Jeannine Delaney

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.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

Detection and quantification of enzymatically active prostate-specific antigen in vivo

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+ LNCaP tumors as compared to PSA− prostate cancer 3 tumors (13.0±3.7 versus 2.8±0.8  pmol, p=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.

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 4939: Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate-Specific Antigen (PSA) is a 237 amino acid, 33 kDa, extracellular serine protease expressed in prostate epithelial tissue. In the normal prostate, high concentrations of PSA are stored in the prostatic ductal network. However, cancer leads to the disruption of normal tissue architecture and subsequent leakage of PSA into the tissue interstitium and circulation. Enzymatically active PSA is only present in prostate tissue and at other sites of prostate cancer growth. In circulation, active PSA immediately forms complexes with the serum protease inhibitor alpha-1-antichymotrypsin (ACT), while the inactive forms remains “free”. Traditional assays relied on testing for the presence of total PSA (mostly inactive and complexed) and do not provide information regarding the amount of enzymatically active PSA, a more biologically relevant tumor biomarker. To fill this gap, we developed a novel near infrared (NIR) fluorescent agent, designed to detect active PSA with no detection of inactive or complexed PSA. This agent, PSA750 FAST, contains a PSA-cleavable peptide sequence labeled with NIR fluorophores (ex/em 750/770 nm) and coupled to a pharmacokinetic modifier designed to improve its plasma availability. In its native state, the agent is nearly completely optically quenched (> 95%); and only becomes fluorescent upon enzymatic cleavage with active PSA, yielding a 300-1800 fold increase in signal as compared to the signal obtained using inactive or complexed PSA. Proteolytic cleavage was also selective for PSA over a large panel of enzymes, including Kallikrein 1, Cathepsin B, MMP-9, MMP-12, MMP-13, uPA, chymotrypsin and thrombin. We hypothesized that the enzymatic activity of PSA could be non-invasively and quantitatively monitored in 3D using Fluorescence Molecular Tomography (FMT), a powerful near-infrared imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals. LNCaP (PSA positive) and PC3 (PSA negative) cells were implanted in the chest area of male nude mice. Real time imaging was performed after systemic administration of PSA750 FAST when tumors reached the desired size. Our results demonstrated a significantly higher fluorescent signal in LNCaP tumors as compared the adjoining muscle (34.1 +/- 3.8 nM versus 7.2 +/- 0.6 nM, p=0.0043). Likewise, fluorescence concentration in tumors was significantly higher in LNCaP as compared to PC3 tumors (34.1 +/- 3.8 nM vs 17.14 +/- 3.48 nM, p=0.0191; 12.99 +/- 3.7 vs 2.8 +/- 0.8 pmoles, p=0.0233). This is the first report that demonstrates the feasibility of non-invasive, real-time, molecular imaging of active PSA in vivo in a relevant tumor model. These findings demonstrate the effectiveness of this agent in conjunction with optical imaging as a functional platform for molecular imaging in the field of prostate cancer. Citation Format: Jeffrey D. Peterson, Guojie Ho, Jeffrey Morin, Jeannine Delaney, Wael Yared, Milind Rajopadhye, Sylvie C. Kossodo. Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4939. doi:10.1158/1538-7445.AM2014-4939

Abstract 4934: Imaging and quantification of bombesin receptor expression in vivo using a NIR-labeled bombesin peptide

Gastrin-releasing peptide (GRP) is one of the mammalian analogs of bombesin-like peptides that function as growth factors in normal and neoplastic tissues upon binding to a family of bombesin-binding G protein-coupled receptors. Bombesin receptors are overexpressed in a variety of cancers, particularly prostate, breast, and gastrointestinal stromal tumors, and as such have been used to develop radiolabeled imaging tracers. As a pre-clinical alternative to using radiolabeled ligands, we developed a novel near infrared (NIR) fluorescent agent, BombesinRSense 680 (BRS680), designed to target and quantify bombesin receptors in vivo. BRS680 was produced from a modified 7 amino-acid GRP analog peptide labeled with a NIR fluorophore (ex/em 660/680 nm) and a pharmacokinetic modifier designed to improve its plasma availability (plasma t1/2 = 1.5 hours). In vitro labeling of human colonic adenocarcinoma HT-29 cells, which express GRP receptors, and blocking the signal by addition of unlabeled native bombesin, demonstrated the specificity of the agent by both fluorescence microscopy and flow cytometry. In vivo receptor expression was quantified by fluorescence tomography after BRS680 (2 nmol/mouse) was injected intravenously into nude mice bearing HT-29 tumor xenografts. HT-29 tumors showed a high level of receptor expression with approximately 30 pmol (1.5% injected dose) of BRS680 quantified in the tumors at 24 hours, and lower fluorescence in other tissues except for pancreas, a tissue known for high receptor expression, and kidneys, indicating renal clearance. In contrast to the fast clearance from circulation, the tumor tissue half-life of BRS680 was shown to be approximately 42 hours. In vivo targeting specificity was confirmed by collecting tumor tissue from injected mice and co-localizing BRS680 fluorescent signal with an anti-GRP receptor antibody on frozen sections. More importantly, treatment of HT-29 tumor-bearing mice with a tumor growth-arresting chemotherapy regime decreased in vivo BRS680 signal. Six days after beginning treatment with 5-fluorouracil and oxaliplatin in mice with established tumors, BRS680 fluorescent signal was significantly decreased in treated mice as compared to control mice (21.55 + 4.89 versus 34.10 + 2.90 pmoles, p=0.043) paralleling the inhibition of tumor growth (74.25 + 7.65 versus 141 + 19.39 mm3, p=0.003). Interestingly, chemotherapy did not consistently affect the fluorescent signal associated with ProSense 750 FAST, an agent that is specifically activated by the cathepsin family of inflammatory proteases, co-injected in the same animals (6.99 +1.68 versus 10.91 + 1.73 pmoles, p=0.104). These studies demonstrate the utility of BRS680 in tracking in vivo expression of bombesin receptors and underscores its potential to serve as an in vivo real-time indicator of anti-tumor treatment efficacy. Citation Format: Jeffrey D. Peterson, Nara Narayanan, Jeannine Delaney, Jeffrey Morin, Milind Rajopadhye, Wael Yared, Sylvie Kossodo. Imaging and quantification of bombesin receptor expression in vivo using a NIR-labeled bombesin peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2014-4934

Abstract LB-511: In vivo imaging of tumor vasculature with a novel near infra-red lectin agent

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 (e=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

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 4557: In vivo near-infrared fluorescent quantification of therapy-induced apoptosis using Annexin-Vivo 750

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Drug-induced ex vivo apoptotic tests are widely used pre-clinically to monitor chemotherapeutic efficacy. Real-time non-invasive in vivo imaging and quantification of therapy-induced tumor apoptosis would impact drug discovery significantly. As a key apoptotic marker, phosphatidylserine (PS) translocated to the outer leaflet of the plasma membrane can be detected by fluorescent-labeled Annexin A5 specifically with high affinity. We synthesized an in vivo imaging contrast agent Annexin-Vivo 750 by conjugating a human recombinant Annexin A5 to a near-infrared fluorophore with em 770 nm. Fluorescence microscopy showed 4h anti-FAS Ab treatment significantly increased the number of Annexin-Vivo 750 positive Jurkat cells, confirming that the agent binds to PS on apoptotic cells. Similarly, flow cytometry showed anti-FAS Ab increased Annexin-Vivo 750 positive cells from 15% to 55%. TUNEL positive cells were increased from 0% to 65.14 ± 3.93% (p=0.0004) by anti-FAS antibody. The TUNEL data correlate very well with flow cytometry data and are consistent with the fluorescence microscopy data. In vivo imaging of Annexin-Vivo 750 was performed in nude mice implanted with human colorectal carcinoma HT-29 cells. Once tumors reached a volume of 142.36 + 8.3 mm3 mice were randomized and treated intraperitoneally with cyclophosphamide (170 mg/kg) or vehicle at time 0h. The agent was injected at 24h with a dose of 2.4 nmol/animal and the tumors were imaged at 26h with an FMT2500 quantitative tomography imager for in vivo real-time 3D Annexin-Vivo 750 quantification. Tumor fluorescence signal was 2.55 fold higher with than without cyclophosphamide (p=0.0002), while tumor volumes were the same (p=0.35). Fluorescent signal decreased significantly by 24h and returned to baseline at 48h post agent injection. Likewise, intratumoral injection of TNFα (2.5 μg) plus IFNγ subcutaneous injection (2500 units) also significantly increased HT29 tumor Annexin-Vivo 750 fluorescence (3 fold compared to control, p=0.0099, 24h after treatment, 2h post-agent injection). TUNEL positive HT-29 cells were increased from 11.16 ± 7.05 % to 41.77 ± 8.78 % (p=0.0004) by cyclophosphomide, indicating apoptosis. These results demonstrate that Annexin-Vivo 750 in vivo imaging can be used for detecting, quantifying and monitoring apoptosis following cancer treatments, underscoring its value in drug discovery and development in cancer and potentially in other clinically important areas, such as cardiovascular-, autoimmune-, inflammatory- and neurodegenerative diseases. 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 4557.

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.