1) Segregate and suspend monolayer cells with 80% confluence in phosphate-buffered saline (PBS).
2) Develop heterotopic models by subcutaneously inoculating cell suspension (5x105 cells or adjusted for different cells) in nude mice.
3) Implant the tumors in both hind limbs of mice and use for experiments when the tumor size reaches 0.5 cm in diameter.
Orthotopic brain cancer models were developed by intracranial injection of D54 cancer cells. Lung tumors developed following tail vein injection of H460 cells. Liver metastases developed splenic injection of HT22 cells.
The phage-displayed peptide library represents 10 million independent clones of phages expressing random nonamer peptides that were displayed on T7 phages as fusion with N-terminus of 10A capsid proteins. There are 415 copies of unique fusion peptide per virion. Briefly,
1) After treating the tumor-bearing mice were treated, administer phage libraries intracardiac injection at 4 hours following irradiation.
2) After 10 minutes of circulation, euthanize the mice and perfuse with 10 ml of PBS into the left ventricle that were recovered from the right atrium. Perfuse with PBS at a rate of 2 ml per minute to remove phages that stayed in circulation but did not bind to blood vessels.
3) Sacrifice mice to remove organs and tumors for quantifying plaque-forming units.
4) Weigh the organs so that the number of phage can be normalized by weight of the organ.
5) Disrupt the tissues using a hand-held homogenizer on ice. To avoid cross contamination, clean the homogenizer with bleach and rinse with PBS between homogenization of different organs.
6) Microcentrifuge the homogenate 5000 rpm and discard the supernatant.
7) Resuspend the pellets and wash 5 times with PBS. Amplify the T7 phages bound to tumors (which are insoluble in pellets) by adding E. coli BL21 into the washed pellets.
8) To determine the total phage output per organ, titrate the resuspended cell pellets in bacterial culture in 15 minutes when the phage infection has occurred but the amplification is not yet complete.
9) Normalize the titers of phages recovered from each tissue with the weight of each tissue.
10) Amplify the phages recovered from the treated tumors at 37 oC with shaking until the culture is lysed (in around 2.5 hours).
11) Centrifuge the cultures 8000 rpm for 15 minutes to have the amplified phages in supernatants. Partially purify the amplified phages by PEG-precipitation and resuspend in PBS for next round of biopanning by repeating these steps.
12) After six rounds of biopanning, isolate single plaques from soft agar, and amplify gene fragments encoding peptides polymerase chain reaction (PCR) following standard protocols.
13) The PCR primers include an upstream primer (5’-AGC GGA CCA GAT TAT CGC TA-3’) and a downstream primer (5’- AAC CCT CAA GAC CCG TTT A-3’). Perform the sequencing reaction with one primer and collect sequence data in an ABI 377 sequencer. Deduce the peptide sequences from the decoded DNA information.
1) Treat or inject tumor-bearing mice with 1×109 pfu of HVGGSSV phage or control phage, respectively.
2) At 48 hours after administration of phages, remove and fix the tumors.
3) Stain phages in tumor tissue with anti-T7 phage polyclonal antibodies (a gift from Dr. Toshiyuki Mori, National Cancer Institute at Frederick, MD).
4) Use a secondary IgG-HRP conjugate (Sigma, St. Louise, MS) to visualize the primary antibody binding by using DAB (3,3'-Diaminobenzidine, Sigma) as substrate for HRP.
5) Counterstain the tissues with Hematoxylin. In detection of use a complex of streptavidin and biotinylated peptide, anti-streptavidin antibody (Sigma) respectively.
6) Perform TUNEL staining as previously described 24.
Near infrared (NIR) imaging:
1) Label the PEG-precipitated phages, synthetic HVGGSSV peptide (Genemed Synthesis. South San Francisco, CA) or streptavidin (Sigma), with amine-reactive Cy7 dye (Amersham) by following the manufacturer’s instructions.
2) Inject labeled phages or the complex of biotinylated peptide and streptavidin-Cy7 conjugate into the circulation by tail vein or jugular vein catheter in tumor-bearing mice that had been treated with IR and/or TKIs.
3) Take near infrared images with IVIS imaging system (Xenogen Corp., Hopkinton, MA) at various time points after injection.
4) Measure radiance (photons/sec/cm2) in the region of interest (ROI) by using the program provided by the Xenogen.
5) While correlating peptide binding (radiance) to tumor response (tumor growth), normalize radiance from peptide within tumors to that of the whole body.
Tumor growth study:
1) Implant tumors in hind limbs of mice.
2) Start treatment when tumor size reached 0.5 cm in diameter. Groups include irradiation (IR, 3 Gy), SU11248 (40 mg/kg), , combined treatment with IR and SU11248, and untreated control. SU11248 was administrated through intraperitoneal injection. Give all treatments once a day for 5 consecutive days.
3) Measure tumor size every other day by use of calipers and calculate fold increase in tumor volume (compared to the tumor size in the first day of treatment) to show tumor responsiveness to the treatment. Include six animals in each group.
Data analysis and Statistics:
Analyze group comparisonwith student t test. Linear correlation of peptide binding and tumor response to treatment was developed by use of Correlation coefficient of tumor growth and radiance data sets (CORREL).