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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC May 15, 2011.
Published in final edited form as:
PMCID: PMC2871283
NIHMSID: NIHMS188889
Near Infrared Heptamethine Cyanine Dye-Mediated Cancer Imaging*
Xiaojian Yang,1,2 Chunmeng Shi,1,3 Rong Tong,4 Weiping Qian,1 Haiyen E. Zhau,1,2 Ruoxiang Wang,1 Guodong Zhu,1 Jianjun Cheng,4 Vincent W. Yang,5 Tianmin Cheng,3 Maged Henary,6 Lucjan Strekowski,6 and Leland W.K. Chung1,2,7
1 Molecular Urology and Therapeutics, Department of Urology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA
2 Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
3 Chongqing Engineering and Technology Center for Nanomedicine, Third Military Medical University, Chongqing 400038, China
4 Department of Material Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
5 Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
6 Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
7 To whom correspondence should be addressed: Leland W. K. Chung, Ph.D., Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
Purpose
Near-infrared (NIR) fluorescence imaging has great potential for noninvasive in vivo imaging of tumors. In this study, we demonstrate the preferential uptake and retention of two hepatamethine cyanine dyes, IR-783 and MHI-148, in tumor cells and tissues.
Experimental Design
IR-783 and MHI-148 were investigated for their ability to accumulate in human cancer cells, tumor xenografts and spontaneous mouse tumors in transgenic animals. Time- and concentration-dependent dye uptake and retention in normal and cancer cells and tissues were compared, and subcellular localization of the dyes and mechanisms of the dye uptake and retention in tumor cells were evaluated using organelle-specific tracking dyes and bromosulfophthalein (BSP), a competitive inhibitor of organic anion transporting peptides (OATPs). These dyes were used to detect human cancer metastases in a mouse model and differentiate cancer cells from normal cells in blood.
Results
These NIR hepatamethine cyanine dyes were retained in cancer cells but not normal cells, in tumor xenografts, and in spontaneous tumors in transgenic mice. They can be used to detect cancer metastasis and cancer cells in blood with a high degree of sensitivity. The dyes were found to concentrate in the mitochondria and lysosomes of cancer cells, probably through OATPs since the dye uptake and retention in cancer cells can be blocked completely by BSP. These dyes, when injected to mice, did not cause systemic toxicity.
Conclusions
These two heptamethine cyanine dyes are promising imaging agents for human cancers and can be further exploited to improve cancer detection, prognosis and treatment.
Keywords: near-infrared cyanine dye, transgenic mouse models, human tumor xenografts, prostate cancer, intestinal tumors, organic anion transporting peptides, cancer detection imaging, targeting
Near-infrared (NIR) excitable fluorescent contrast agents offer unique possibilities for in vivo cancer imaging. These agents show little autofluorescence in aqueous solution, and upon binding to macromolecules in cells, NIR dyes display drastically increased fluorescence due to rigidization of the fluorophores (1). The most common NIR fluorophores are polymethine cyanine dyes. In clinical practice, pentamethine and heptamethine cyanines comprised of benzoxazole, indole, and quinoline are of great value and interest (2, 3). These organic dyes are characterized by high extinction coefficients and relatively large Stokes' shifts. With emission profiles at 700–1000 nm, their fluorescence can be readily detected from deep tissues by commercially available imaging modalities (4-6).
Application of organic dyes in cancer detection and diagnosis has yet to be fully explored (5). The conventional approach to tumor imaging is through designed delivery of NIR fluorophores, mostly by chemical conjugation to tumor-specific ligands including metabolic substrates, aptamers, growth factors, and antibodies (7-10). A number of surface molecules have been tested as targets, including membrane receptors, extracellular matrices, cancer cell-specific markers and neovascular endothelial cell-specific markers (11-13). One limitation of these approaches is that the NIR moieties only detect specific cancer cell types with well-characterized surface properties, whereas tumors are notorious for their heterogeneity (14, 15). In addition, chemical conjugation may alter the specificity and affinity of the targeting ligands (3). A simpler and more straightforward strategy is needed to broaden the use of NIR dyes for non-invasive tumor imaging.
We identified a class of NIR fluorescence heptamethine cyanines as dual imaging and targeting agents, and present our results with IR-783 and MHI-148, two prototypic heptamethine cyanine dyes. These organic dyes are spontaneously taken up and accumulated in cancer but not normal cells, providing the advantage of tumor-specific targeting that does not require chemical conjugation of the imaging dyes. Administration of the organic dyes to tumor-bearing mice, combined with non-invasive NIR imaging, enabled us to detect a panel of human and mouse tumors in various experimental settings. Exposure of human cancer cells to these dyes allowed us to differentiate normal from cancer cells and detect cancer cells in human blood with a high degree of sensitivity. These dyes were found to be non-toxic when administered to mice. The dual imaging and targeting property of these organic dyes could be further exploited as improved modalities of cancer detection, diagnosis and therapy. These two heptamethine cyanine dyes have almost identical imaging and targeting properties. In this report, we used IR-783 to demonstrate their many exploitable features.
Chemicals
The heptamethine cyanine dye IR 783 (2-[2-[2-Chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium) was purchased from Sigma-Aldrich (St. Louis, MO) and purified by the published methods. The heptamethine cyanine dye MHI-148, (2-[2-[2-Chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(5-carboxypentyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium bromide), was synthesized and purified as described previously (16-18). The other heptamethine cyanine dyes and their derivatives (see Supplement Table 1) were also prepared by published procedures. All materials were dissolved in DMSO diluted with appropriate vehicles, filtered through 0.2 μm filters and stored at 4°C before use.
Cell lines and cell culture
Human cancer cells used in this study were: prostate cancer (LNCaP, C4-2, C4-2B, ARCaPE, ARCaPM, and PC-3), lung cancer (H358), breast cancer (MCF-7), cervical cancer (HeLa), leukemia (K562), renal cancer (SN12C, ACHN), bladder cancer (T24) and pancreatic cancer (MIA PaCa-2). As controls, normal human bone marrow stroma cells (HS-27A), normal human prostate epithelial cells (P69 and NPE), normal human prostate fibroblasts (NPF), human vascular endothelial cells (HUVEC-CS), and human embryonic kidney cells (HEK293) were used. LNCaP, ARCaP, and their lineage-derived cells (C4-2 and C4-2 B) were established by our laboratory and cultured in T-medium as described (19, 20). Human prostate epithelial cells, NPE, and human prostate fibroblasts, NPF, were derived from the normal areas of prostatectomy specimens by our laboratory using an Emory University approved protocol and were maintained in T-medium as described (21). SN12C was obtained from a patient with renal clear cell carcinoma (22) and was cultured in T-medium. Unless otherwise specified, all of the other human cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in ATCC recommended media, with 5% fetal bovine serum (FBS) and 1× penicillin/streptomycin at 37°C with 5% CO2. In this study, we also investigated the dye uptake by mouse pancreatic cancer cell lines, PDAC2.3, PDAC3.3, BTC3 and BTC4, derived from transgenic mice, kindly provided to us by Dr. Douglas Hanahan from University of California at San Francisco. These cells were also cultured in T-medium.
Cell and tissue uptake study using NIR heptamethine cyanine dyes
Cells (1×104/well) were seeded on vitronectin-coated four-well chamber slides (Nalgen Nunc, Naperville, IL) and incubated with T-medium containing 5% FBS for 24 h. After the cells had attached to the chamber slides, the cells were washed with PBS (Phosphate Buffered Saline) and exposed to the cyanine dye at a concentration of 20 μM in T-medium. The slides were incubated at 37°C for 30 min, washed twice with PBS to remove excess dyes, and cells were fixed with 10% formaldehyde at 4°C. The slides were then washed twice with PBS and covered with glass coverslips with an aqueous mounting medium (Sigma-Aldrich, St. Louis, MO). Images were recorded by confocal laser microscopy (Zeiss LSM 510 META, Germany) using a 633 nm excitation laser and 670-810 nm long pass filter, or a fluorescence microscope (Olympus 1× 71; Olympus, Melville, NY) equipped with a 75 W Xenon lamp and an Indo-Cyanine Green filter cube (excitation 750-800nm; emission 820-860 nm) (Chroma, Rockingham, VT).
To determine dye uptake in tissues, tissues isolated from tumor bearing mice (see below) were placed in OTC medium and frozen at -80°C. Frozen 5 μm tissue sections were prepared for histopathologic observation using the microscope as described above.
Assessment of cyanine dye uptake into the mitochondria and lysosomes of cancer cells
Cells were plated on live-cell imaging chambers (World Precision Instrument, Sarasota, FL) overnight. Cells were exposed to cyanine dyes at different concentrations and dye uptake was evaluated by a Perkin-Elmer Ultraview ERS spinning disc confocal microscope. This system was mounted on a Zeiss Axiovert 200m inverted microscope equipped with a 37°C stage warmer, incubator, and constant CO2 perfusion. A 63× or 100× Zeiss oil objective (numerical aperture, 1.4) was used for live cell images and a Z-stack was created using the attached piezoelectric z-stepper motor. The 633 nm laser line of an argon ion laser (set at 60% power) was used to excite the cyanine dyes. Light emission at 650 nm, while not optimal for these dyes, was detected and was found to correlate directly with the dye concentrations in the cells (Fig. S-1). For comparative studies, the exposure time and laser intensity were kept identical for accurate intensity measurements. Pixel intensity was quantified using Metamorph 6.1 (Universal Imaging, Downingtown, PA) and the mean pixel intensity was generated as grey level using the Region Statistics feature on the software (23). To determine the dye uptake by the mitochondria, the mitochondrial tracking dye Mito Tracker Orange CMTMROS (Molecular Probes, Carlsbad, CA) was used. To determine the dye localization in lysosomes, a lysosome tracking dye, Lyso Tracker Green DND-26 (Molecular Probes, Carlsbad, CA), was selected. Imaging of mitochondrial and/or lysosome localization of the cyanine dye was conducted under confocal microscopy (24).
To determine if the cyanine dye uptake and accumulation in cancer cells was dependent upon OATPs, we preincubated cells with 250 μM bromosulfophthalein (BSP), a competitive inhibitor of organic anion transporting peptides (OATPs) (25), for 5 minutes prior to incubating the cells with cyanine dyes. The uptake and accumulation of cyanine dyes in the presence and absence of BSP were conducted in the stage warmer incubator for a period of 35 minutes. The levels of cyanine dye taken up and accumulated in normal prostate (P69) and prostate cancer (ARCaPM) cells were determined and compared on a real-time basis.
Uptake and accumulation of cyanine dyes in tumors in live mice
Human cancer cells were implanted (1 × 106) either subcutaneously, orthotopically, or intraosseously into 4 to 6 week old athymic nude mice (National Cancer Institute, Frederick, MD) according to our previously published procedures (26, 27). All animal studies were conducted under the Emory University Animal Care and Use Committee guidelines. When tumor sizes reached between 1-6 mm in diameter, as assessed by X-ray or by palpation, mice were injected i.v or i.p with cyanine dyes at a dose of 0.375 mg/kg or 10 nmol/20 g mouse body weight. Whole body optical imaging was taken at 24 h using a Kodak Imaging Station 4000 MM (New Haven, CT) equipped with fluorescent filter sets (excitation/emission, 800/850 nm). The field of view (FOV) was 120 mm in diameter. The frequency rate for NIR excitation light was 2 mW/cm2. The camera settings included maximal gain, 2 × 2 binning, 1024 × 1024 pixel resolution, and an exposure time of 5 sec. In some instances, live mice were also imaged by an Olympus OV100 Whole Mouse Imaging System (excitation 762 nm; emission 800nm) (Olympus Corp., Tokyo, Japan), containing a MT-20 light source (Olympus Biosystems, Planegg, Germany) and DP70 CCD camera (Olympus). Prior to imaging, mice were anesthetized with ketamine (75mg/kg). During imaging, mice were maintained in an anesthetized state.
We also studied the spontaneous metastasis of ARCaPM tumor cells stably transduced with an AsRed2 red fluorescence protein (RFP) (Clontech, Mountain View, CA) by injecting these cells orthotopically in mice. ARCaPM-RFP metastasis was determined by the same procedures described above for capturing cyanine dye tumor imaging after IR-783 i.p injection at a dose of 10 nmol/20 g. In addition, at the time of sacrifice both frozen and paraffin embedded tissue sections were obtained for RFP and confocal fluorescence imaging. Positive identification of ARCaPM-RFP cells was accomplished by fluorescence microscopy and validated by subculturing ARCaPM-RFP cells directly from bone metastasis tissue specimens.
The uptake of cyanine dyes by the TRAMP mouse prostate model and the ApcMin/+ mouse-adenoma model (obtained from The Jackson Laboratory, Bar Harbor, ME) was assessed by a similar protocol as described above. We also utilized the Olympus OV100 imaging system to detect adenoma in the ApcMin/+ mouse model. In brief, mice were injected intraperitoneally with IR-783 dye at a dose of 10 nmol/20 g body weight and animals were subjected to total body cyanine dye imaging as described above. Animals were sacrificed at 48 hrs after dye administration and tumors were dissected and subjected to NIR imaging. The presence of tumor cells in tissue specimens was confirmed by histopathologic analysis.
Assessments of heptamethine cyanine dye biodistribution in normal and tumor-bearing mice in vivo
To assess tissue distribution of these dyes, athymic mice without tumor implantation were sacrificed at 0, 6 and 80 hrs (N=3 each) after i.v injection of IR-783 dye at a dose of 10 nmol/20 g. Dissected organs were subjected to NIR imaging by a Kodak Imaging Station 4000 MM. In another study the mice bearing orthotopic ARCaPM tumors were subjected to NIR imaging at 0.5, 24, 48, 72 and 96 hrs after IR-783 i.v administration at a dose of 10 nmol/20 g. In some cases, we also assessed the biodistribution of NIR dye by a spectral method in tissues harvested from athymic mice bearing subcutaneous ARCaPM tumors (N=6). Tumors and normal host organs were homogenized in PBS, centrifuged at 15,000 × g for 15 minutes to recover the supernatant fraction after the mice were injected i.p with IR-783 at a dose of 10 nmol/20 g. The presence of the organic dyes (parental IR-783 and its metabolites) in tissues was estimated spectrophotometrically at an emission wavelength of 820 nm by a PTI Near Infrared Fluorometer QuantaMaster™ 50 (PTI, Birmingham, NJ) equipped with a 75-watt xenon arc lamp under 500 to 1700 nm InGaAs detector using known concentrations of IR-783 as the standard (28). In other cases, tumor tissues harvested from mice were stored in formalin from 1 week to 3 months, and fluorescence images were obtained and compared.
Detection of cancer cells in human blood
An experimental model of evaluating human prostate cancer cells in blood was developed. In brief, heparinized whole blood from human volunteers was collected according to an Emory University approved IRB protocol. A known number of human prostate cancer cells (10-1,000) were added to 1 ml of whole blood, mixed gently with 20 μM IR-783 and incubated for 30 minutes at 37°C. The mononuclear cells and cancer cells were recovered by gradient centrifugation using Histopaque-1077 (Sigma, St. Louis, MO). The isolated live cells were observed under a confocal fluorescence microscope.
Assessment of systemic toxicity of IR-783 in mice
We investigated the systemic toxicity of IR-783 in C57BL/6 mice (National Cancer Institute, Frederick, MD) by injecting the dye by an i.p route. The mice (N=8 per group) were subdivided into 4 groups and received PBS as control and IR-783 i.p injection daily at the following doses: 0.375 mg/kg (imaging dose), 3.75 mg/kg and 37.5 mg/kg. They were weighed daily and their physical activities were observed for one month following dye injection. The histomorphologic appearance of their vital organs was assessed at the time of sacrifice.
Data processing and statistics
The statistical significance of all data was determined by Student's t-test. Data were expressed as the average ± standard error of the mean of the indicated number of determinations. The statistical significant difference was assigned as P<0.05.
Structural requirement of heptamethine cyanine dyes for tumor-specific uptake and retention
Using human cancer and normal human cell lines to study dye uptake and retention, we found that IR-783 and MHI-148 were unique in that they had both tumor imaging and targeting properties (Supplement Table 1). A comparative analysis also uncovered several common structural features of heptamethine cyanine dyes accounting for their preferential uptake and retention by cancer cells. We classified the dyes operationally as active and inactive based upon their specific uptake and retention in cancer but not normal cells. A rigid cyclohexenyl ring in the heptamethine chain with a central chlorine atom maintains photostability, increases quantum yield, decreases photobleaching, and reduces dye aggregation in solution (1). Chemical substitution of the central chlorine atom with a thio-benzyl-amine group on the cyclohexenyl ring dramatically reduced the fluorescence intensity and eliminated their uptake by cancer cells and tumor xenografts, and so would a substitution of the side chain with hydroxyl, an ester, or an amino group rather than a charged carboxyl (i.e. MHI-148) or sulfonic acid (i.e. IR-783) moiety (see Supplement Table 1, Fig. S-2 and Fig. S-3). In this report we focus on characterizing the tumor-specific uptake and retention of two cyanine dyes, IR-783 (available commercially) and MHI-148 (available by chemical synthesis, see above).
Preferential uptake and retention of NIR fluorescence dyes by human cancer cells and tumor xenografts
Human cancer and normal cell studies
Cancer cell surface properties and surrounding leaky vasculatures have been exploited for the delivery of imaging agents (29-32). IR-783 and MHI-148 were tested for their ability to detect cancer cells (Fig. 1A). The two dyes were found not to accumulate in normal human bone marrow cells (HS-27A), vascular endothelial cells (HUVEC-CS), embryonic fetal kidney cells (HEK293), a primary culture of human prostate epithelial cells (NPE), or normal prostate fibroblasts (NPF) (Fig. 1B). These dyes, however, were found to be retained in cancerous cells of human origin including the prostate (C4-2, PC-3, and ARCaPM), breast (MCF-7), lung (H358), cervical (HeLa), liver (HepG2), kidney (SN12C), pancreas (MIA PaCa-2), and leukemia (K562) (Fig. 1C). These dyes were also found to be taken up by other malignant cells from both human and mouse, including human bladder cancer cell (T-24), renal cancer cell (ACHN), and mouse pancreatic cancer cell lines (PDAC2.3, PDAC3.3, BTC3 and BTC4 derived from transgenic mouse) (Fig. S-4). There was no discernible difference in the amount and specificity of uptake of these two heptamethine cyanine NIR dyes by cancer cell lines. In this report, we focused predominately on the uptake and retention of IR-783 in cancer cells and tumor xenografts.
Fig. 1
Fig. 1
Active uptake of hepatamethine cyanine dyes by human cancer cells but not normal cells in culture. (A), The chemical structures of two hepatamethine cyanine dyes, IR-783 and MHI-148. (B), Normal human cells including bone marrow stromal cells (HS-27A), (more ...)
We next compared the kinetics of IR-783 uptake by cultured human prostate cancer ARCaPM versus P69 cells, a normal human prostate epithelial cell line (Fig. 2A). This study revealed a differential time-dependent uptake and retention of IR-783 by ARCaPM and P69 cells (Fig. 2B). Uptake and retention of IR-783 in ARCaPM cells occurred in two phases, an early phase completed in 12 minutes, and a late phase completed in 30 minutes. In the control P69 cells, the uptake and retention of IR-783 only began at 12 minutes, with a much lower plateau. Interestingly the uptake and accumulation of IR-783 could be abolished by bromosulfophthalein (BSP), a competitive inhibitor of the organic anion transporting polypeptides (OATPs) (25) (Fig. 2C). These results are consistent with the observation that IR-783 uptake into cancer cells was high at 37°C but none at 0°C (data not shown). These results confirmed that the cancer cell-specific uptake was an energy-dependent active process, most probably mediated by members of the OATP family.
Fig. 2
Fig. 2
Kinetics and subcellular localization of the NIR dyes. (A), Confocal imaging shows significant uptake of IR-783 dye in ARCaPM cells but not in normal human prostate epithelial P69 cells at 630× magnification. (B), Histogram shows differential (more ...)
We then evaluated the subcellular compartments where IR-783 was retained. Based on the dye co-localization using the tracking dyes, the NIR signal appeared to condense on mitochondrial and lysosomal organelles, with homogenous staining also detected throughout other cytoplasmic and nuclear compartments (Fig. 2D). These heptamethine cyanine NIR dyes apparently localized primarily within mitochondrial and lysosomes but can bind to a host of other intracellular proteins.
Human tumor xenograft studies
IR-783 was injected intraperitoneally (i.p) or intravenously (i.v) in athymic mice bearing human bladder tumors (T-24, subcutaneously), pancreas tumors (MIA PaCa-2, subcutaneously), prostate tumors (ARCaPM, orthotopically), and kidney tumors (SN12C, intraosseouslly to tibia). The animals were imaged non-invasively with a NIR small animal imaging system (Fig. 3). Successive observations at different time points revealed that after the initial systemic distribution and clearance, intense signals were clearly associated with the tumors implanted at various anatomical sites, with no background interfering fluorescence from the mice. The presence of tumor cells in the tissue specimens was confirmed by histopathology analysis with tissue sections stained with H/E and positive fluorescence imaging in cancer cells in frozen sections (data not shown).
Fig. 3
Fig. 3
Preferential uptake and retention of the hepatamethine cyanine dyes in human tumor xenografts. Mice bearing human prostate (ARCaPM, orthotopic prostate tumor, p.o), bladder (T24, subcutaneous, s.c), pancreatic (MIA PaCa-2, subcutaneous), and renal (SN12C, (more ...)
Human cancer metastasis studies
To investigate if NIR dye could detect spontaneously metastasized tumors and to confirm if the NIR dye is associated with prostate cancer bone metastasis, we inoculated mice orthotopically with ARCaPM cells that were stably tagged with AsRed2 RFP (Fig. 4A-a). On signs of cachexia at 3 months, the animals were subjected to non-invasive whole body NIR imaging with IR-783 (Fig. 4A-b). In addition to the presence of localized orthotopic tumors (see thick arrow), RFP-tagged ARCaPM tumors also appeared in mouse bone (see thin arrow). Upon ex vivo imaging, we detected both the primary tumor and the metastases in mouse tibia/femur. The presence of tumor cells in the mouse skeleton was confirmed by histopathologic evaluation and by the presence of RFP-tagged cells upon subculture of cells derived from the skeletal metastasis specimens (Fig. 4A-c and 4A-d).
Fig. 4
Fig. 4
Detection of tumor metastasis in mice and spontaneous tumors in transgenic animals. (A) Confirmation of the presence of bone metastatic prostate tumors in mice by NIR imaging after IR-783 i.p injection at a dose of 10 nmol/20g. (a), The ARCaPM human prostate (more ...)
Detection of spontaneous prostate and intestinal tumors in transgenic mouse models
To investigate if IR-783 could be used to detect spontaneously developed tumors, we adopted two transgenic mouse models that were known to display high degrees of tumor penetrance, the TRAMP mouse model for prostate cancer and the ApcMin/+ mouse model for colon cancer (33, 34). Since the TRAMP and ApcMin/+ mouse models represent the development of adenocarcinoma/neuroendocrine prostate tumors and adenoma of the intestine, respectively, this study also allowed us to assess if IR-783 could detect the early stage of tumor development (i.e. adenoma). IR-783 could detect tumor in both the TRAMP mice and the ApcMin/+ mice (Fig. 4B and 4C). Specific detection of tumor but not normal cells was also confirmed by histopathologic analysis of the tumor specimens (Fig. 4B-f, and Fig. 4C-c and 4C-d). An additional advantage of IR-783 imaging was its optical stability, even after prolonged tissue fixation. TRAMP tumor specimens retain heptamethine cyanine NIR fluorescence even after being stored in neutralized formalin solution for 3 weeks (Fig. 4B-e).
NIR dye tissue distribution studies
Hepatamethine cyanine dye tissue distribution studies were conducted in normal and tumor-bearing mice. Time-dependent dye clearance from normal mouse organs is shown in Fig. 5A. At 6 hrs, NIR dye IR-783 was found to accumulate in mouse liver, kidney, lung and heart. By 80 hrs, dye was cleared from all mouse vital organs. The dye, however, was found to accumulate in tumor tissues at 24 hrs with minimal background autofluorescence. Tumors retained IR-783 dye even at 4 days (or 96 hrs, see Fig. 5B). In both in vivo whole body and ex vivo analysis, we detected signal to noise ratios exceeding 25 in tumor specimens; however, normal organs, liver, lung, heart, spleen and kidneys displayed very low signals (Fig. 5C). In these studies, NIR dyes in tumor implants could be retained for as long as 15 days after dye administration (data not shown).
Fig. 5
Fig. 5
Distribution of heptamethine cyanine dye IR-783 and its metabolites in tissues; time-course and concentration-dependent studies in normal and tumor-bearing mice. (A) Normal organs dissected at 0, 6 and 80 hrs after IR-783 iv injection at a dose of 10 (more ...)
Prior to the quantification of the heptamethine cyanine dyes in excised tumors and normal organs, we established a standard curve by monitoring the emission profile of IR-783 at 820 nm (28, 35). Within concentration ranges from 0–40 μM, a linear correlation (r=0.9991) was found between the concentration of IR-783 and its emission intensity (left panel, Fig. 5D). Using this standard curve, we estimated spectrophotometically the apparent concentrations of the dye and its metabolites in tissues. Fig. 5D (right panel) shows that the apparent concentrations of the NIR dye and its metabolites (defined here as light emission intensity at 820 nm) in tumors were significantly higher than those in normal tissues with a difference approaching 10-fold (P<0.05, data are expressed as average ±SEM of 3 determinations). This fluorescence emission could be contributed by the parental dye, its metabolites and their binding to nucleic acids and proteins (36).
In dye systemic toxicity study, we observed no systemic toxicity of IR-783 dye in normal C-57BL/6 mice and this dye also did not affect body weights of the mice. No abnormal histopathology was seen in vital organs harvested from mice at the time of sacrifice.
Detection of cancer cells in human blood
Since IR-783 was confirmed to detect human cancer but not normal cells, we then tested whether this dye could be further exploited to detect circulating cancer cells in the blood using an experimental model. Fig. 6A shows that cancer cells can be clearly visualized after mixture with human blood cells by IR-783 NIR imaging. We estimate that this dye is sufficiently sensitive to detect as few as 10 cancer cells per milliliter in whole blood (Fig. 6B).
Fig. 6
Fig. 6
Detection of human prostate cancer cells in human blood. (A), ARCaPM cells mixed with human blood were incubated with IR-783 and the particulate fractions containing normal healthy mononuclear cells and cancer cells were isolated using gradient centrifugation. (more ...)
Chemically conjugated cyanine dyes have proved to be useful for measuring blood flow and cardiac output, as well as imaging tumors (2, 3, 37). The chemical structures of water-soluble pentamethine and heptamethine cyanine dyes have recently been modified to increase their chemical stability, photostability, and quantum yield (1). IR-783 and MHI-148 are two such new dyes, modified with a rigid cyclohexenyl substitution in the polymethine linker. The present study describes for the first time that these NIR dyes can be actively taken up and accumulated by cancer cells but not by normal cells. The salient features of these newly discovered dual imaging and targeting NIR dyes are: (1) Detecting cancer cells and cancer metastases directly without the requirement of chemical conjugation. (2) Detecting many other tumor types and tumor cell populations under cell culture and in vivo conditions. The cancer-specific uptake and retention of these dyes is likely to be mediated by OATPs since the transport of these dyes into cancer cells can be antagonized by BSP, an OATP competitive inhibitor (38, 39). (3) Serving as potential carriers for drug payloads or radioactive agents to increase the specificity and reduce the toxicity of therapeutic agents by preferential uptake and accumulation in cancer cells but not in normal cells.
The cyanine dyes are water soluble, so they have rapid clearance and are unlikely to be trapped in the reticular endothelium of the liver, lung or spleen. They were found to be superior for cancer detection to other cyanine dyes such as indocyanine green and non-cyanine dyes such as rhodamine 123 (data not shown). Imaging with NIR dyes can yield much higher signal/noise ratios with minimal interfering background fluorescence. The fluorescence efficiency of cyanine dyes can increase by ~1,000-fold upon binding to proteins and nucleic acids (36). The stable binding together with the shift toward increased fluorescence could be highly beneficial, accounting for the “trapping” of the NIR signals in cancer cells for prolonged periods (>5 days) and allowing tumor detection in live animals with high signal/noise ratios. The stability of these cyanine dyes after formalin fixation raises the possibility of developing new and sensitive means of detecting cancer cells in whole blood and in harvested surgical specimens by injecting the cyanine dyes prior to sampling at the time of surgery. In practice, these could help physicians and pathologists follow up patients with possible circulating cancer cells in blood and assess surgical margins at the time of surgery. Our study suggests the differential dye uptake and retention by cancer and normal cells and tissues can be demonstrated robustly by the use of a variety of detecting devices including Zeiss LSM 510 META, Kodak 4000MM, and Olympus OV100 systems. We adopted these different detecting methodologies based on their sensitivity and capability of allowing merging of images obtained via different detection modalities (e.g. X-ray and NIR imagings). The wide range of detecting devises used in our study supports the conclusion that IR-783 is preferentially taken up and retained by cancer but not normal cells.
The mechanisms by which these cyanine dyes cross the cytoplasmic membranes of cancer cells but not normal cells were investigated. We concluded that the uptake was mediated by proteins of the OATP family, because the active uptake could be effectively blocked by BSP. OATPs are well-recognized as channels for the transport of a diverse group of substrates including bile acids, hormones, xenobiotics and their metabolites (40-42). Results from this study are consistent with published reports which indicate differences in the type and levels of OATPs between cancer and normal cells(43-46). Moreover certain members of OATPs have recently been shown to be overexpressed in various human cancer tissues as well as in cancer cell lines (47-50) and the confirmation of OATPs as the key mediator of heptamethine cyanine dye uptake and retention in tumor cells warrants further investigation.
The ability of mouse tumors to accumulate these cyanine dyes is of great significance. This will facilitate the use of these dyes in immune-intact syngenic and transgenic mouse models to study the fundamentals of cancer biology, metastasis and therapy. Since these dyes can be further explored as generalized ligands for all malignant cells, the synthesis of dye-antineoplastic drug conjugates, dye-radiolabeled drug conjugates and dye-toxin conjugates could immensely facilitate the development of new therapeutics to treat cancer and pre-cancerous conditions.
In summary, two heptamethine cyanine dyes were demonstrated to selectively target cancer but not normal cells, irrespective of their species and organ of origin. This class of dual imaging and targeting cyanine dyes holds great promise for novel therapeutics for future cancer therapy and imaging. Future application of NIR fluorescent dyes in the clinic could lead to important progress in the management of cancer patients on an individual basis.
Translational Relevance
Cancer mortality can be reduced by the development of non-invasive and effective imaging technologies that can detect tumors at metastatic sites and cancer cells in biologic fluids. We report our discovery of two heptamethine cyanine dyes with near-infrared fluorescence emission profiles that can detect the presence of human tumors grown in mice, spontaneous prostate and intestinal tumors in transgenic animals, and tumor cells in human blood, without the necessity of chemical conjugation. These unique heptamethine cyanine dyes can be further exploited for the detection of tumor cells in histopathologic specimens, circulating tumor cells in blood, and differentiating surgical margins in clinical specimens for improved diagnosis, prognosis and treatment of cancer patients.
1: Table 1. Structural requirement of heptamethine cyanine dyes actively uptake by cancer but not normal cells
2: Fig. S-1. Correlation of NIR fluorescence intensity and concentration of cyanine dye in cancer cells
Prostate cancer cells (ARCaPM) were plated on live-cell imaging chambers and imaged 30 min after adding IR-783 at the following concentrations: 1μM, 10μM, 20 μM and 50 μM using a Perkin-Elmer disc confocal microscope as described (see Methods). Images were acquired at 650 nm emission using a 63× objective. The mean pixel intensity of IR-783 in cells was quantitated using Metamorph 6.1 software. (A), Images of IR-783 dye uptake in ARCaPM cells at concentration of 1μM, 10μM, 20 μM and 50 μM. (B), The fluorescence emission intensity was measured and correlated with the concentrations of IR-783 in ARCaPM cells (r=0.997).
3: Fig. S-2. Structural requirement of heptamethine cyanine dyes for cancer cell uptake and retention
We screened a series of heptamethine cyanine dyes and their derivatives in cultured MIA PaCa-2 human pancreatic cancer cells with parental MHI-148 served as a positive control. Results showed that IR-1, IR-2, IR-3 (modifications of MHI-148) and IR-4 (4-aminothiophenol derivative of IR783) were found to be devoid of any uptake and retention by this cancer cell line when compared with MHI-148.
5: Fig. S-3. Structural requirement of the uptake and retention of heptamethine cyanine dyes by tumor tissues in mice
Mice bearing human renal cancer SN12C at subcutaneous sites were injected i.p. with MHI-148, IR-1, IR-2, IR-3 and IR-4 heptamethine cyanine dyes at a dose of 10 nmol per mouse. Whole-body optical imaging was taken at 24 hrs using a Kodak Imaging System. Strong signals were visualized in tumors after MHI-148 injection (see white arrows) while background fluorescence signals were observed in mice injected with IR-1, IR-2, IR-3 or IR-4 (see black arrows).
4: Fig. S-4. The uptake and retention of IR-783 dye by human and mouse cancer cells
Human bladder cancer cell (T-24), renal cancer cell (ACHN), and mouse pancreatic cancer cell lines (PDAC2.3, PDAC3.3, BTC3 and BTC4) showed significant uptake after incubating with 20 μM IR-783 dye. The images show IR-783 staining (NIR), cell morphology (BF) and a merger of the two images (Merge). All the images were acquired at 400 × magnification.
Acknowledgments
This work is supported in part by research funds from NIH 1P50 CA128301, NIH 1U54 CA119338, and Dr. Leland W. K. Chung's a Georgia Cancer Coalition Distinguished Cancer Scholar research fund.
Abbreviations
BSPbromosulfophthalein
FBSfetal bovine serum
OATPsorganic anion transporting peptides
NIRnear-infrared
NPEnormal prostate epithelial cell
NPFnormal prostate fibroblasts
RFPred fluorescence protein
TRAMPtransgenic adenocarcinoma mouse prostate

Footnotes
*This work was reported in abstract form by Chung LWK, Cheng J, Hanahan D, and Kattti KV, Tackling metastasis through team science: Cancer biologists lead the charge synergizing their discoveries behind common nanotechnology platforms. NCI Alliance for Nanotechnology in Cancer Bulletin 1: 1-4, 2008 and Yang X, Shi C, Wang R, Zhau HE, Henary M, Strekowski L and Chung LWK, New near infrared heptamethine cyanine fluorescence dyes improved detection and treatment of human and mouse prostate tumors. Journal of Urology, 2009; 181 suppl 4: 708.
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