Kevin Groves

Hybrid In Vivo FMT-CT Imaging of Protease Activity in Atherosclerosis With Customized Nanosensors

Objective—Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap, and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address 2 major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomic information), we compared protease sensors (PS) of variable size and pharmacokinetics and coregistered FMT datasets with computed tomography (FMT-CT). Methods and Results—Coregistration of FMT and CT was achieved with a multimodal imaging cartridge containing fiducial markers detectable by both modalities. A high-resolution CT angiography protocol accurately localized fluorescence to the aortic root of atherosclerotic apoE−/− mice. To identify suitable sensors, we first modeled signal kinetics in-silico and then compared 3 probes with oligo-l-lysine cleavage sequences: PS-5, 5 nm in diameter containing 2 fluorochromes, PS-25, a 25-nm version with an elongated lysine chain and PS-40, a polymeric nanoparticle. Serial FMT-CT showed fastest kinetics for PS-5 but, surprisingly, highest fluorescence in lesions of the aortic root for PS-40. PS-40 robustly reported therapeutic effects of atorvastatin, corroborated by ex vivo imaging and qPCR for the model protease cathepsin B. Conclusions—FMT-CT is a robust and observer-independent tool for noninvasive assessment of inflammatory murine atherosclerosis. Reporter-containing nanomaterials may have unique advantages over small molecule agents for in vivo imaging.

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors.

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.

Noninvasive In Vivo Quantification of Neutrophil Elastase Activity in Acute Experimental Mouse Lung Injury

We developed a neutrophil elastase-specific near-infrared fluorescence imaging agent, which, combined with fluorescence molecular tomographic imaging, allowed us to detect and quantify neutrophil elastase activity in vivo, in real time, and noninvasively in an acute model of lung injury (ALI). Significantly higher fluorescent signal was quantified in mice with LPS/fMLP-induced ALI as compared to healthy controls, correlating with increases in the number of bronchoalveolar lavage cells, neutrophils, and elastase activity. The agent was significantly activated ex vivo in lung sections from ALI but not from control mice, and this activation was ablated by the specific inhibitor sivelestat. Treatment with the specific inhibitor sivelestat significantly reduced lung signal in mice with ALI. These results underscore the unique ability of fluorescence molecular imaging to quantify specific molecular processes in vivo, crucial for understanding the mechanisms underlying disease progression and for assessing and monitoring novel pharmacological interventions.

Quantitative Longitudinal Imaging of Vascular Inflammation and Treatment by Ezetimibe in apoE Mice by FMT Using New Optical Imaging Biomarkers of Cathepsin Activity and αvβ3 Integrin

Inflammation as a core pathological event of atherosclerotic lesions is associated with the secretion of cathepsin proteases and the expression of α v β 3 integrin. We employed fluorescence molecular tomographic (FMT) noninvasive imaging of these molecular activities using cathepsin sensing (ProSense, CatB FAST) and α v β 3 integrin (IntegriSense) near-infrared fluorescence (NIRF) agents. A statistically significant increase in the ProSense and IntegriSense signal was observed within the chest region of apoE−/− mice (P < 0.05) versus C57BL/6 mice starting 25 and 22 weeks on high cholesterol diet, respectively. In a treatment study using ezetimibe (7 mg/kg), there was a statistically significant reduction in the ProSense and CatB FAST chest signal of treated (P < 0.05) versus untreated apoE−/− mice at 31 and 21 weeks on high cholesterol diet, respectively. The signal of ProSense and CatB FAST correlated with macrophage counts and was found associated with inflammatory cells by fluorescence microscopy and flow cytometry of cells dissociated from aortas. This report demonstrates that cathepsin and α v β 3 integrin NIRF agents can be used as molecular imaging biomarkers for longitudinal detection of atherosclerosis, and cathepsin agents can monitor anti-inflammatory effects of ezetimibe with applications in preclinical testing of therapeutics and potentially for early diagnosis of atherosclerosis in patients.

Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET in early detection of sorafenib-induced changes in tumor metabolism

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.

Abstract 2444: Non-invasive FMT quantification of folate receptor expression in mouse tumor xenografts with a new near-infrared fluorescent folate agent

Folate receptors (FR) are overexpressed on the surface of many different human tumor cells, including ovarian, breast, cervical, renal, colorectal and nasopharyngeal cancer cells, with little expression in normal tissues. As such, folic acid has been successfully exploited as a cancer specific targeting moiety for the efficient delivery of chemotherapeutic agents, drug carriers, photosensitizers and diagnostic reporters. Critical to the success of such agents is the determination of the level of FR expression for a given tumor, since weak FR-expressing cancers will not respond well to folate-targeted therapies. There is therefore a need for specific and quantitative imaging agents and methods for the determination of FR expression in vivo. Optical imaging in the near-infrared (NIR) range allows efficient penetration of photons through living tissue and minimizes interference from tissue autofluorescence. Combined with quantitative fluorescence molecular tomography (FMT), NIR fluorescent agents have emerged as invaluable tools for quantitative, deep tissue imaging across a range of important areas of disease research including oncology. We have developed a new, near-infrared fluorescent folate targeted agent, VM3244, and used it to noninvasively quantify FR expression in vivo by FMT of mouse tumor xenografts. Two cell types, KB and HeLa with different degrees of FR expression were employed in the present study. In vitro, FR expression levels of each cell type were visualized with anti-FR antibody by microscopy, showing high FR expression in KB cells and lower expression in HeLa cells. The binding of VM3244 to FR was quantified by flow cytometry, confirming high and low FR expression in KB and HeLa cells, with good agreement between VM3244 and the antibody. Specificity of the agent was demonstrated by blocking of the signal by addition of an excess of unlabeled folic acid. In vivo quantification was performed by injection of 2 nmol of VM3244 into nude mice mice bearing KB or HeLa tumor xenografts and imaging by FMT at 4 to 24 h post injection. KB tumors showed a high level of FR expression with approximately 200 pmol (10% injected dose) quantified in the tumors, with little fluorescence in other tissues except the kidneys at 4 h post injection biodistribution study. Consistent with the in vitro profile, HeLa tumors had less tumor fluorescence, with about 75 pmol (3.8% injected dose) quantified in the tumors. Both tumor cell lines showed significant (∼80%) knockdown of signal by co-injection of the mice with unlabeled folic acid and ex vivo colocalization with FR antibody on tumor slices, confirming the in vivo specificity of the agent. Thus, we have demonstrated the ability of VM3244 to quantify FR expression both in vitro and in vivo and, combined with FMT, to noninvasively distinguish between high and lower FR expressing tumors. 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 2444. doi:1538-7445.AM2012-2444

Abstract 3949: In vivo imaging of tumor hypoxia by a new near-infrared fluorescent carbonic anhydrase IX-targeted agent

Carbonic anhydrase IX (CA IX), a cell surface enzyme involved in tumor cell acidification, is induced during tumor hypoxia (lack of oxygen) in many different types of tumors. CA IX expression is correlated to tumor cell proliferation and metastasis, poor prognosis, and resistance to therapeutic intervention, making CA IX an important biomarker in the study of hypoxia, tumor cell proliferation, and therapy. Therefore, determination of CAIX expression and identification of hypoxic tumors in vivo are critical for cancer research and treatments. We have designed and synthesized a near-infrared (NIR) fluorescent agent (VM3219) for in vivo detection and quantification of a cell surface biomarker of hypoxia, CA IX based on a well-known CA IX inhibitor, acetazolamide. VM3219 was characterized extensively in HeLa cells and in mouse HeLa tumor xenografts. Preliminary chemical and biochemical characterization of VM3219 showed excellent CA IX selectivity and affinity. In vitro, VM3219 detected 4-7-fold up-regulation of CA IX in live hypoxic HeLa cells as visualized by fluorescence microscopy and quantified by flow cytometry. VM3219 fluorescence signals were blocked by 80% when cells were pre-incubated with unlabeled acetazolamide. Fluorescent signal of VM3219 was low in normoxic HeLa cells and was comparable to the signal that were detected by using a non-binding control agent (VM3182) incubated with normoxic or hypoxic cells. In vivo imaging by fluorescence molecular tomography (FMT TM ) showed that approximately 5% of the injected dose (2 nmol VM3219) was retained in the tumor tissues after 24 h (100 -120 pmol), 10-20-fold more than measured with a non-binding control agent. Tumor fluorescence was blocked >65% by prophylactic acetazolamide treatment. Maintenance of tumor-bearing mice on low oxygen (8%), as compared to normally housed tumor bearing mice (20% oxygen), induced a >2X (∼250 pmol) increase in VM3219 tumor fluorescence signal suggesting that CAIX expression was up-regulated within tumors by low oxygen. In tissue sections, VM3219 signal was shown to be specifically co-localized in the tumor hypoxic regions with CA IX antibody and Hypoxyprobe (pimonidazole), and the signal was away from non-hypoxic regions as determined by Hoechst stain. In conclusion, VM3219 is a new non-invasive tumor hypoxia imaging agent that offers a tool for specific imaging of a biologically important hypoxia biomarker, CA IX. 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 3949. doi:1538-7445.AM2012-3949

Abstract 4888: Development of a novel FAST cathepsin-activatable NIR agent for tumor imaging

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Fluorescence molecular tomography (FMT) and near-infrared (NIR) imaging agents enable the quantification of kinetic changes in tumor biology in vivo, information useful for the detection, staging, and monitoring of chemotherapeutic efficacy. In particular, imaging agents that become fluorescent upon protease activation in vivo have emerged as valuable tools in oncology research due to the important role of proteases in tumor progression and invasion. Our objective was to develop a novel pan-cathepsin agent with the capability of fast activation kinetics and the potential for early imaging time points in monitoring tumor progression in vivo. To develop this agent, we compared it to an established and well characterized pan-cathepsin activatable imaging agent (ProSenseTM750, a high molecular weight, PEG-decorated NIR-labeled polylysine), which has a long circulation half-life of approximately 20h and offers a long and stable imaging window from 24 to 48h post-injection. ProSenseTM750 FAST is a newly developed 22 kDa molecule based on a novel scaffold consisting of a discrete pair of fluorophores separated by a cleavable sequence and attached to a small pharmacokinetic modifier. To compare selectivity profiles, the agents were assessed for activation by a variety of proteases, including cathepsins and matrix metalloproteases (MMPs). Based on their profiles, both agents show broad pan-cathepsin activation, cleaved by cathepsins B, S, L, and K, with little or no cleavage by MMPs. Consistent with its small architecture, ProSense750 FAST is efficiently activated, approximately 5X faster than the larger ProSense750, offering protease assay readouts as early as 1-2h. In mouse macrophages and 4T1 mouse breast adenocarcinoma cells, both agents were effectively up-taken and activated within the lysosomes for fluorescence microscopy assessment. Nearly complete inhibition of agent activation was achieved using the cell-permeable calpain/cysteine inhibitor E64d. In vivo, FMT imaging of ProSense750 FAST in a 4T-1 tumor model revealed clearly detectable tumor fluorescence as early as 2h after injection, reflecting its short pharmacokinetics (t1/2 ∼ 30 min) and quick in vitro activation profile, with quantification of peak signal around 6h and wash out by 72h. Animals can be optimally imaged at 6 or 24h + 1h, requiring scheduled injection/imaging times. The larger ProSense750 agent, in contrast, shows later optimal imaging (24-48h) but allows greater flexibility in imaging times for large animal cohorts (+ 3h). Careful assessment of organ biodistribution in 4T1 tumor-bearing mice showed different metabolic routes for the two agents, with ProSense750 cleared predominantly through the liver and ProSense750 FAST by renal clearance. These agents offer powerful tools for predicting and monitoring changes in tumor biology in preclinical models of disease. 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 4888. doi:10.1158/1538-7445.AM2011-4888

Abstract 4556: Fast-activating cathepsin B optical agent for quantitative in vivo imaging of tumor and therapeutic response

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cathepsin (Cat) B, a lysosomal cysteine protease, is strongly overexpressed in cancers and premalignant lesions and associated with cancer growth, neo-vascularization and metastasis. Cat B is also expressed in tumor-associated macrophages (TAM), inflammatory cells that comprise a significant proportion of tumor mass and are reported to be important for cancer progression. We evaluated a novel, highly selective, near-infrared fluorescence (NIRF) imaging agent, Cat B 750 FAST, for specific and rapid detection of tumor-associated Cat B activity. The agent consists of a pair of quenched fluorophores separated by a highly selective Cat B substrate, which is further conjugated to a pharmacokinetic modifier to achieve ∼2h plasma half-life. Upon in vitro proteolysis the agent de-quenches and becomes highly fluorescent (35-fold) with em=770 nm. To assess selectivity of activation, a variety of cathepsins and other tumor associated proteases were incubated with the agent under appropriate conditions for each enzyme. Cat B FAST was selectively cleaved and activated in vitro by Cat B. At 5 h, the fluorescence released by Cat B was 3-fold greater than by Cat S, 15-fold greater than by Cat L and >40-fold greater than by Cat K, Cat D or Legumain (p<0.05). It was rapidly activated following uptake into endosomes/lysosomes of isolated mouse macrophages, which was inhibited potently by Cat B inhibitors (p<0.05). Cat B 750 FAST agent signal accumulateed as early as 2-6 h in relevant tissues, seen both ex vivo in spontaneously-arising cancerous polyps in the intestines of APCmin mice maintained on high fat diet and in vivo in human colorectal HT-29 tumors implanted in nude mice. In vivo real-time 3D imaging and quantification with the FMT 2500 Fluorescence Molecular Tomography (FMT) system yielded quantifiable signal and good tumor definition as early as 2h, increased tumor fluorescence at 6h, and wash-out of fluorescence by 48h. Fluorescence microscopy at 6h showed more Cat B 750 FAST signal at the tumor periphery than the center, which co-localized with Cat B immunostaining. Cyclophosphamide treatment (170 mg/kg initial and 40 mg/kg daily thereafter) for 10 days slowed HT-29 tumor growth significantly (188 + 45 versus 353 + 83 mm3 in control, p=0.04), increased the overall tumor Cat B 750 FAST signal (p<0.05) and increased (p<0.05) macrophage specific anti-MAC3 immuno-fluorescent labeling in the ex vivo tumors, indicating increased TAM infiltration. In conclusion, a selective Cat B agent was developed with rapid pharmacokinetics for early and repeated imaging, enabling the in vivo FMT quantitative tomographic measurement of intra-tumoral Cat B activity and changes attributed to both cancer cells and tumor associated macrophages. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4556.