Abstract 4556: Fast-activating cathepsin B optical agent for quantitative in vivo imaging of tumor and therapeutic response
Authors: Groves Kevin , Morin Jeff , Zhang Jun , Handy Emma , Delaney Jeannine , Kossodo Sylvie , Bao Bagna , Cuneo Gary , Peterson Jeffrey D. , Rajopadhye Milind , Madden Karen , Gao David Z. 
Published: February 2011

Abstract
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 &gt;40-fold greater than by Cat K, Cat D or Legumain (p&lt;0.05). It was rapidly activated following uptake into endosomes/lysosomes of isolated mouse macrophages, which was inhibited potently by Cat B inhibitors (p&lt;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&lt;0.05) and increased (p&lt;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.