a basic understanding of the photothermal interaction needed to control the thermal damage inside a tumor using interstitial laser treatment. Our work may lead to an optimal protocol for future cancer treatment using interstitial phototherapy in conjunction with immunotherapy.

Molecular MRI assessment of vascular endothelial growth factor receptor-2 in rat C6 gliomas.

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide-based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti-VEGFR2 monoclonal antibody, shown by varied increases in T(1) signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin-Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.

Multiparametric assessment of the anti-glioma properties of OKN007 by magnetic resonance imaging.

PURPOSE: To demonstrate that OKN007, a disulfonyl derivative of phenyl-tert-butyl nitrone (PBN), has anti-glioma activity in the clinically relevant C6 rat glioma model using multi-parametric magnetic resonance imaging. MATERIALS AND METHODS: Twenty-one rats were intracerebrally implanted with C6 cells and administered OKN007 or kept as controls. Animals were monitored with MRI at 7 Tesla (T), using morphologic, diffusion-weighted and perfusion imaging, followed by histology and Western blots of angiogenesis and inflammatory markers. RESULTS: OKN007 was found to decrease tumor volumes and increase survival. The glioma tissues of OKN007-treated rats were found to have longitudinal apparent diffusion coefficients (ADC(z)) of 0.76 +/- 0.06 x 10(-3) mm(2)/s, similar to the contralateral tissue and significantly smaller than untreated gliomas (0.97 +/- 0.13 x 10(-3) mm(2)/s). They had higher perfusion rates (66 +/- 4 mL/100 g.min) than untreated gliomas (26 +/- 7 mL/100 g.min). All examined molecular markers were decreased in OKN007-treated rat gliomas, compared with elevated levels in untreated rats. CONCLUSION: MRI assessment was successfully used to monitor a decrease in tumor growth, and corresponding alterations in ADC and perfusion rates in rat C6 gliomas treated with the anti-glioma agent, OKN007.

Assessment of in vivo oxidative lipid metabolism following acute microcystin-LR-induced hepatotoxicity in rats.

Oxidative lipid metabolism as a result of acute cyanobacterial toxin-induced hepatotoxicity was monitored in male Sprague-Dawley rats using electron spin resonance (ESR) spectroscopy and image-guided proton nuclear magnetic resonance (1H-NMR) spectroscopy. ESR spectroscopy, coupled with spin trapping, was used to trap and detect lipid-derived radicals, formed in rat livers after acute in vivo exposure (LD50) to the cyanobacterial toxin, microcystin-LR (MCLR). A statistically significant increase in the levels (spectral peak integrals) of lipid radicals was detected in MCLR-treated livers (p < 0.05) (n = 8), in comparison to control livers (n = 6). In order to monitor lipid metabolism, before and for a period of 3 h, following toxin exposure, in vivo proton image-guided NMR spectroscopy was used. A statistically significant decrease in the levels of lipid methylene hydrogen resonances (spectral peak integrals) was observed from MCLR-treated livers (n = 6) 2 and 3 h post-exposure (p < 0.05), in comparison to controls (n = 6). Image-guided NMR spectroscopy was also used to detect significant decreasing levels of in vivo glutamine/glutamate, following exposure to MCLR. Biochemical assessment of perchloric extracts of liver glutamine and glutamate levels correlated with NMR spectroscopy results. Lactate levels measured as perchloric acid extracts, were also found to significantly decrease. In addition, assessment of serum enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were used to confirm hepatotoxicity (n = 20). This study strongly suggests that oxidative stress related processes are involved in in vivo microcystin-induced hepatotoxicity in mammals, and may play an integral role in MCLR-induced toxicity.

In vivo assessment of nodularin-induced hepatotoxicity in the rat using magnetic resonance techniques (MRI, MRS and EPR oximetry).

Acute nodularin-induced hepatotoxicity was assessed in vivo, in rats using magnetic resonance (MR) techniques, including MR imaging (MRI), MR spectroscopy (MRS), and electron paramagnetic resonance (EPR) oximetry. Nodularin is a cyclic hepatotoxin isolated from the cyanobacterium Nodularia spumigena. Three hours following the intraperitoneal (i.p.) administration of nodularin (LD50), a region of 'damage', characterized by an increase in signal intensity, was observed proximal to the porta hepatis (PH) region in T2-weighted MR images of rat liver. Image analysis of these regions of apparent 'damage' indicated a statistically significant increase in signal intensity around the PH region following nodularin administration, in comparison with controls and regions peripheral to the PH region. An increase in signal intensity was also observed proximal to the PH region in water chemical shift selective images (CSSI) of nodularin-treated rat livers, indicating that the increased signal observed by MRI is an oedematous response to the toxin. Microscopic assessment (histology and electron microscopy) and serum liver enzyme function tests (aminotransferase (ALT) and aspartate ALT (AST)) confirmed the nodularin-induced tissue injury observed by MRI. In vivo and in vitro MRS was used to detect alterations in metabolites, such as lipids, Glu+Gln, and choline, during the hepatotoxic response (2-3 h post-exposure). Biochemical assessment of perchloric acid extracts of nodularin-treated rat livers were used to confirm the MRS results. In vivo EPR oximetry was used to monitor decreasing hepatic pO2 (approximately 2-fold from controls) 2-3 h following nodularin exposure. In vivo MR techniques (MRI, MRS and EPR oximetry) are able to highlight effects that may not have been evident in single end point studies, and are ideal methods to follow tissue injury progression in longitudinally, increasing the power of a study through repeated measures, and decreasing the number of animals to perform a similar study using histological or biochemical techniques.