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.