In vivo breast cancer characterization imaging using two monoclonal antibodies activatably labeled with near infrared fluorophores

Panitumumab (Pan), a fully human IgG₂ monoclonal antibody (mAb) directed against the extracellular domain of the human EGFR (HER1), was purchased from Amgen (Thousand Oaks, CA, USA). Trastuzumab (Tra), a recombinant humanized mAb directed against the human HER2, was purchased from Genentech Inc. (South San Francisco, CA, USA). ICG-Sulfo-OSu was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Alexa680-NHS ester was purchased from Invitrogen Co. (Carlsbad, CA, USA). All other chemicals used were of reagent grade.

Panitumumab (0.5 mg, 3.4 nmol) was incubated with Alexa680-NHS ester (39.3 μg, 34 nmol) in 0.1 M Na₂HPO₄ (pH 8.6) at room temperature for one hour, followed by the purification with a size exclusion column (PD-10; GE Healthcare, Piscataway, NJ, USA). ICG labeling of trastuzumab was also performed by reacting mAb with ICG at a ratio of 1:6 in the same manner as Pan-Alexa680. The concentrations of Alexa680 and ICG were calculated by measuring the absorption with the UV-Vis system (8453 Value UV-Vis system; Agilent Technologies, Santa Clara, CA, USA) to confirm the number of fluorophore molecules conjugated with each antibody molecule. The protein concentration was also determined by measuring the absorption at 280 nm with a UV-Vis system. The number of Alexa680 and ICG per antibody was adjusted to approximately 4.0 to 4.5 and 0.7 to 1.0, respectively. As a comparison, Pan conjugated with approximately one Alexa680 (always-on type; Pan-Alexa680 (ON)) was also synthesized.

The quenching abilities of each conjugate were investigated by denaturing them with 1% SDS as described previously [9]. Briefly, the conjugates were incubated with 1% sodium dodecyl sulfate (SDS) in PBS for 15 minutes at room temperature. As a control, the samples were incubated in PBS. The fluorescence signal intensity of Pan-Alexa680 was measured with a fluorescence spectrometer (Perkin-Elmer LS55, Perkin-Elmer, Shelton, CT, USA). The change in fluorescence intensity of ICG was investigated with an in vivo imaging system (Maestro, CRi Inc., Woburn, MA, USA) using 710 to 760 nm excitation and 800 nm long-pass emission filters.

The EGFR+/HER2- breast cancer cell line, MDA-MB-468, and the EGFR-/HER2+ cell line, NIH/3T3 (3T3/HER2), were used. Both cell lines were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Life Technologies), 0.03% L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin in 5% CO₂ at 37°C.

MDA-MB-468 cells or 3T3/HER2 cells (1 × 10⁴) were plated on a covered glass-bottomed culture well and incubated for 16 hours. Pan-Alexa680 (ON or self-quenched; SQ) and Tra-ICG (10 μg/mL) were then added to MDA-MB-468 cells and 3T3/HER2 cells, respectively. The cells were incubated for either one or eight hours followed by washing once with PBS, and fluorescence microscopy was performed using an Olympus BX81 microscope (Olympus America, Inc., Melville, NY, USA) equipped with the following filters: excitation wavelength 590 to 650 nm and 672.5 to 747.5 nm, emission wavelength 662.5 to 747.5 nm and 765 to 855 nm for Alexa680 and ICG, respectively. Transmitted light differential interference contrast images were also acquired. To validate the specific binding of the antibody, excess antibody (100 μg) was used to block 10 μg of dye-antibody conjugates.

Fluorescence signal from cells after incubation with Pan-Alexa680 (ON, SQ) or Tra-ICG was measured using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and CellQuest software (BD Biosciences). Briefly, MDA-MB-468 and 3T3/HER2 cells (1 × 10⁵) were incubated with Pan-Alexa680 (ON or SQ) and Tra-ICG at 37°C for one or eight hours, respectively. To validate the specific binding of the antibody, excess antibody (50 μg) was used to block 0.5 μg of dye-antibody conjugates.

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 National Cancer Institute Animal Care and Use Committee. Six- to eight-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick, Frederick, MD, USA). During the procedure, mice were anesthetized with isoflurane. MDA-MB-468 cells (2 × 10⁶ cells) were injected subcutaneously into the right mammary pads of the mice, and two weeks later (because they grow faster), 3T3/HER2 cells (2 × 10⁶ cells) were injected into the left mammary pads. The experiments were conducted at six days after 3T3/HER2 cell injection.

A mixture of Tra-ICG and Pan-Alexa680(SQ or ON) (each 50 μg) was injected via the tail vein into tumor-bearing mice (MDA-MB-468 and 3T3/HER2). The mice were anesthetized with intraperitoneally administered 10% sodium pentobarbital, and fluorescence images were obtained for four days with a flurescence camera (Pearl Imager LI-COR Biosciences using the 700 and 800 nm fluorescence channel or Maestro in vivo Imaging System CRi) using two filter sets). The red filter sets were used to image Alexa680 fluorescence and the NIR filter sets were used for ICG fluorescence. The red filter set uses a band-pass filter, which ranges between 615 to 665 nm (excitation) and a long-pass filter over 700 nm (emission); the NIR filter set uses a band-pass filter from 710 to 760 nm (excitation) and a long-pass filter over 800 nm (emission). The tunable emission filter was automatically stepped in 10 nm increments from 650 to 950 nm for the red and NIR filter sets at constant exposure. The spectral fluorescence images consist of autofluorescence spectra and the spectra from Alexa680 and ICG, which were then unmixed, based on their spectral patterns using commercial software (Maestro software; CRi). Mice were sacrificed with carbon dioxide immediately after in vivo imaging. The tumors were excised and ex vivo imaging was performed using Pearl Imager and Maestro in vivo imaging system.

The quenching capacities measured by adding 1% SDS to dye-conjugated antibody were 50-, 5.2- and 1.2-fold for Tra-ICG, Pan-Alexa680(SQ), and Pan-Alexa680(ON), respectively.

In the microscopy studies (Figure 1), the fluorescence signals from Pan-Alexa680(ON) and Pan-Alexa680(SQ) were observed on the surface of MDA-MB-468 cells one hour after incubation. Both probes also showed fluorescent signal within the cells eight hours after incubation, and the fluorescent dots were brighter for Pan-Alexa680(SQ) than for Pan-Alexa680(ON). These signals were completely blocked by the addition of excess panitumumab. Similarly, fluorescence signal from Tra-ICG was observed within the 3T3/HER2 cells. While signal was minimal at one hour after incubation because of greater quenching magnitude of Tra-ICG than Pan-Alexa680, many bright intracellular foci were present by eight hours, and these signals were blocked by excess trastuzumab.

FACS studies were similar to microscopy studies, that is, Pan-Alexa680(ON, SQ) and Tra-ICG exhibited specific binding to MDA-MB-468 and 3T3/HER2 cells, respectively (Figure 2). Pan-Alexa680(SQ) showed progressively brighter signal over time compared with Pan-Alexa680(ON) where the signal was lower and constant.

Figure 3A shows the images of tumor-bearing mice (MDA-MB-468 and 3T3/HER2) injected with the mixture of Pan-Alexa680(ON or SQ) and Tra-ICG, which was obtained with the Pearl Imager. Pan-Alexa680(ON) detected the targeted tumor (MDA-MB-468, EGFR+); however, high background and non-specific tumor uptake (3T3/HER2) are evident on Day 1 through Day 3 post injection. At Day 4, relatively selective images of EGFR positive (MDA-MB-468) tumors were obtained. In contrast, Pan-Alexa680(SQ) clearly visualized MDA-MB-468 with high tumor-to-background ratios as early as Day 2 and at all later time points, although a slight signal was detected in the 3T3/HER2 tumor and bladder at Day 1. On the other hand, Tra-ICG specifically visualized the HER2 positive 3T3/HER2 tumors, although slight fluorescent signals were noticed in the liver and intestine. Tumor-to-background (non-targeted tumors, liver, and neck) ratios were significantly higher (P < 0.01) for Pan-Alexa680(SQ) compared with Pan-Alexa680(ON) for four days (Figure 3B). Tumor-to-non-targeted tumor, tumor-liver and tumor-neck ratios were 8.1, 10.3, and 8.8 for Pan-Alexa680(SQ) at Day 4; whereas, they were 2.7, 3.9, and 4.5 for Pan-Alexa680(ON). In contrast, tumor-to-background ratios with Tra-ICG were not significantly different between the two groups (Figure 3C). The Maestro spectral fluorescence imager provided similar images to the Pearl imager (Figure 4). For instance, tumor-to-non-targeted tumor, tumor-liver and tumor-neck were 8.1, 6.7, and 7.9 for Pan-Alexa680(SQ) at Day 4, whereas, these same ratios were 2.8, 3.0, and 4.2 for Pan-Alexa680(ON). Tumor-to-background ratios on Tra-ICG were not significantly different between both groups.