Probe Synthesis and Characterization
The uMUC-1-targeted MN-EPPT probe and the scrambled control probe, MN-SCR, were synthesized and characterized as described (1
). Peptide sequences were as follows: EPPT= C-AHA-A-R-E-P-P-T-R-T-F-A-Y-W-G-K(FITC); SCR= C-AHA-A-E-G-R-P-TF-P-T-R-A-Y-W-K(FITC). Synthesis resulted in a triple labeled nanoparticle, consisting of FITC on EPPT peptide (for fluorescence microscopy), superparamagnetic iron oxide (MN, a magnetic label for MRI), and Cy5.5 dye (Amersham Biosciences, Piscataway, NJ) attached to the MN (for NIRF optical imaging). Iron concentration and peptide/FITC, Cy5.5 payloads were determined as described in (1
). The resultant probe had an average iron concentration of 7.3±0.16 mg/ml, 2.1 Cy5.5 molecules per nanoparticle, and 4.43 peptides per nanoparticle.
In vitro Cell Treatment
To validate the observation that treatment with doxorubicin downregulates uMUC-1 in BT-20 breast adenocarcinoma cells, cells were incubated with a 0.4 µM of doxorubicin-HCl (Sigma, St. Louis, MO) for 48 hrs, as previously described (17
). PBS-treated cells served as controls. Following incubation, the cells were analyzed for uMUC-1 expression by quantitative RT-PCR analysis and Western blot, as described below. Alternatively, to assess the relative accumulation of MN-EPPT in these cells, following treatment with doxorubicin, the cells were incubated at 37°C overnight with MN-EPPT or the scrambled control probe, MN-SCR, washed, fixed in 2% paraformaldehyde, suspended in 0.5 ml PCR tubes, and imaged by MRI, as described below. Following the MR imaging session, the cells were transferred to FACS tubes and analyzed by flow cytometry.
Real-Time Quantitative RT-PCR
Total RNA was extracted from breast adenocarcinoma cells and tumor tissues, using the Rneasy Mini kit, according to the manufacturer’s protocol (Qiagen Inc., Valencia, CA). Relative levels of uMUC-1 mRNA were determined by real-time quantitative RT-PCR (TaqMan protocol). TaqMan analysis was performed using an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). The PCR primers and TaqMan probe specific for MUC-1 mRNA were designed using Primer express software 1.5. Primer and Probe sequences were as follows:
Forward primer, 5’-ACAGGTTCTGGTCATGCAAGC-3’ (nucleotides 64–84 in the 5’ non-repetitive region);
Reverse primer, 5’-CTCACAGCATTCTTCTCAGTAGAGCT-3’ (nucleotides 139–164 in the 5’ non-repetitive region);
TaqMan Probe, 5’-FAM-TGGAGAAAAGGAGACTTCGGCTACCCAGA-TAMRA-3’ (nucleotides 96–124 in the 5’ non-repetitive region).
Eukaryotic 18S rRNA TaqMan PDAR Endogenous Control reagent mix (PE Applied Biosystems, Foster City, CA) was used to amplify 18S rRNA as an internal control, according to the manufacturer’s protocol.
For Western blot, total cell extracts were prepared by solubilization of BT-20 breast adenocarcinoma cells with RIPA lysis and extraction buffer (Pierce, Rockford, IL) with added Halt™ Protease Inhibitor Cocktail (Pierce, Rockford, IL).
Protein concentration in the extracts was determined using BCA Protein Assay kit (Pierce, Rockford, IL). Samples were incubated with 1% SDS and 3% 2-mercaptoethanol at 100°C for 5 min. and centrifuged. Approximately 100 µg of total protein was separated on a 12% polyacrylamide gel (SDS-PAGE). Precision Plus Protein Standards/Kaleidoscope (Bio-Rad, Hercules, CA) were used as molecular weight standards. Separated proteins were transferred to PVDF membrane and developed with One-Step TM Advanced Western Kit for mouse primary antibody (Genscript Corp., Piscataway, NJ). Primary anti-Muc-1 mouse monoclonal antibodies specific for the backbone peptide APDTRPAP (VU4H5 clone, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Tubulin mouse monoclonal antibodies as an internal standard (V10178, Biomeda, Foster City, CA) were used at a 200- and a 10,000-fold dilution, respectively.
MR Imaging of Cell Phantoms
In order to show that the specificity of MN-EPPT for uMUC-1 can be used to probe for the downregulation of uMUC-1 by doxorubicin, we performed MR imaging of cell phantoms prepared as described in the previous section. Imaging was performed using a 9.4T Bruker horizontal bore scanner (Billerica, MA) equipped with ParaVision 3.0 software. The imaging protocol consisted of coronal T2 weighted spin echo (SE) pulse sequences with the following parameters: SE TR/TE = 3000/ [8, 16, 24, 32, 40, 48, 56, 64]; FOV = 40 ×40 mm; matrix size = 128 × 128 pixels; slice thickness = 0.5 mm; in-plane resolution = 312×312 µm. Image reconstruction and analysis were performed using Marevisi 3.5 software (Institute for Biodiagnostics, National Research Council, Canada). T2 maps were constructed according to established protocol by fitting the T2 values for each of the eight echo times (TE) to a standard exponential decay curve.
T2 relaxation times were calculated by manually segmenting out the cell pellet on MR images.
Flow cytometry was performed in order to confirm if the MN-EPPT specificity for uMUC-1 translates in reduced probe accumulation in breast adenocarcinoma cells, following uMUC-1 downregulation by doxorubicin. Flow cytometry was performed on cells treated as described in the “In vitro Cell Treatment” section, using a FACSCalibur (Becton Dickinson, San Diego, CA, USA) equipped with the Cell Quest software package (Becton Dickinson, San Diego, CA).
In order to establish a pre-clinical orthotopic tumor model of human breast cancer, 5–6 wk old female nu/nu
mice (n = 10; Massachusetts General Hospital Radiation Oncology breeding facilities) were inoculated in the right mammary fat pad with the uMUC-1-positive human breast adenocarcinoma cell line, BT-20 (American Type Culture Collection, Manassas, VA), as previously described (18
All animal experiments were performed in compliance with institutional guidelines and according to the animal protocol approved by the Subcommittee on Research Animals Care (SRAC) at Massachusetts General Hospital.
In order to establish a clinically relevant treatment model, we utilized the standard chemotherapeutic agent, doxorubicin (DOX). The treatment protocol involved intravenous injections of 7 mg/kg of DOX in saline solution once weekly for two consecutive weeks beginning ~8 d after tumor implantation once tumors had reached a diameter of 0.5 cm as previously suggested (17
). Saline solution-injected animals served as non-treated controls. As previously described, in order to evaluate tumor response to chemotherapy, in vivo imaging was performed one day before the beginning of treatment and one day following the completion of treatment (3
In vivo MR Imaging
MRI was performed before and 24-hrs after intravenous injection of MN-EPPT or the scrambled control probe, MN-SCR (10 mg Fe/kg), using a 9.4T Bruker horizontal bore scanner (Billerica, MA) equipped with ParaVision 3.0 software using sequences as for in vitro MRI. Image reconstruction and analysis were performed as described for in vitro MRI.
Tumor volumes and tumor T2 relaxation times were calculated by manually segmenting out the tumor on MR images. Quantitative evaluation of differential tumor growth by MRI was based on multislice T2-weighted images. The volume was estimated according to the formula for the volume of an ellipsoid: V = 4/3 π(abc) where a and b are the equatorial radii (along the x and y axes) and c is the polar radius (along the z-axis). For T2 map analysis of relaxation times, the terminal slices were not included in the analysis in order to avoid interference from partial volume effects. Relative MN-EPPT accumulation in the tumors was estimated based on the formula: T2 before injection minus T2 after injection (delta-T2, ms).
In vivo and ex vivo Optical Imaging
In vivo near-infrared fluorescence (NIRF) optical imaging was performed immediately after each MR imaging session. Animals were placed into a whole-mouse imaging system (Imaging Station IS2000MM, Eastman Kodak Company, New Haven, CT) and imaged in the Cy5.5 channel. At the end-point of each experiment following the last imaging session, mice were sacrificed, tumors excised, placed in the optical imaging system and imaged ex vivo. Image analysis was performed using the Kodak 1D™ 3.6.3 Network software. The actual volumes of excised tumors were determined by measuring tumor dimensions ex vivo using calipers.
Immunohistochemistry and in situ apoptosis detection
To detect the accumulation of MN-EPPT in tumors at the microscopic level, we performed correlative immunohistochemistry. Tumors were embedded in Tissue-Tek O.C.T. Compound (Sakura Fineteck, Japan) and snap-frozen in liquid nitrogen. Tumors were then cut into 7-µm frozen sections, fixed in 2% paraformaldehyde, washed, counterstained with VECTASHIELD Mounting Medium with DAPI (Vector) and analyzed by fluorescence microscopy. Microscopy was performed using a Nikon Eclipse 50i fluorescence microscope equipped with an appropriate filter set (Chroma Technology Corporation, Rockingham, VT). Images were acquired using a CCD camera with near infrared sensitivity (SPOT 7.4 Slider RTKE; Diagnostic Instruments, Sterling Heights, MI) and analyzed using SPOT 4.0 advanced version software (Diagnostic Instruments, Sterling Heights, MI). Fluorescence was collected in the green channel for detection of the FITC label on EPPT peptides, blue channel for DAPI and in the NIR channel for detection of the Cy5.5 label on MN nanoparticles.
To evaluate levels of apoptosis in tumor cells, we performed a terminal deoxynucleotidyl transferase –mediated dNTP nick end-labeling (TUNEL) assay (Apoptag Fluorescein In Situ Apoptosis Detection kit, Chemicon International, Temecula, CA) according to the manufacturer's protocol. The nuclei were counterstained with DAPI and examined under the fluorescence microscope.
All data were represented as means ±SEM. Statistical analysis was performed using two-tailed Student’s t test and linear regression where indicated. A p-value ≤0.05 was considered statistically significant.