NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.
Physical measurement of tumor volume reduction is the most commonly used approach to assess tumor progression and treatment efficacy in mouse tumor models. However, it is relatively insensitive, and often requires long treatment courses to achieve gross physical tumor destruction. As alternatives, several non-invasive imaging methods such as bioluminescence imaging (BLI), fluorescence imaging (FLI) and positron emission tomography (PET) have been developed for more accurate measurement. As tumors have elevated glucose metabolism, 18F-fludeoxyglucose (18F-FDG) has become a sensitive PET imaging tracer for cancer detection, diagnosis, and efficacy assessment by measuring alterations in glucose metabolism. In particular, the ability of 18F-FDG imaging to detect drug-induced effects on tumor metabolism at a very early phase has dramatically improved the speed of decision-making regarding treatment efficacy. Here we demonstrated an approach with FLI that offers not only comparable performance to PET imaging, but also provides additional benefits, including ease of use, imaging throughput, probe stability, and the potential for multiplex imaging. In this report, we used sorafenib, a tyrosine kinase inhibitor clinically approved for cancer therapy, for treatment of a mouse tumor xenograft model. The drug is known to block several key signaling pathways involved in tumor metabolism. We first identified an appropriate sorafenib dose, 40 mg/kg (daily on days 0–4 and 7–10), that retained ultimate therapeutic efficacy yet provided a 2–3 day window post-treatment for imaging early, subtle metabolic changes prior to gross tumor regression. We then used 18F-FDG PET as the gold standard for assessing the effects of sorafenib treatment on tumor metabolism and compared this to results obtained by measurement of tumor size, tumor BLI, and tumor FLI changes. PET imaging showed ~55–60% inhibition of tumor uptake of 18F-FDG as early as days 2 and 3 post-treatment, without noticeable changes in tumor size. For comparison, two FLI probes, BombesinRSense™ 680 (BRS-680) and Transferrin-Vivo™ 750 (TfV-750), were assessed for their potential in metabolic imaging. Metabolically active cancer cells are known to have elevated bombesin and transferrin receptor levels on the surface. In excellent agreement with PET imaging, the BRS-680 imaging showed 40% and 79% inhibition on days 2 and 3, respectively, and the TfV-750 imaging showed 65% inhibition on day 3. In both cases, no significant reduction in tumor volume or BLI signal was observed during the first 3 days of treatment. These results suggest that metabolic FLI has potential preclinical application as an additional method for detecting drug-induced metabolic changes in tumors.
In mouse pharmacokinetic (PK) studies, current standard methods often require large numbers of animals to support collection of blood samples serially over a defined time range. We have developed and validated a noninvasive fluorescence molecular tomography (FMT) heart imaging approach for blood PK quantification that uses small numbers of mice and has the advantage of repeated, longitudinal live imaging. This method was validated using a variety of near infrared (NIR) fluorescent-labeled molecules, ranging in size from 1.3 to 150 kDa, that were assessed by microplate blood assays as well as by noninvasive FMT 4000 imaging. Excellent agreement in kinetic profiles and calculated PK metrics was seen for the two methods, establishing the robustness of this noninvasive optical imaging approach. FMT heart imaging was further assessed in the challenging application of inulin-based glomerular filtration rate (GFR) measurement. After a single bolus injection of an NIR fluorescent-labeled inulin probe in small cohorts of mice (n 5 5 per group), 2-minute heart scans (at 2, 6, 15, 30, and 45 minutes) were performed by FMT imaging. GFR was calculated using two-compartment PK modeling, determining an average rate of 240 6 21 ml/min in normal mice, in agreement with published mouse GFR ranges. Validation of GFR assessment in unilaterally nephrectomized mice and cyclosporin A-treated mice both measured ∼50% decreases in GFR. Imaging results correlated well with ex vivo plasma microplate assays for inulin blood kinetics, and the decreases in GFR were accompanied by increases in plasma creatinine and blood urea nitrogen.
Abstract. Assays for blood levels of prostate-specific antigen (PSA), performed in prostate cancer detection, measure mostly inactive/complexed PSA and do not provide information regarding enzymatically active PSA, which is biologically more relevant. Thus, we designed and synthesized an enzymatically cleavable peptide sequence labeled with near-infrared (NIR) fluorophores (ex/em 740∕770 nm) and coupled it to a pharmacokinetic modifier designed to improve its plasma kinetics. In its native state, the agent, PSA750 FAST™ (PSA750), is optically quenched (>95%) and only becomes fluorescent upon cleavage by active PSA, yielding a significant increase in signal. This activation is highly selective for PSA relative to a large panel of disease-relevant enzymes. Active PSA was detected in tumor frozen sections using PSA750 and this activity was abolished in the presence of the inhibitor, alpha-1 anti-chymotrypsin. In vivo imaging of tumor-bearing mice using fluorescence molecular tomography demonstrated a significantly higher fluorescent signal in PSA þ LNCaP tumors as compared to PSA − prostate cancer 3 tumors (13.0 AE 3.7 versus 2.8 AE 0.8 pmol, p ¼ 0.023). Ex vivo imaging of tumor sections confirms PSA750-derived NIR signal localization in nonvascular tissue. This is the first report that demonstrates the feasibility and effectiveness of noninvasive, real time, fluorescence molecular imaging of PSA enzymatic activity in prostate cancer. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
Tumors induce significant blood vessel development in order to support their aggressive growth and progression, and the extensive and disordered nature of tumor vasculature impairs drug delivery and efficacy. Destroying the tumor vasculature and/or inhibiting neo-vascularization, alone or in combination with traditional chemotherapies, has become a well-accepted and proven cancer treatment strategy. A well-known tool for studying tumor angiogenesis and measuring microvessel density is tomato (Lycopersicon esculentum) lectin, a single polypeptide glycoprotein that readily binds to sugar-containing proteins present on the endothelium. Our objective was to develop a near infra-red (NIR) tomato lectin agent in order to non-invasively assess tumor vasculature in vivo. A NIR fluorophore, VivoTag 680 XL (ε=210,000 M/cm; abs/em max 665/688 nm), was conjugated to tomato lectin to produce an imaging agent, tLectin 680. The conjugation was carried out by addition of the fluorophore in a DMSO solution to lectin in aqueous sodium bicarbonate. Yields of greater than 95% were achieved, based on absorbance with an average loading of 2 dyes per lectin. In vitro, tLectin 680 preferentially labeled primary endothelial cells from human umbilical veins. Specificity of binding was confirmed by control experiments using free dye and competitive blockade with unlabeled excess tomato lectin. In vivo, we used tLectin 680, in combination with Fluorescence Molecular Tomography (FMT), for non-invasive imaging and quantification of tumor neo-vasculature in Lewis Lung Carcinoma tumors implanted in nude mice. FMT imaging quantified a statistically significant difference between the concentrations of localized tLectin 680 in tumors implanted in the flank of nude mice versus in control (non-tumor) contra-lateral flanks (50.96 +12 versus 2.32 +1 pmol), as early as 6 hours after intravenous delivery. Specific localization of the agent to the tumor vasculature was confirmed by fluorescence microscopy of frozen tumor sections and by comparison to FITC-labeled CD31. In addition vessel counts performed ex vivo in frozen sections of different tumor cell lines by fluorescence microscopy, showed a good correlation (R2= 0.99) between CD31 and tLectin 680 signal: Lewis Lung Carcinomas (27.7 vessels/sample tLectin vs. 32 vessels/sample CD-31), HT-29 (13.4 vessels/sample vs. 15.4 vessels/sample), and matrigel plugs (5.5 vessels/sample vs. 7 vessels/sample). In vivo tLectin 680 signal was also shown to correlate with ex vivo microscopy (R2= 0.90) in these tumors (Lewis Lung Carcinomas 177.6 + 15 nM, 27.7 vessel/sample, HT-29 118.1 + 6 nM, 13.4 vessel/sample), and matrigel plugs 73.6 + 9 nM, 5.5 vessel/sample). These results underscore the potential of tLectin 680 combined with FMT imaging in assessing vascularity in vivo and in real time, improving the early detection and monitoring of anti-angiogenic treatments in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-511. doi:1538-7445.AM2012-LB-511
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