- PMC4177906
Published online Oct 29, 2013. doi: 10.1021/bc400328m
In Vivo Type 2 Cannabinoid Receptor-Targeted Tumor Optical Imaging Using a Near Infrared Fluorescent Probe
Abstract
The type 2 cannabinoid receptor (CB2R) plays a vital role in carcinogenesis and progression and is emerging as a therapeutic target for cancers. However, the exact role of CB2R in cancer progression and therapy remains unclear. This has driven the increasing efforts to study CB2R and cancers using molecular imaging tools. In addition, many types of cancers overexpress CB2R, and the expression levels of CB2R appear to be associated with tumor aggressiveness. Such upregulation of the receptor in cancer cells provides opportunities for CB2R-targeted imaging with high contrast and for therapy with low side effects. In the present study, we report the first in vivo tumor-targeted optical imaging using a novel CB2R-targeted near-infrared probe. In vitro cell fluorescent imaging and a competitive binding assay indicated specific binding of NIR760-mbc94 to CB2R in CB2-mid delayed brain tumor (DBT) cells. NIR760-mbc94 also preferentially labeled CB2-mid DBT tumors in vivo, with a 3.7-fold tumor-to-normal contrast enhancement at 72 h postinjection, whereas the fluorescence signal from the tumors of the mice treated with NIR760 free dye was nearly at the background level at the same time point. SR144528, a CB2R competitor, significantly inhibited tumor uptake of NIR760-mbc94, indicating that NIR760-mbc94 binds to CB2R specifically. In summary, NIR760-mbc94 specifically binds to CB2R in vitro and in vivo and appears to be a promising molecular tool that may have great potential for use in diagnostic imaging of CB2R-positive cancers and therapeutic monitoring as well as in elucidating the role of CB2R in cancer progression and therapy.
INTRODUCTION
Cannabinoid receptors belong to the G protein-coupled receptor (GPCR) family and are involved in the regulation of fundamental cellular functions throughout the body.1 To date, two types of cannabinoid receptors, type 1 (CB1R) and type 2 (CB2R), have been cloned and characterized.2 In general, CB1R is expressed at high levels in the brain and at much lower levels in peripheral tissues, whereas CB2R is predominantly a peripheral receptor abundantly expressed by immune cells.3,4 Recent studies have shown that CB2R plays a vital role in cancer evolution and progression and that CB2R ligands may offer therapeutic potential for cancer treatment.5,6 However, the exact role of CB2R in cancer remains unclear. This has driven the increasing efforts to study CB2R and cancers using molecular imaging tools.
CB2R represents a unique type of target for cancer research. CB2R expression is high only in the spleen, tonsils, thymus, macrophages, and leucocytes, and it is low or even undetectable in the brain, thyroid, retina, placenta, skeletal muscle, kidney, liver, adrenal gland, heart, prostate, and ovary.7,8 However, many types of cancers overexpress CB2R, such as prostate, skin, liver, and breast cancers, and the expression levels of CB2R appear to be associated with tumor aggressiveness.9–13 Such upregulation of the receptor in cancer cells provides opportunities for CB2R-targeted imaging with high contrast and for therapy with low side effects. However, the field of CB2R-targeted imaging, particularly in oncology, is largely unexplored.
To date, only a limited number of CB2R-targeted contrast agents have been reported for noninvasive positron emission tomography (PET) imaging of CB2R in cancers,14–17 and few studies have been reported with other imaging modalities, such as magnetic resonance imaging (MRI), optical imaging, and ultrasound.18 Despite the advantages of high sensitivity and deep tissue penetration, PET has many limitations, such as relatively low resolution, narrow time window, high instrument cost, and injection of radioactive agents. Optical imaging, however, serves as an alternative low-cost approach with relatively high sensitivity and resolution. The major disadvantage of limited tissue penetration can be partially resolved by adapting near-infrared (NIR) light (650–900 nm), under which tissues have a relatively low absorption, scattering, and autofluorescence.19 As such, NIR optical imaging is suitable for both cellular and in vivo imaging applications.
Recently, we reported the first NIR CB2R-targeted probe, NIR-mbc94, and validated its selective binding to CB2R in vitro.20Building upon our previous study, we now report the development, in vitro characterization, and in vivo cancer imaging of a novel CB2R-targeted NIR probe, NIR760-mbc94. To the best of our knowledge, this is the first in vivo optical imaging study using a CB2R-targeted NIR probe. Such a probe may serve as a valuable tool to image cancers and to elucidate the role of CB2R in cancer progression and therapy.
EXPERIMENTAL SECTION
Preparation of CB2R-Targeted NIR Probe NIR760-mbc94
The solvents used were of commercial grade. The ligand mbc94 was synthesized using a previously described procedure.8 Flash column chromatography was performed on a Teledyne ISCO (combiflash RF) purification system with silica gel (standard grade, 60A, Sorbtech) or C18-reversed-phase silica gel (20–40 µm, RediSepRf). 1H and 13C NMR spectra were recorded on a Bruker Avance III 400 MHz. Mass spectra were recorded on a Waters LCT Premier mass spectrometer. UV-vis spectra were recorded on a Cary 100 Bio UV-vis spectrophotometer, and fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer.
NIR760
The synthetic method is similar to the reported procedure.21Overall yield (two steps): 42%. 1H NMR (DMSO-d6): δ = 8.14 (d, 2H, J = 8 Hz), 7.55–7.58 (m, 4H), 7.32 (d, 2H, J = 8 Hz), 7.26 (d, 2H, J = 8.4 Hz), 7.09 (d, 2H, J = 14.4 Hz), 6.41 (d, 2H, J = 14.4 Hz), 4.26 (br.s, 4H), 2.71 (br.st, 4H), 2.54–2.55 (m, 4H), 1.94–1.97 (m, 6H), 1.1 (s, 12H). MS (ESI): m/z (M + 3H)+ calcd for C43H48N2O14S4, 944.20; found, 944.44.
NIR760-mbc94
A mixture of NIR760 (10 mg, 10 µmol), HBTU (5 mg, 13 µmol), and HOBt (1.8 mg, 13 µmol) in dry DMF (2 mL) was stirred at room temperature for 5 min. DIEA (3 µL, 17 µmol) was then added, and the mixture was stirred for another 10 min. After that, mbc94 (6 mg, 10 µmol) in dry DMF (1 mL) was added to the dye solution. The resulting mixture was stirred at room temperature under an atmosphere of Argon in the absence of light for 24 h. The solvent was removed, and the resulting solid was purified over a C18-reversed-phase silica gel column using H2O/MeOH (20% MeOH to 75% MeOH) as eluent, resulting in NIR760-mbc94 (4 mg, 25%) as a green solid. 1H NMR (DMSO-d6): δ = 8.62 (t, 1H, J = 4.8 Hz), 8.13 (d, 2 H, J = 8 Hz), 7.59 (d, 2H, J = 8 Hz), 7.46–7.51 (m, 5H), 7.38 (d, 2H, J = 8 Hz), 7.35 (d, 2H, J = 8 Hz), 7.29 (d, 1H, J = 8 Hz), 7.09 (d, 1H, J = 9.6 Hz), 7.01–7.06 (m, 3H), 6.91 (d, 1H, J = 9.2 Hz), 6.44 (d, 2H, J = 14.4 Hz), 5.5 (s, 2H), 4.27 (m, 4H), 4.11 (s, 2H), 3.69 (d, 1H, J = 8.8 Hz), 2.9 (t, 2H, J = 7.6 Hz), 2.72 (br.s, 4H), 2.52–2.54 (m, 4H), 2.33 (s, 3H), 1.92–1.97 (m, 6H), 1.62–1.73 (m, 6H), 1.23–1.34 (m, 8H), 1.08 (s, 12H), 1.01 (s, 3H), 0.84 (t, 3H, J = 7.2 Hz), 0.78 (s, 3H). 13C NMR (DMSO-d6): δ = 171.82, 165.71, 162.04, 146.39, 144.94, 142.80, 140.44, 138.16, 136.69, 134.27, 131.87, 131.84, 131.70, 130.88, 129.80, 129.66, 128.57, 128.10, 127.37, 110.70, 63.19, 48.48, 48.47, 48.22, 48.07, 31.31, 27.56, 26.22, 25.57, 25.40, 23.76, 21.59, 20.05, 20.01, 16.96, 14.23. MS (ESI): m/z (M + H)+ calcd for C78H95ClN7O14S4, 1516.55; found, 1516.6.
Viability Assay
The cell viability assay was performed using a mouse malignant astrocytoma cell line transfected with CB2R, CB2-mid DBT, which expresses CB2R at endogenous levels.22 CB2-mid DBT cells were cultured in DMEM containing 10% fetal bovine serum, 4 mM glutamine, 100 units/ml of penicillin, and 100 µg/mL of streptomycin. CB2-mid DBT cells were treated with various concentrations of NIR760-mbc94 ranging from 0 to 100 µM, with indocyanine green (ICG) (Sigma-Aldrich) serving as a negative control and tamoxifen (COSH Healthcare LTD) as a positive control23 for 24 h in a water-jacketed incubator (37 °C, 5% CO2). Cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) as per the manufacturer’s instructions.
Stability Study
To compare the stability of NIR760 and IRDye-800CW, solutions of the respective dyes in PBS with the same initial absorbance of 0.65 at the maximum absorption wavelength were kept in capped clear glass vials and exposed to ambient light. The fluorescence intensities at the maximum emission wavelength of the dyes were recorded in 1 cm quartz cuvettes (3 mL) over time.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
For CB2-mid DBT cell samples, CB2-mid DBT cells were seeded onto 3.5 mm dishes and cultured at 37 °C for 24 h. To collect CB2-mid DBT tumor samples, 1 × 106 CB2-mid DBT cells were subcutaneously implanted into the right flank of a 6–8 week old female nu/nu mouse. After approximately 10 days of tumor growth, mice were sacrificed, and CB2-mid DBT tumors were isolated and rapidly frozen by liquid nitrogen for storage. Total RNA was extracted from both CB2-mid DBT cells and CB2-mid DBT tumor samples using RNAzol Reagent (Invitrogen). Single-strand cDNA was synthesized from total RNA using the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen). For quantitative real-time PCR assays, primers for CB2R and GAPDH (housekeeping gene) were obtained from Integrated DNA Technologies. The sequences used for CB2R were 5′-tcctatcatttacgccctgc-3′ and 5′-ggctcctaggtggttttcacatcagcctc-3′,19 and for GAPDH (as housekeeping gene), the primer sequences were 5′-tgaacgggaagctcactggcat-3′ and 5′-tgcctgcttcaccaccttcttg-3′. First-strand cDNAs were synthesized using the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen). Amplifications were run using Platinum Taq DNA polymerase (Invitrogen) and consisted of 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C.
Western Blot
CB2-mid DBT cell and tumor samples were prepared using the same method described above. Cell lysates and tumor lysates were extracted with RIPA buffer with Halt Protease Inhibitor Cocktail (Thermo Scientific). Thirty micrograms of whole CB2-mid DBT cell and tumor protein extracts was resolved on a 10% SDS-PAGE gel before electrophoretic transfer onto a nitrocellulose membrane. After blocking with 5% nonfat milk in TBS-T (Tris-buffered solution pH 7.6 with 0.005% Tween 20), the membranes were incubated overnight at 4 °C with anti-CB2 primary monoclonal antibodies (Sigma-Aldrich) diluted in blocking buffer. The primary antibody reaction was followed by a 1 h incubation with the appropriate secondary antibodies. Immunocomplexes were detected using an enhanced chemiluminescence detection system and membrane exposure using the Chemi Doc MP Imaging System (Bio-Rad).
In Vitro Saturation Binding Assay of NIR760-mbc94
We carried out an intact cell saturation binding assay to determine CB2R binding affinity to NIR760-mbc94.24 Briefly, CB2-mid DBT cells were seeded onto 96-well optical bottom plates and incubated for 24 h. HBSS with 1 mM Mg2+, 0.1% BSA, and 0.1% NaN3 was used as the binding medium. Cells were preincubated with or without 100 nM of the blocking agent SR144528 (Cayman Chemical) for 30 min (nonspecific binding or total binding) and were then incubated for 1 h with an increasing concentration of NIR760-mbc94 at 4 °C. Cells were then rinsed, and fluorescence at 790 nm (relative fluorescence units, RFU) was read with a SynergyTM H4 Hybrid Multi-Mode Microplate Reader. DRAQ-5 (Cell Signaling) was used to normalize cell numbers from RFU. The specific binding was obtained by the subtraction of nonspecific binding from the total binding. The dissociation constant (Kd) and receptor density (Bmax) were estimated from the nonlinear fitting of specific binding versus NIR760-mbc94 concentration using Prism software (GraphPad).
Cell Fluorescent Imaging of NIR760-mbc94
CB2-mid DBT cells were treated with 5 µM NIR760-mbc94 or NIR760 at 37 °C for 30 min with or without 30 min of pretreatment with 10 µM of SR144528 as the CB2R competitor. After washing three times with serum-free medium, the cells were fixed with 4% paraformaldehyde/PBS for 20 min at room temperature. Cells were then permeabilized with 0.1% Triton X-100. The cell nucleus was stained with 1 µg/mL of DAPI for 15 min at room temperature. Cells were mounted and then imaged using the Zeiss Axio Observer fluorescent microscope with the ApoTome 2 imaging system. NIR760-mbc94 or NIR760 fluorescent images were captured with a NIR camera under ICG filters (excitation/emission: 750–800/820–875 nm). Nuclear images were obtained with DAPI filters (excitation/emission: 335–383/420–470 nm). Differential interference contrast (DIC) images were obtained through Trans light DIC.
We used a multiplate reader system for the quantitative CB2R binding assay. CB2-mid DBT cells were grown to 90% confluence in T75 flasks, harvested, and seeded onto 96-well optical plates followed by incubation in a water-jacketed incubator (37 °C, 5% CO2) for 24 h prior to the assay. On the day of the assay, the culture medium was aspirated and replaced with 100 µL of serum-free media with or without 10 µM of SR144528 for 30 min at 37 °C. Next, 5 µM NIR760-mbc94 or NIR760 or DMSO as the vehicle was added in culture medium, and the cells were incubated for an additional 30 min. Prior to reading the plate, the cells were washed three times with serum-free medium. A Synergy H4 Hybrid Multi-Mode Microplate Reader was used for reading fluorescence at 740/790 nm (excitation/emission). Assays for each group were done in triplicate.
In Vivo Optical Imaging and Competitive Blocking Studies
We conducted our animal experiments in accordance with the guidelines for the Care and Use of Laboratory Animals of the Medical Research Council of University of Pittsburgh. CB2-mid DBT cells (1 ×106) were subcutaneously implanted into the right flank of 6–8 week old female nu/nu mice. Experiments with tumor-bearing mice were performed 10 days after the injection of tumor cells. Mice were divided into three groups, and the mice were injected with the following agents (dissolved in 100 µL of saline) via the tail vein: three mice received 10 nmol of NIR760-mbc94, three received 10 nmol of free NIR760, and three mice received 100 nmol of SR144528 that was administered 1 h before the injection of 10 nmol of NIR760-mbc94 (SR144528 + NIR760-mbc94). Mice were anesthetized with 2.5% isoflurane, and tumor CB2R-targeted images were obtained preinjection and at 0.02, 1, 2, 6, 12, 24, 48, and 72 h postinjection with a charge-coupled device camera-based bioluminescence imaging system (IVIS Lumina XR; excitation filter, 745 nm; emission filter, 800 nm; exposure time, 1 s; binning, small; field of view, 12; f/stop, 2; open filter). The signal was displayed as radiant efficiency ([photons/s/cm2/sr]/[µW/cm2]). Images were acquired and analyzed using Living Image 2.5 software (Xenogen). For determining tumor contrast, the radiant efficiency of the tumor area at the right flank of the animal (T) and of the area at the left flank [normal tissue (N)] was calculated by the region of interest (ROI) function in the Living Image software. Dividing T by N yielded the contrast between the tumor and normal tissue.
Ex Vivo Imaging, Biodistribution, and Histological Study
At 72 h postinjection of NIR760-mbc94, NIR760, or SR144528 + NIR760-mbc94, CB2-mid DBT tumor-bearing nude mice were sacrificed. Tumors as well as organs of interest (blood, heart, lung, liver, spleen, pancreas, kidney, muscle from left leg, and brain) were extracted. Ex vivo imaging was carried out, and biodistribution was analyzed. For histological study, tumor samples were embedded in Tissue-Tek OCT compound mounting medium (Sakura Finetek U.S.A. Inc.), frozen in liquid nitrogen, and cryostat-sectioned (5 µm) using a Leica Jung CM3000. Histological sections from cryostat blocks were stained with hematoxylin and eosin (H&E).
Data Processing and Statistics
All of the data are given as the mean ± standard deviation (SD) of nindependent measurements. Statistical analysis was performed using a two-tailed unpaired Student’s t test (IBM SPSS Statistics version 21), with p values <0.05 considered to be statistically significant.
RESULTS
Characterization of Properties of NIR760-mbc94
To develop the NIR CB2R probe, NIR760-mbc94, we synthesized a functional CB2R ligand, mbc94, and a NIR dye, NIR760. Mbc94 was then coupled to NIR760 using an amide-coupling reaction (Figure 1a,b). Both the free dye, NIR760, and the probe, NIR760-mbc94, show intense absorption in the NIR region in aqueous solutions (Figure 1c). NIR760 has a maximum absorption peak at 760 nm, which was red-shifted to 766 nm after mbc94 was coupled to NIR760. The molar extinction coefficients of NIR760 and NIR760-mbc94 are 2.4 × 105 and 1.44 × 105 M−1 cm−1, respectively. NIR760 and NIR760-mbc94 are highly emissive, and they exhibit strong fluorescence with maxima at 781 and 785 nm, respectively. Their relative fluorescence quantum yields in water were measured with indocyanine green (ICG) as the standard.21 The quantum yield of NIR760 is 11.7%, and the quantum yield of NIR760-mbc94 is about 15.2%.
NIR760-mbc94 Has Low Toxicity to Living Cells
We used the CellTiter-Glo Luminescent Cell Viability Assay kit to evaluate cell toxicity. The assay uses a homogeneous method of determining the number of viable cells in culture based on a luminescent quantitation of the ATP present, an indicator of metabolically active cells. As shown in Figure 2, CB2-mid DBT cells treated with NIR760-mbc94 exhibited a comparably low cytotoxicity to those treated with ICG, the only FDA approved NIR fluorescent dye. Tamoxifen, a commonly used chemotherapy drug, served as the positive control. As expected, the number of viable cells dramatically decreased after treatment with 50 and 100 µM tamoxifen.
RT-PCR and Western Blot
RT-PCR was performed using primers for the CB2 receptor or GAPDH, with expected amplicons of 165 and 124 base pairs (bp), respectively. CB2R-positive bands at 165 bp were detected in both CB2-mid DBT cells and tumors using RT-PCR (Figure 3a). GAPDH positive band at 124 bp were also found in CB2-mid DBT cells and tumors samples. Therefore, CB2-mid DBT cells and tumors revealed positive mRNA expression of CB2R. In western blots of protein homogenates from CB2-mid DBT cells and tumors, we found major bands at 55 kDa, which indicated positive expression of CB2R at the protein level. β-Actin (loading control)-positive bands at 42 kDa were also present in CB2-mid DBT cells and tumor protein samples (Figure 3b).
NIR760-mbc94 Has Nanomolar Level Binding Affinity with CB2R
To determine NIR760-mbc94’s binding affinity to CB2R in intact cells, we used an in vitro saturation binding assay to measure the equilibrium dissociation constant (Kd) and the maximum specific binding (Bmax). A large excess of SR144528 was added to a parallel set of cells to saturate the receptor binding sites and to account for nonspecific binding. A representative saturation binding curve is shown in Figure 4. The data show that NIR760-mbc94 binds to CB2R with a Kd of 26.9 nM (±3.7) and Bmax of 259.8 pmol/mg (±11.9).
NIR760-mbc94 Specifically Binds to CB2R in Vitro
To evaluate the specificity and imaging potential of NIR760-mbc94 in vitro, we incubated CB2-mid DBT cells with 5 µM of NIR760-mbc94 or NIR760 at 37 °C for 30 min with or without a 30 min pretreatment with 10 µM of SR144528, a well-known CB2R ligand. After washing, no significant fluorescence signal was observed from cells incubated with the free dye (NIR760) control, whereas cells incubated with NIR760-mbc94 showed a strong fluorescence signal, primarily localized in cytoplasm. In addition, SR144528 significantly reduced NIR760-mbc94 uptake in CB2-mid DBT cells (Figure 5a), indicating specific binding of NIR760-mbc94 to CB2R. To evaluate quantitatively the specificity of NIR760-mbc94 for CB2R, we performed CB2-mid DBT cell binding assays using a multiwell plate reader system (Figure 5b). Cells incubated with NIR760-mbc94 showed a 4-fold higher fluorescence signal than those incubated with NIR760 (459.67 ± 27.54 vs 116.33 ± 15.37, p< 0.001). When challenged with SR144528 (10 µM), CB2-mid DBT cells incubated with 5 µM of NIR760-mbc94 exhibited significantly lower (~40%) fluorescence intensity (from 459.67 ± 27.54 to 276.33 ± 57.62, p = 0.008), whereas NIR760- or vehicle (DMSO)-treated cells did not show a significant change in fluorescence intensity after the challenge.
In Vivo Tumor CB2R-Targeted Optical Imaging with NIR760-mbc94
To study the potential of NIR760-mbc94 in CB2R-targeted cancer imaging in living systems, in vivo optical imaging was performed in a CB2-mid DBT mouse tumor model. In vivo imaging experiments were initiated approximately 10 days postinoculation of CB2-mid DBT tumor cells subcutaneously. Mice were divided into three groups (three mice in each group): NIR760-mbc94 (10 nmol), free NIR760 (10 nmol), and 10-fold SR144528 (100 nmol) administered 1 h before NIR760-mbc94 (10 nmol) injection. The tumor sizes in all mice showed no significant difference (data not shown). Figure 6a shows the time-dependent fluorescence images of one representative mouse from each group. Because all images are displayed on the same scale, a direct visual comparison can be made among the tumor profiles of NIR760-mbc94, free NIR760 dye, and NIR760-mbc94 + SR144528. The free dye control mouse demonstrated an intense fluorescent signal throughout the whole body immediately after injection, but the strong signal only lasted for roughly 2 h. In addition, no obvious contrast enhancement in the tumor region was observed. However, the tumor of the mouse injected with NIR760-mbc94 showed a visible signal contrast immediately after the injection, and the uptake gradually increased over time and reached maximum at 24 h postinjection. Tumor uptake gradually cleared 24 h postinjection, but the tumor contrast was visible even at 48 and 72 h postinjection. Mice treated with both NIR760-mbc94 and SR144528 showed less tumor uptake than NIR760-mbc94-treated mice at nearly all time points (Figure 6a).
To account for the nonspecific binding of NIR760-mbc94 in normal tissues, we normalized NIR760-mbc94 signals in the tumor area (T, right flank) with that in the normal area (N, same ROI in left flank) to generate time-dependent contrast enhancement (T/N) profiles (Figure 6b). In NIR760-treated mice, the peak T/N ratio appeared at 24 h postinjection, with only a 1.74-fold contrast enhancement, which gradually decreased to the baseline level at 72 h postinjection. By contrast, the T/N ratio of NIR760-mbc94-treated mice increased gradually over time, with a 2.04-, 2.86-, and 3.73-fold contrast enhancement at 24, 48, and 72 h postinjection, respectively. Importantly, challenged mice treated with both NIR760-mbc94 and SR144528 showed decreased T/N ratios as compared with unchallenged mice, with a 1.80-, 2.40-, and 2.56-fold contrast enhancement at 24, 48, and 72 h postinjection, respectively. A significant difference in the T/N ratio was found between challenged and unchallenged mice at the 48 (16% blockage effect, p = 0.007) and 72 h (31% blockage effect, p = 0.001) postinjection time points (Figure 6c). Similar to our in vitro competitive binding study, SR144528 partially inhibited tumor uptake of NIR760-mbc94 in vivo, indicating that NIR760-mbc94 labeled tumor tissues through specific binding to CB2R.
Ex Vivo Imaging, Biodistribution, and Histological Study
After the last imaging data point, all CB2-mid DBT tumor-bearing mice were sacrificed. The tumor and organs of interest (blood, heart, lung, liver, spleen, pancreas, kidney, muscle from left leg, and brain) were harvested and imaged with the IVIS imaging system (Figure 7a). The fluorescent intensities in the tumor and organs were measured, and the signal contrast (fluorescence signal from tumor or organ/signal from muscle) values are presented in Figure 7b,c. The NIR760-mbc94-treated mice showed an ex vivo T/N ratio as high as roughly 10 (9.77 ± 2.10), whereas SR144528-challenged mice showed a significantly lower T/N ratio of roughly 6 (6.32 ± 0.24 vs 9.77 ± 2.10, p = 0.048), and free dye control mice showed a T/N ratio of almost 1 (1.23 ± 0.16 vs 9.77 ± 2.10, p < 0.001). The significant signal contrast in the liver and kidney from unchallenged (liver/N = 12.53 ± 1.80 and kidney/N = 4.05 ± 0.92) and challenged (liver/N = 15.70 ± 2.35 and kidney/N = 6.10 ± 1.45) mice indicate that NIR760-mbc94 was cleared from both the liver and kidney. H&E staining (Figure 7d) from CB2-mid DBT tumor tissues revealed that the tumor nuclei were irregular-shaped with hyperchromatin, polymorphism, and increased mitotic activity.
DISCUSSION
Targeting receptors that cancers overexpress remains a common strategy for molecular imaging of the disease. Examples of upregulated receptors include somatostatin, integrin, cholecystokinin-2, translocator protein, epidermal growth factor, human epidermal growth factor 2, vascular endothelial growth factor, folate, and estrogen receptors.25 However, many of these receptors, although at lower levels, are expressed in normal tissues as well.26–34 As a result, high-contrast imaging is challenging. CB2R is a superior target for cancer-imaging studies. Under basal conditions, CB2R is expressed mainly in cells of the immune system, and the expression in other types of cells is low to undetectable.20,35 The high upregulation of CB2R in certain cancer cells and the undetectable expression in corresponding normal tissues allows for high-contrast cancer imaging. In addition, CB2R-targeted imaging could help to elucidate the exact role of CB2R in cancer progression as well as its potential as a therapeutic target and could be applied to cancer diagnosis, therapeutic monitoring, and high-throughput drug screening.20
Among all imaging modalities, NIR optical imaging stands out as a promising and low-cost technique with high sensitivity and resolution. NIR optical imaging has been widely used to image cancer both in vitro and in vivo.25 In our previous study, we developed the first NIR CB2R-targeted probe, NIR-mbc94, and validated its selective binding to CB2R in CB2-mid DBT cells.20 To advance the scope of our CB2R-targeted NIR imaging probe to in vivo applications, we synthesized a novel CB2R-targeted NIR probe, NIR760-mbc94, and investigated its potential in tumor CB2R-targeted imaging both in vitro and in vivo. NIR-mbc94 and NIR760-mbc94 share the same targeting moiety, whereas the NIR fluorophores are different. IRDye-800CW, the fluorophore used in NIR-mbc94, is a commercially available dye with high hydrophilicity and intense absorption and emission in the desired NIR region. However, IRDye-800CW is a high-cost asymmetric dye whose synthesis and purification is rather challenging. In addition, previous studies indicate that heptamethine cyanine dyes with an enol ether linkage, such as IRDye-800CW, have significant stability issues.21 NIR760 is a new NIR fluorescent dye developed in our lab with high hydrophilicity and intense NIR absorption and emission. Moreover, NIR760 is a symmetric dye that can be easily synthesized with low cost. Furthermore, NIR760 overcomes the stability issue of IRDye-800CW with a more robust C-C linkage at the meso position of the polymethine chain. The spectroscopic properties and overall reaction yields of NIR760 and IRDye-800CW are summarized in Table 1. NIR760 has a comparably high molar extinction coefficient (ε = 2.14 × 105 M−1 cm−1) to that of IRDye-800CW (ε = 2.37 × 105M−1 cm−1),36 and it has a significantly higher fluorescence quantum yield (Φ = 20.1 vs 14.2% for IRDye-800CW). In addition, NIR760 can be synthesized with only two steps and an overall yield of 42%, whereas the synthesis of IRDye-800CW (with terminal carboxylic acid group) requires four reaction steps with an overall yield of only 13%.37 To compare the stability of these two dyes, we exposed two dye samples in PBS with the same initial absorption intensity to the ambient light and monitored the change of fluorescence intensity over time. As shown in Figure S1, after 2 h of exposure, the fluorescence intensity of IRDye-800CW decreased by 55%, whereas that of NIR760 decreased by only 37%. Overall, NIR760 appears to be a superior NIR dye. The attachment of the CB2R targeting moiety (mbc94) red-shifted the maximum absorption of NIR760 from 760 to 766 nm (Figure 1b). NIR760-mbc94 has a high molar extinction coefficient (ε = 1.44 × 105 M−1 cm−1) and strong emission at 785 nm with a quantum yield (Φ = 15.2% in water) that is more than 50 times higher than that of ICG (Φ = 0.28% in water38), the only FDA-approved NIR fluorescent dye.
NIR760-mbc94 specifically binds to CB2R in vitro, as evidenced by studies from cell saturation binding assays, fluorescent microscopy, and a multiplate reader system. As shown in Figure 4, the Kd value for NIR760-mbc94 bound to CB2R was 26.9 nM (±3.7) and the Bmax over Kd value was roughly 10, indicating favorable binding affinity for in vivo imaging. Furthermore, cellular fluorescence imaging (Figure 5a) showed that NIR760-mbc94 was internalized and localized to the cytoplasm of CB2-mid DBT cells and that SR144528 partially (40%) inhibited NIR760-mbc94 uptake. Cells treated with free dye control did not show significant fluorescence signal. These results indicate specific binding of NIR760-mbc94 to the target receptor and are consistent with our previous cellular imaging studies using another fluorescent CB2R probe.8,20, As a more quantitative approach, a multiplate reader system was used to analyze cellular uptake of NIR760-mbc94 (Figure 5b). Cells treated with NIR760-mbc94 showed a 4-fold higher fluorescence signal than those with free NIR 760 dye (p < 0.001), and blocking with SR144528 (10 µM) resulted in an approximately 40% decrease in fluorescence intensity for cells with NIR760-mbc94 (p < 0.01).
Encouraged by the promising in vitro imaging results, we evaluated the CB2R-targeted imaging potential of NIR760-mbc94 in CB2-mid DBT tumor-bearing mice. Free NIR760 showed quick bioclearance from the bodies without tumor contrast, and no significant fluorescence signal was observed at 6 h postinjection. Conversely, we observed significant tumor contrast in the mice injected with NIR760-mbc94, and the T/N ratio increased gradually over time, with a 2.04-, 2.86-, and 3.73-fold of contrast enhancement at 24, 48, and 72 h postinjection, respectively. The high tumor uptake and slow clearance of NIR760-mbc94 may involve CB2R specific binding as well as a rebound effect (GPCR can translocate from cytoplasm back to the cell surface after internalizing the ligand), which is a typical internalization mechanism observed in GPCRs.39
To validate further the CB2R specificity of NIR760-mbc94 in vivo, the blocking effect was studied by injecting mice with blocking agent SR144528 1 h before the NIR760-mbc94 injection. We observed a 31% decrease in the T/N ratio at the 72 h postinjection time point (p < 0.05), which was consistent with the in vitro blocking study where 40% blocking was shown. The partial inhibition indicates nonspecific binding of NIR760-mbc94, which may result from its inherent net negative charge. Recent studies have shown that the nonspecific binding of imaging probes can be minimized by reducing the net charge.40 Future study will involve the development of optimized CB2R probes with reduced nonspecific binding to allow for enhanced imaging contrast at the target.
CONCLUSIONS
We have developed a novel CB2R-targeted NIR probe that demonstrated specific binding both in vitro and in vivo. This study reports the first in vivo CB2R-targeted optical imaging using a molecular probe. Given the important but unclear role of CB2R in cancer progression and therapy, such a targeted probe may be of great value to the CB2R and cancer research communities. Specifically, NIR760-mbc94 may have great potential for noninvasive diagnostic imaging of cancer, monitoring therapeutic effects, high-throughput screening of CB2R ligands, and defining the relationship between CB2R and cancer.
ACKNOWLEDGMENTS
We thank Dr. Nephi Stella at the University of Washington for providing DBT cells and technical advice. We also thank Dr. Xiangqun Xie at the University of Pittsburgh for providing support to this project. This work was supported by the startup fund provided by the Department of Radiology, University of Pittsburgh. This project used the UPCI imaging facilities supported, in part, by award P30CA047904.
Footnotes
ASSOCIATED CONTENT
Supporting Information
Stability of NIR760 and IRDye-800CW in PBS. This material is available free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
REFERENCES
