Reagents
Panitumumab, a fully humanized IgG2 monoclonal antibody (mAb) directed against human EGFR was purchased from AMGEN Inc. (Thousand Oaks, CA). A water soluble,silicon-based-phthalocyanine dye derivative, IRDye 700DX NHS ester (IR700; C74H96N12Na4O27S6Si3, molecular weight of 1954.22), was obtained from LI-COR Bioscience (Lincoln, NE). All other chemicals used were of reagent grade.
Synthesis of IR700-conjugated Panitumumab
Panitumumab (1 mg, 6.8 nmol) was incubated with IR700 (66.8 μg, 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na2HPO4 (pH 8.5) at room temperature for 1 h. Then the mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). The protein concentration was determined with a Coomassie Plus protein assay kit (Pierce Biotechnology, Rockford, IL) by measuring the absorption at 595 nm (8453 Value System; Agilent Technologies, Santa Clara, CA). The concentration of IR700 was measured by its absorption to confirm the number of fluorophore molecules conjugated to each Panitumumab molecule. The number of IR700 per antibody was approximately 4 for the 1:4.5 reaction conditions. The resulting compound, Pan-IR700, was kept at 4°C as a stock solution.
Cell line
EGFR-expressing MDA-MB-468luc, [6] stable luciferase-transfected cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in tissue culture flasks using a humidified incubator at 37°C in an atmosphere of 95% air and 5% carbon dioxide.
Fluorescence microscopy
To detect the antigen specific localization of Pan-IR700, fluorescence microscopy was performed (BX61; Olympus America, Melville, NY). MDA-MB-468luc was seeded on cover-glass-bottomed dishes and incubated for 16 h. Pan-IR700 conjugate (10 μg/mL) was added to the culture medium and incubated for 6 h at 37°C, followed by washing with PBS. The filter was set to detect IR700 fluorescence with a 590–650 nm excitation filter and a 665–740 nm band pass emission filter.
Phototoxicity assay
Cytotoxicity of PIT was determined by measuring luciferase activity and by quantitative flow cytometry using propidium iodide (PI) as a stain for dead cells. For the luciferase activity assay, d-luciferin (150 μg/ml, Gold Biotechnology, St. Louis, MO) was added to the culture medium and bioluminescence (signal intensity) was detected (Photon Imager; Biospace Lab, Cambridge, MA). For the flow cytometry assay, cells were trypsinized after treatment, washed with PBS and 1 μL of PI (5 mg/mL) was added to the cell suspension which was vortexed. Cells were analyzed on a flow cytometer (FACS Calibur; BD BioSciences, San Jose, CA).
In vitrophotoimmunotherapy
Cells were seeded on a 96 well plate or 35 mm cell culture dishes and incubated for 8 h at 37°C. The culture medium was refreshed and 10 μg/mL of Pan-IR700 was added over night. After washing with PBS, phenol red free culture medium was added. Then, cells were irradiated with a red light-emitting diode (LED), which emits light at 670 to 710 nm wavelength (L690-66-60; Marubeni America Co., New York, NY), controlled by FluorVivo software (INDEC Systems, Santa Clara, CA) at a current of 100 mA (continuous-wave; CW), 400 mA (CW) and 1733 mA (pulse-wave; PW). The pulse wave duration was 0.2 ms separated by 0.8 ms so that the pulse occurred every 1 ms (Figure 1). The power density of the LED was 12.5 mW/cm2 at 100 mA CW and 25 mW/cm2 at 400 mA CW and 1733 mA PW as measured with an optical power meter (PM 100; Thorlabs, Newton, NJ).
MDA-MB-468luc cells were irradiated at 0.2, 0.5, 2 and 5 J/cm2 using all 3 power settings (100 mA CW, 400 mA CW and 1733 mA PW). Cell viability was analyzed with flow cytometry and bioluminescence imaging.
Pan-IR700 was added to cells at concentrations of 0.3, 1, 3, 10 μg/mL. Cells were incubated for 8 h followed by washing once with PBS and restoration of phenol red free culture medium. The cells were then irradiated with the LED light of 400 mA (CW) at a total dose of 2 J/cm2. In vitro treatments for cells were performed using the following combinations of Pan-IR700 and NIR light dose: (1) 3 μg/mL and 0.5 J/cm2, (2) 1 μg/mL and 1.5 J/cm2, and (3) 0.3 μg/mL and 5 J/cm2.
Tumor model
All procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the local Animal Care and Use Committee. Six- to eight-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick, Frederick, MD). During the procedure, mice were anesthetized with isoflurane. MDA-MB-468luc cells (2 × 106 cells) were injected subcutaneously into the right mammary pads of the mice. The experiments were conducted 2 weeks after MDA-MB-468luc cell implantation.
In vivophotoimmunotherapy with different power levels of LED light
Orthotopic breast tumors were irradiated at all three power settings, 100 mA (CW), 400 mA (CW) and 1733 mA (PW). Total irradiation doses were 30 J/cm2 with power density of 200 mW/cm2. Mice images were acquired with a fluorescence imager (Pearl Imager; LI-COR Biosciences) for detecting IR700 fluorescence, and Photon Imager for BLI. BLI was used for evaluation of PIT effects. Regions of interest (ROIs) were placed over the entire tumor and photon numbers were counted for each ROI.
Statistical analysis
Statistical analysis was performed using a statistics program (GraphPad Prism6, GraphPad Software, La Jolla, CA). A one-way analysis of variance (ANOVA) was used to compare differences in responses to level of light exposure among the three groups. Pearson’s correlation coefficient was used to analyze the correlation between the dead cell ratio and the concentrations of Pan-IR700. Values of p < 0.05 were considered statistically significant.