uMuc-1 Tumor Antigen As An Imaging And Therapeutic Target
Zdravka Medarova, Young Kim, George Dai, Anna Moore
Athinoula A. Martinos Center For Biomedical Imaging • Department Of Radiology • Massachusetts General Hospital • Harvard Medical School
INTRODUCTION
One of the early hallmarks of tumorigenesis in the majority of epithelial cell adenocarcinomas is the overexpression and underglycosylation of the MUC-1 antigen (uMUC-1). As a result of underglycosylation, core peptide epitopes on uMUC-1, which are cryptic in the non-neoplastic state, become exposed and can be targeted for imaging and therapeutic purposes. In order to target the uMUC-1 tumor antigen, we synthesized a multimodal imaging probe (CLIO-EPPT) utilizing the magnetic resonance (MR) and optical imaging modalities. In addition, the probe was modified with EPPT peptides specific for the uMUC-1 antigen. CLIO-EPPT- a targeted multimodal imaging probe CLIO: Magnetic Resonance Imaging (MRI) Cy5.5: Near Infrared Fluorescence Optical Imaging (NIRF) EPPT: C-AHA-Y-C(ACM)A-R-E-P-P-T-R-T-FA-Y-W-G-K(FITC) uMUC-1: APDTRP peptide core repeat domain Physical Properties of CLIO-EPPT Table 1
Compound CLIO-NH2 CLIO-EPPT Size(nm) 29.5 35.8 R1(s-1mM-1) 22.54 26.43 R2(s-1mM-1) 43.69 53.44 Peptides per particle none 7 Cy 5.5 per particle none 1
RESULTS: CLIO-EPPT binding with uMUC-1 + and uMUC-1 - cell lines (fluorescence microscopy)
III. In vivo MR/NIRF imaging and therapeutic assessment in an orthotopic model of pancreatic cancer MATERIALS AND METHODS In vivo MR and optical imaging of uMUC-1 tumor antigen. a. Animals with orthotopic tumors (uMUC-1 positive CAPAN-2 pancreatic adenocarcinoma) b. Intravenous injection of CLIO-EPPT (10 mg Fe/kg). c. MR imaging was performed before and 24 h after injection of the probe. d. Optical imaging was performed 24 post injection immediately after MR imaging e. Correlative fluorescence microscopy on frozen tumor sections.
uMUC-1 positive cell lines: BT-20 CAPAN-2 ChaGo-K-1 HT-29 LS174 T uMUC-1 negative cell lines: 293 MCF10-A
Treatment model: 5-fluorouracil i.p. (30mg/kg), once a day for 5 days. Imaging was performed 1d before the beginning of treatment and 1d after the completion of treatment. RESULTS: CLIO-EPPT - utility for tumor delineation in a pre-clinical model of pancreatic adenocarcinoma (MRI) SE TR/TE 3000/15, 30, 45, 60; FoV 30x30 mm; Matrix size 128x128; Slice thickness 0.5mm; Resolution 375x375 µm; 9.4T Bruker horizontal bore scanner (Billerica, MA); ParaVision 3.0 software Image reconstruction Marevisi 3.5 software (Institute for Biodiagnostics, National Research Council, Canada) RESULTS: CLIO-EPPT - utility for tumor delineation in a preclinical model of pancreatic adenocarcinoma (NIRF) a. In vivo: white-light (left), NIRF (middle), color-coded NIRF (right). b. Ex vivo pancreas: white-light (left), NIRF (middle), color-coded NIRF (right). c. Histology tumor: Cy5.5, FITC, overlay, H&E (arrows point to blood vessels). RESULTS: In vivo MR/NIRF tracking of tumor response to therapy Imaging: MRI: SE TR/TE = 3000/ 8, 16, 24, 32, 40, 48, 56, 64; FoV = 40x40 mm; Matrix size 128x128; Slice thickness = 0.5mm; 9.4T Bruker horizontal bore scanner (Billerica, MA); ParaVision 3.0 software NIRF: Exposure time: 20.05 sec; F-stop 2.8; FOV 100mm; resolution 260dpi RESULTS: CLIO-EPPT is a reliable tool for monitoring tumor response to therapy - Ex vivo evidence
II. In vivo MR/NIRF imaging of subcutaneous tumor models using CLIO-EPPT MATERIALS AND METHODS 1) 2) 3) 4) 5) Animals with bi-lateral tumors (uMUC-1 positive and uMUC-1 negative) Intravenous injection of CLIO-EPPT (10 mg Fe/kg). MR imaging was performed before and 24 h after injection of the probe. Optical imaging was performed 24 post injection immediately after MR imaging Correlative fluorescence microscopy on frozen tumor sections.
RESULTS: In vivo MR imaging of tumor bearing mice injected with CLIOEPPT (U87/LS174T) SE TR/TE = 3000/ 8, 16, 24, 32, 40, 48, 56, 64 FoV = 40x40 mm Matrix size 128x128 Slice thickness = 0.5mm (total 5 slices) Resolution 312x312 µm Imaging time 12min 48 sec 9.4T Bruker horizontal bore scanner (Billerica, MA) ParaVision 3.0 software Image reconstruction Marevisi 3.5 software (Institute for Biodiagnostics, National Research Council, Canada)
I. Specificity of CLIO-EPPT MATERIALS AND METHODS 1) Evaluation of uMUC-1 expression by quantitative RT-PCR 2) Evaluation of specific CLIO-EPPT uptake by a panel of adenocarcinoma cell lines: a. cell binding assay b. fluorescence microscopy
RESULTS: MUC-1 relative expression (quantitative RT-PCR)
RESULTS: In vivo NIRF imaging of tumor bearing mice injected with CLIO-EPPT (U87 brain/LS174T colon)
uMUC-1uMUC-1+
Ex vivo NIRF: Signal persists even after treatment TUNEL: Tumor response to therapy is reflected by differences in apoptosis rates between treated and non-treated animals
Light image
NIRF image
Color-coded image
RESULTS: CLIO-EPPT binding to uMUC-1-positive and uMUC-1-negative cell lines - cell binding assay
RESULTS: Ex vivo NIRF imaging and histology of tumors from mice injected with CLIO-EPPT
Light NIRF
CONCLUSIONS
We have developed a targeted imaging probe (CLIO-EPPT) which is specific for the underglycosylated mucin 1 (uMUC-1) tumor antigen and displays In vivo selectivity for uMUC-1-expressing adenocarcinomas. By combining the high spatial resolution and unlimited depth penetration of MRI with the high sensitivity and low cost of NIRF optical imaging, the multimodal strategy described here permits detailed high-throughput assessment of changes in tumor volume in response to therapy. The described imaging strategy has numerous implications, including but not limited to, optimization of existing cancer treatment regiments, testing of novel therapeutic paradigms, development of personalized medicine protocols, and detailed analysis of the mechanisms behind cancer progression In vivo.
Moore A et al, Cancer Res, 2004, 64:1821-1827.