Probe For Early Detection Research Articles

Synthesis, Radiolabeling, and Biological Evaluation of 68Ga-Mucin1 and Its Folate Hybrid Peptide Conjugates for the Diagnosis of Ovarian Cancer

Because of our interest in developing new hybrid peptide radioconjugates with suitable biochemical properties for multiple-receptors targeting properties that are overexpressed on many human cancers especially ovarian cancer, we have synthesized 68Ga-NODAGA-MUC1 and 68Ga-NODAGA-MUC1-FA hybrid peptide conjugates using a straightforward and one-step simple reaction. Radiochemical yields were found to be higher than 95% (decay corrected), with a total synthesis time of less than 20 min. Radiochemical purities were always higher than 95% without HPLC purification. In vitro studies on KB cancer cells showed that substantial amounts of the radioconjugates were associated with cell fractions and held great affinities and specificities toward the KB cell line. In vivo characterization in normal female Balb/c mice revealed rapid blood clearance of these radioconjugates with excretion predominantly by the urinary system. Biodistribution studies in nude mice bearing human KB cell line xenografts demonstrated significant tumor uptake and favorable biodistribution profile for 68Ga-NODAGA-MUC1-FA hybrid peptide conjugate compared to the 68Ga-NODAGA-MUC1 peptide monomeric counterpart. The uptake in the tumors was blocked by the excess injection of hybrid peptide, suggesting a receptor-mediated process. These results demonstrate that 68Ga-NODAGA-MUC1-FA hybrid peptide conjugate may be useful as a molecular probe for early detection and staging of folate and MUC1 receptor-positive cancers such as ovarian cancer and their metastasis as well as monitoring tumor response to treatment.

Development of nanobody and nanofiber-based immunoassay for sensitive detection of human cystatin C

become important diagnostic tools. MRI offers excellent soft tissue contrast and anatomic information. PET has high sensitivity and can perform metabolic imaging. In this context, the combination of PET and MRI has drawn many attentions around the world recent years, which is regarded as a better alternative for PET/CT. Currently, PET/MRI integrated system is available for clinical use. In this context, in this study, we develop PET/MRI dual modality, αvβ3 and EGFR dual targeting probe for early detection of lung cancer. Materials and Methods: The probe was prepared by coupling RGD peptide (c (RGDyK) and D4 peptide (N-LARLLT) onto ultra small iron oxide nanoparticle (USPIO) simultaneously and consequently labeling the particle with fluorine-18 by click chemistry. USPIO was synthesized by polyol method using FeCl3 as iron source in the presence of polyacrylic acid. As-prepared particles were first modified by but-3-yn-1-amine. c(RGDyK) and N-LARLLT peptides were then coupled onto USPIO by EDC/NHS condensation reaction. For labeling F-18 by click chemistry, F-18 labeling precursor 2-azidoethyl-4-toluenesulfonate was first synthesized. Next, through the nucleophilic [F] fluorination, versatile F-labeled 2-[F]fluoroethyl azide ([F]FEA) was prepared. Click reaction of but-3-yn-1-amine modified USPIO and [F]FEA was performed in the presence of CuSO4/NaVc. Results:The procedure for preparation of the probewas shown in Figure 1. The size of USPIO was around 5 nm, which was very stable in physiological condition and demonstrated good T1 relaxivity (11.20 s mM) and reasonable T2 relaxivity (20.14 s mM). The chemical purity of 2-azidoethyl-4-toluenesulfonate was around 95% (Figure 2). The labeling efficiency of [F]FEA was N99%, and the radiochemical purity was N99%, with a total synthesis time of approximate 40 min and RCY of 71% (decay-corrected). The labeling efficiency of but-3-yn-1-amine modified USPIO with [F]FEA by click chemistry was 84%. The probe was collected with a magnet and stable in PBS (pH 7.4).

In vivofluorescence imaging of atherosclerotic plaques with activatable cell-penetrating peptides targeting thrombin activity

Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL-ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL-ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL-ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL-ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques.

Abstract 5285: Novel antibody-based probes for the detection and study of pre-malignant breast cancer cells

Abstract Breast cancer is the most prevalent cancer in women. Every year in the US over 192,000 new cases are diagnosed with ∼62,000 of these women presenting at clinics with stage 0 ductal carcinoma in situ (DCIS), of whom 12-15% will develop an invasive tumor within 10 years. Currently there is no reliable method of identifying these women causing high morbidity due to over-treatment and high mortality from under-treatment. Recently our team discovered a population of variant human mammary epithelial cells (vHMECs) which show the correct phenotype for progression to malignancy. Through gene profiling we identified a specific cell surface antigen (SA) for targeting vHMECs, allowing the development of imaging probes able to provide information on the behavior of these cells in vivo as well as forming the basis of a clinically useful probe for early detection and prevention of breast cancer. We based our imaging probes on a proprietary antibody (anti-SA) specific to the cell surface antigen identified during our gene profiling studies. Using a combination of fluorescence microscopy and flow cytometry we optimized its labeling with a near-IR fluorophore, Alexafluor-680 (AF-680), to produce an optical probe that retained its high affinity for vHMECs. Following successful in vitro evaluation of the probe it was tested in vivo. Nude mice were implanted in one mammary fat pad with luciferase-transduced vHMECs (2.5e5-1e6) and in the contra-lateral pad with an equal number of a SA-ve cell line. Bioluminescence imaging was used to confirm successful implantation and 48 hours after cell implantation the probe (∼1.5 nM based on AF680) was administered via tail vein injection. The mice were then imaged using reflectance and tomographic techniques. To further assess the specificity of the probe xenograft models where mice were implanted with both basal (MDA-MB-231, SUM-159-PT, MDA-MD-468; all SA+ve) and luminal (SKBR3, SUM-52-PE, MDA-MB-453; all SA-ve) breast cancer cell lines in the contra-lateral fat pads were used to ascertain the probe's ability to discriminate between these two breast cancer sub-types. The probe effectively labeled vHMECs in vitro and in vivo. The presence of the vHMECs was clearly discerned upon surgical exposure of the mammary fat pad 24 hours after probe injection. In contrast the signal from the SA-ve implant was 40% lower. When tested in mice implanted with both basal and luminal breast cancer cell lines, the fluorescence signal measured from the basal cells was significantly higher (195% higher; student's t-test, one tailed, p=0.0029). Our optical probe is able to selectively label SA+ve cells in vivo, fulfilling our initial goal of developing an imaging probe to facilitate the study of vHMECs. In addition our results indicate that it can be used to discriminate the more aggressive basal breast cancer sub-type from the luminal sub-type, pointing to its future clinical utility in personalized breast cancer diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5285. doi:10.1158/1538-7445.AM2011-5285