PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biomed Opt. Author manuscript; available in PMC Aug 3, 2010.
Published in final edited form as:
PMCID: PMC2914597
NIHMSID: NIHMS181769
Near infrared fluorescent optical imaging for nodal staging
Lakshmi Sampath, Wei Wang, and Eva M. Sevick-Muraca
Lakshmi Sampath, Baylor College of Medicine, Division of Molecular Imaging, Department of Radiology and Molecular Physiology and Biophysics and The Baylor Breast Center, Houston, Texas 77030;
Address all correspondence to: Eva M. Sevick-Muraca, Ph.D., One Baylor Plaza, BCM 360, Baylor College of Medicine, Houston, Texas 77030. Tel: 713-798-3684; Fax: 713-798-2749; evas/at/bcm.edu
Current techniques to assess lymph node metastases in cancer patients include lymphoscintigraphy after administration of a nonspecific radiocolloid in order to locate and resect lymph nodes for pathological examination of harbored cancer cells. Clinical trials involving intradermal or subcutaneous injection of antibody-based nuclear imaging agents have demonstrated the feasibility for target-specific, molecular imaging of cancer-positive lymph nodes. The basis for employing near-infrared (NIR) optical imaging for assessing disease is evidenced by recent work showing functional lymph imaging in mice, swine, and humans. We review antibody-based immunolymphoscintigraphy with an emphasis on the use of trastuzumab (or Herceptin) to target human epidermal growth factor receptor-2 (HER2) overexpressed in some breast cancers. Specifically, we review in vitro and preclinical imaging data from our laboratory that show how the dual-labeled agent (111In-DTPA)n-trastuzumab-(IRDye800)m utilizes the high photon count provided by an NIR fluorescent dye, IRDye 800CW, and the radioactive signal from a gamma emitter, Indium-111, for possible detection of HER2 metastasis in lymph nodes. We show that the accumulation and clearance of (111In-DTPA)n-trastuzumab-(IRDye800)m from the axillary nodes of mice occurs 48 h after intradermal injection into the dorsal aspect of the foot. The requirement for long clearance times from normal, cancer-negative nodes presents challenges for nuclear imaging agents with limited half-lives but does not hamper NIR optical imaging.
Keywords: fluorescence imaging, breast cancer, human epidermal growth factor receptor-2, (HER2), lymph node staging, intradermal administration
Nodal staging plays a critical role in the evaluation of disease progression and design of therapeutic strategies in the treatment of most cancer patients. For example, in breast cancer, intradermal (i.d.) or subcutaneous (s.c.) administration of a nonspecific 99mTc-radiocolloid is used to identify the tumor draining or sentinel lymph node for resection. Once the surgical pathologist confirms the presence of cancer cells in the resected sentinel lymph node, additional nodes around the axilla are removed to better assess the extent of lymph node involvement. The accuracy of nodal staging is critical for judicious selection of effective therapy. The inability to surgically resect inaccessible tumor-draining lymph nodes, such as the internal mammary or supraclavicular lymph nodes in breast cancer patients, or the pelvic lymph nodes that drain the prostate in prostate cancer patents, can compromise oncologists’ ability to accurately stage and select proper therapy. Noninvasive, diagnostic imaging of cancer-positive lymph nodes could positively impact the manner in which tumor-node-metastasis (TNM) staging is performed.
Molecular imaging approaches for early-stage diagnosis of metastasis in the lymphatics were first explored by Weinstein et al., who used radio-labeled monoclonal antibodies that were administered (i.d.) through the footpad in preclinical studies.1,2 Since then, several investigators have used immunolymphoscintigraphy as a tool to study lymphatic delivery of agents. Steller et al.3 demonstrated the dose dependence of iodinated monoclonal antibody biodistribution following i.d. administration in a murine animal model, while Wahl et al.4 employed iodinated intact and fragmented antibodies to assess the kinetics of their clearance from the lymphatic system in response to ambulation. Additional reports have used immunolymphoscintigraphy to study lung cancer metastases to pulmonary and mediastinal lymph nodes in dogs.5 The i.d. route of imaging agent delivery may be extremely favorable for nodal staging because antibodies and small molecules drain primarily from the lymph plexus into the lymphatic system. Small molecules pass readily through one or more nodes before exiting the lymphatic circulation via the blood circulatory system, while larger agents may be retained longer within lymph nodes. Some advantages for using a direct lymphatic approach include: (1) high lymph node uptake of antibodies, which enables significantly lower doses of labeled antibodies than that required for intravenous (i.v.) administration, (2) more rapid lymph node uptake, enabling minimal time between i.d. antibody administration and imaging, and (3) increased target-to-background ratios due to reduced nonspecific binding, which would otherwise occur in systemic i.v. administration. A more comprehensive review of lymphatic uptake and transport of proteins following intralymphatic administration can be found in an article by Porter and Charman.6
In addition to animal studies, imaging-based clinical applications of intralymphatic administration have also been explored by several investigators, mostly using radio-labeled monoclonal antibodies in their intact or fragmented form to detect tumor-draining lymph nodes in a spectrum of cancer patients ranging from melanoma, T-cell lymphoma, breast, and prostate cancers. One of the first investigations of lymph node imaging was reported by Order et al. in 1975, in which they demonstrated accumulation of 131I-labeled antiferrin immunoglobulin in lymph nodes of patients with breast carcinoma and lymphoma.7
Following that, DeLand et al. conjugated 131I-labeled antibodies to carcinoembryonic antigen (CEA) and showed high sensitivity in detecting nodal metastases after interdigital administration in patients.8 In breast cancer and T-cell lymphoma, immunolymphoscinitigraphy was performed by several investigators with 131I-, 111In-, and 99mTc-labeled monoclonal antibodies to noninvasively assess regional lymph nodes.912 Pelvic nodal metastases in prostate cancer have been investigated13 by bipedal intralymphatic administration of 111In-PAY 276, while antimelanoma antibodies that have been radio-labeled for lymphoscintigraphy have also been reported in the literature.1416 Most injections were well tolerated, but some patients have reported pain at the injection site. Most studies have reported a high sensitivity (80 to 100%) using this route of administration, but nonspecific uptake by normal lymph nodes continues to be problematic.
Although lymphoscintigraphy, or the administration of nonspecific 99mTc sulfur colloid, is the “gold standard” for clinically evaluating lymph mapping, some limitations suffered by this imaging technique include the need for long (~20 min) gamma camera integration times, poor temporal and spatial resolution, low signal-to-noise ratios (SNRs), and the finite physical half-life of the tracer [t1/2(99mTc)=6 h]. In contrast, near-infrared (NIR) fluorophores provide an attractive opportunity that outweighs some of these disadvantages inherent in nuclear techniques. Since fluorescent reporters do not have a physical half-life, they can be repeatedly activated using appropriate excitation light. Theoretically, for a fluorophore with a nanosecond fluorescent lifetime and a quantum efficiency of 0.1, 108 photons can be generated per second per molecule. When compared to the one-photon imaging event resulting from the radioactive decay of a radionuclide, the potential advantages of NIR fluorescent optical imaging with NIR fluorophores becomes evident. The increased photon yield can considerably improve signal-to-noise and significantly reduce image acquisition time for molecular imaging, if tissue attenuation does not significantly reduce the number of detected fluorescent photons. While NIR fluorophores also have an absorption cross section, their administration in trace doses does not provide sufficient absorption contrast for in vivo. Hence, NIR contrast may be best provided from the collection of NIR fluorescent photons rather than from the attenuation of excitation photons, which may occur from endogenous chromophores as well as from the exogenous fluorophores.
Herein, we present our work on a dual-labeled conjugate — (111In-DTPA)n-trastuzumab-(IRDye800)m — which targets the human epidermal growth factor receptor-2 (HER2). Overexpression of HER2 is associated with poor prognosis in about 20 to 30% of breast cancer patients.17 We have previously shown specificity in vivo in subcutaneous xenograft models.18 In this report, we first review recent results from our group describing lymphatic imaging with nonspecific optical agents. Next, we review our prior results showing tumor targeting of dual-labeled (111In-DTPA)n-trastuzumab-(IRDye800)m and enhanced performance of optical over nuclear imaging. We additionally show trafficking of the labeled antibody into the lymphatics and its subsequent drainage into lymph nodes using NIR optical imaging.
Recently, Sharma et al. illustrated the ability to quantitatively image lymph function in a swine model after administering an i.d. injection of a nonspecific NIR fluorophore, IC-Green.19 Their work was the first to noninvasively image pulsatile lymph flow velocities of 0.23 to 0.75 cm/s, averaging approximately 3.3 pulses/min along lymph channels ranging 2 to 16 cm in length. A similar approach was employed by Sevick-Muraca et al., who used i.d. microdoses (10 to 100 μg) of IC-Green to image lymph propulsion in the breast and axilla of breast cancer patients undergoing sentinel lymph node mapping.20 They observed mean velocities ranging between 0.08 and 0.32 cm/s. The image acquisition time for optical imaging in both swine and humans ranged from 200 to 800 ms. Upon further reducing the integration time to 100 ms, Kwon and Sevick-Muraca have been able to rapidly image lymph trafficking in the limbs and tail of mice.21 The average lymph flow velocity ranged from 0.28 to 1.35 mm/s.
Figure 1 depicts representative optical images showing “packets” of IC-Green solution traversing the lymphatic channels after i.d. administration in human (b), swine (c), and mouse (d).1921 The stick-diagram with a white light image (a) illustrates the right breast view of a breast cancer patient with four covered i.d. injection sites. The corresponding optical image from this patient (b) reveals two lymph channels and a region of lymph “pooling” through which IC-packets were propelled into the axilla. The swine model shows lymph propulsion from the hind limb up to the middle iliac node, while the mouse model depicts IC-Green trafficking to the axillary lymph node after i.d. administration into the footpad of the forepaw. These studies demonstrate the feasibility of noninvasive NIR optical imaging to qualitatively and quantitatively contribute toward the standard of care in nodal staging in preclinical and clinical studies. Most importantly, the human studies confirm the ability to use microdose administration (i.e., between 10 and 100 micrograms) of IC-Green, suggesting that the instrumentation is capable of assessing NIR fluorescent signals for molecular imaging after trace administration of molecular imaging conjugate.
Fig. 1
Fig. 1
(a) shows the while light image of a right breast from a patient who has been injected with IC-Green (100 μg) around the areola region. The injection sites have been covered to prevent oversaturation of the camera. (b) is the corresponding optical (more ...)
Other investigators have also conducted NIR fluorescence imaging of IC-Green. Frangioni and coworkers have intraoperatively imaged the fluorescence from IC-Green and other NIR fluorescent conjugates administered intradermally to intraoperatively identify sentinel lymph nodes in animal models.2224 Yodh and coworkers25 also measured fluorescence in humans after intravenous administration of 0.125 mg/kg. In their work, as well as in the work of others described earlier, NIR fluorescence was measured using an integrating CCD camera but was coupled with time-resolved absorbance measurements made in the frequency domain in order to perform fluorescence optical tomography from the fluorescence CW measurements. To date, there have been no reports of fluorescence enhanced optical tomography using PMT-based, time-dependent measurements of fluorescence as reported in the companion review paper by Sevick-Muraca and Rasmussen.26 Fluorescence tomography using gain modulation of an intensified CCD camera has been demonstrated in phantoms by Godavarty et al.,27,28 Roy et al.,29 and Joshi et al.,30 however, using mM amounts of IC-Green. Before fluorescence tomography measurements can be conducted using microdoses in clinical relevant tissue volumes, two requirements must be met: the “noise floor” of fluorescence measurements must be reduced,26 and a molecularly targeting NIR optical imaging agent specific to disease must be used. Herein, we review and present new work on a dual-labeled imaging agent for planar NIR fluorescence imaging validated with nuclear imaging techniques.
Imaging agents that are designed with both nuclear and optical tracers combine complementary advantages from each modality and enhance the potential for detection of disease. Dual-labeled agents provide us with a unique opportunity to cross-validate NIR and nuclear imaging modalities. Some peptide-based dual-labeled imaging agents developed include a cyclopentapeptide, c(KRGDf), which is known to target alpha(v)beta3 integrins,31,32 and a cyclic peptide, c(CGRRAGGSC), which is known to target IL-11 Ref. 33. Quantitative comparison between planar scintigraphy and optical imaging in in vivo xenograft models injected with the RGD peptide have revealed comparable sensitivities between the two modalities, but NIR fluorescence shows improved signal-to-noise ratios.32 Herein, we employ the dual-labeling strategy to image antibodies in the lymphatics.
3.1 Labeled Trastuzumab for Metastasis and Nodal Staging
The human epidermal growth factor receptor (HER) family consists of transmembrane receptor tyrosine kinases that regulate complex intracellular signaling networks to control normal and pathological cellular growth, differentiation, and survival.34 The second member, HER2, is of particular importance in breast cancer because overexpression or gene amplification of HER2 is closely associated with aggressive tumor progression and poor prognosis. Since a significant percentage of breast cancer patients are affected by the aberrant HER2 signaling, this family of receptors represents an excellent target for therapeutics. Additionally, the presence of HER2 on the plasma membrane of cells has also made it a potential target for diagnostic biomarkers. Trastuzumab (Herceptin, Genentech, San Francisco) is a humanized anti-HER2 antibody35 that interferes with HER2 signaling and is approved clinically as a therapeutic for breast cancer.36,37 Evidence also suggests that with metastasis, HER2 is conserved or elevated with respect to that expressed on the primary tumors.38,39
Several investigators have labeled intact, derived, and fragments [(Fab, F(ab′)2)] of trastuzumab antibody with a number of radioactive nuclear agents such as Ga-68 (Ref. 40), Y-90 (Refs. 4143), In-111 (Refs. 4143), and Tc-99m (Ref. 44) to image HER2 overexpressing breast cancer xenografts, as shown in Table 1. Smith-Jones et al.40 showed that sequential positron emission tomography (PET) imaging using 68Ga-labeled F(ab′)2 fragments of trastuzumab can be used to quantify the loss and recovery of HER2 induced by HSP90 inhibitors in animals bearing BT-474 human breast tumors, while Tang et al.43,44 have developed In-111 and Tc-99m based trastuzumab Fab conjugates to detect xenografts using whole-body scintigraphy. Huh et al.46 and Artemov et al.47 have conjugated nanocrystals and iron-oxide-based nanoparticles to detect HER2 expression with magnetic resonance imaging (MRI). Optical imaging–based strategies include intact trastuzumab labeled with a red fluorescent dye to image HER2-overexpressing human breast cancer xenografts, as shown by Hilger et al.,49 while recently Koyama et al. applied spectral fluorescence imaging methods with rhodamine-green conjugated trastuzumab to visualize pulmonary metastases.50 Tada et al. have used a confocal scanner unit coupled with an EMCCD camera to track single quantum dot particles, which have been conjugated to trastuzumab, from a capillary vessel to a cancer cell in a live animal.51
Table 1
Table 1
Summary of molecular imaging studies using trastuzumab.
The first use of trastuzumab as a diagnostic imaging agent was reported by Behr et al.53 This was followed by a study by Perik et al. In which In-111-labeled trastuzumab was clinically translated for scintigraphy and systemic administration in patients (n=17) to help identify late-stage metastases with SPECT/CT imaging that was not previously identified.54
3.2 Dual-Labeled Trastuzumab for Cancer Imaging
Recently, we have synthesized a dual-labeled antibody — (111In-DTPA)n-trastuzumab-(IRDye800)m — and demonstrated imaging specificity for HER2 in vitro and in vivo. The reader is referred to Ref. 18 for details of synthesis and imaging. Figure 2 represents a schematic of the imaging agent. Since DTPA dianhyride and the NIR dye, IRDye 800CW, bind at the lysine residues of the antibody, we see a range in the ratios of binding between each of these molecules with trastuzumab, with m and n typically ranging between 7 and 10. The agent is stable at 4 °C for extended periods of time, but DTPA dianhydride shows serum-based instability with degradation occurring at the rate of 6% per day. As an alternative, we have since employed more stable forms of DTPA, such as p-SCN-Bz-DTPA, for chelation of In-111, a gamma emitter with a half life of 2.8 days.
Fig. 2
Fig. 2
Schematic of (111In-DTPA)n-trastuzumab-(IRDye800)m.
The binding of (DTPA)n-trastuzumab-(IRDye800)m to a single SKBR3 cell that overexpresses HER2 is shown in Fig. 3. Sytox green stains the nucleus, while the fluorescent signal from IRDye 800CW indicating local expression of HER2 is present only along the outer rim of the cell, demonstrating extracellular binding. In Fig. 3 the nuclear stain is pseudo-colored green and (DTPA)n-trastuzumab-(IRDye800)m binding is represented in red. Prior incubation with unlabeled trastuzumab obliterates (DTPA)n-trastuzumab-(IRDye800)m cell binding, indicating the molecular specificity of the agent.18 Figure 3 also illustrates the usefulness of optical labeling in molecular imaging, as in vitro specificity can be readily visualized from microscopy, an advantage not present in nuclear imaging techniques.
Fig. 3
Fig. 3
Confocal image of one SKBr3 cell after incubation with (DTPA)n-trastuzumab-(IRDye800)m. Nuclear staining is by sytox green and is pseudo-colored in green, while fluorescence from IRDye 800 is represented in red. (Color online only.)
Both nuclear and planar NIR fluorescence imaging was used to evaluate the in vivo specificity of (111In-DTPA)n-trastuzumab-(IRDye800)m. Figure 4 shows a series of whole-body fluorescence (a and b), planar scintigraphy (c), and SPECT/CT (d) images from athymic nude mice with HER2-overexpressing SKBr3 human breast cancer xenografts after i.v. injection of (111In-DTPA)n-trastuzumab-(IRDye800)m. In vivo fluorescence imaging was accomplished by illuminating the animal with light from a laser diode (85 mA and 80 mW for 785-nm light; DL7140-201; Sanyo) that expanded to a circular area approximately 8 cm in diameter. The reemitted fluorescent light was collected with an EMCCD camera in CW mode. Filter sets used in this study included a bandpass filter with an 830-nm center wavelength and an optical density of greater than 3 at 785 nm (ANDV8483; Andover Corp.) for collecting IRDye 800CW fluorescence and a holographic filter with an optical density of greater than 6 (HSP 785.0; Kaiser Optical Systems) for rejecting backscattered and reflected excitation light. Image acquisition was accomplished with V11 software to collect raw fluorescence images, and with these images, data processing and analysis were accomplished with ImageJ software. The integration time for white-light and fluorescence images was 800 ms. SPECT and CT images were acquired consecutively with a MicroCAT II scanner (Siemens Medical Solutions). The SPECT scan was acquired for 20 projections over 360 day for a scan time of 1 min per frame. SPECT and CT tomographic images were coegistered by geometric transformation and rendered to make the fused images with Amira (version 3.1; Konrad-Zuse-Zentrum fur Informationstechnik). Planar scintigraphy was performed by removing the 3-mm pinhole collimator of the SPECT scanner, adding a 1.22-mm planar collimator, and integrating for 10 min.
Fig. 4
Fig. 4
(a) and (b) are representative white light and whole-body fluorescence images of an athymic nude mouse inoculated with SKBr3-luc xenografts, taken 48 h after i.v. administration of (111In-DTPA)n-trastuzumab-(IRDye800)m. High agent uptake is visible in (more ...)
The fluorescence and nuclear signal obtained at the tumor region appear significantly higher than the rest of the body. Comparison of tumor uptake between mice after administration of dye alone and dual-labeled trastuzumab revealed significant accumulation of the dual-labeled agent in the tumor, as opposed to dye alone (p-value: 0.0011). Antibody interaction with the hepatocytes (which represent routes for degradation and clearance) increased agent accumulation in the liver and is expected with i.v. administration of an antibody-based imaging agent. Liver uptake is observed in both imaging modalities, consistent with the integrity of the dual-labeled agent.
The target-to-muscle ratios (TMRs) calculated from the planar fluorescence and planar scintigraphy images according to the approach described by Houston et al.32 are found to be comparable at 2.11 and 2.43, respectively, and consistent with the dual labeling on a single imaging agent. The signal-to-noises ratio (SNRs) calculated from the planar fluorescence and planar scintigraphy images are found to be dramatically different at 16.82 and 4.22, respectively. The differences can be attributed to the increased photon count rates of fluorescence imaging when compared to scintigraphy. Upon dissection of major organs and evaluation of gamma and fluorescent photon counts, the TMRs were calculated for both modalities. When optical TMR is normalized by area (or pixels) and nuclear TMR by tissue weight (in grams), no significant change is observed within each modality over time, but the signal obtained from optical imaging is statistically higher compared to that from nuclear imaging. In contrast, when both TMRs are calculated based on tissue weight, the statistical significance disappears, as represented by Fig. 5, consistent with the origin of both nuclear and fluorescent signals from a single imaging agent. In addition, the standard error observed in optical imaging is smaller than nuclear imaging, consistent with the reduced SNR also observed in the case of the cyclic RGD peptide.32
Fig. 5
Fig. 5
Plot of nuclear versus optical tumor-to-muscle ratios (TMRs), normalized per gram of tissue. No statistical differences have been observed, but optical imaging with smaller error bars indicates higher signal-to-noise ratios (SNRs). Reproduced from Ref. (more ...)
Further, we assessed the potential of antibody-based optical imaging for lymph node imaging using i.d. administration. Upon injecting 1 μg (5.4 pmole) of a 1 μg/μl solution into the footpad of athymic nude mice, we were able to image trafficking of (In-DTPA)n-trastuzumab-(IRDye800)m into the axillary nodes and popliteal nodes [Figs. 6(a) and 6(b), respectively]. A closer look at the popliteal lymph nodes, revealed lymphatic channels that picked up the agent from the injection site and drained it into the lymph node as indicated by the arrows in Fig. 6(c). Both images were obtained within 1 h of injection. It is of interest to note that the volume injected in our preclinical study is considerably less when compared to the 50 to 200 μl injections as reported with nuclear imaging.2,3
Fig. 6
Fig. 6
Intradermal injection trastuzumab-(IRDye800)m in the footpads of mice show trafficking to the axillary nodes (a) and popliteal nodes (b). The injection site has been covered in (b). A higher magnification of the region around the popliteal nodes shows (more ...)
Figure 7(b) shows a sequence of optical images taken after an i.d. injection of (In-DTPA)n-trastuzumab-(IRDye800)m (1 μg, 5.4 pmole) into the footpad of an athymic mouse. The lymph node targeted was the axillary node. The corresponding sample white light image is shown in Fig. 7(a). The accumulation and clearance of the antibody-based imaging agent in the lymph node is slower than that observed by IC-Green or IRDye 800CW. IRDye 800CW has a molecular weight of 1166 Da, which is significantly smaller than antibodies (MW: 185kDa), enabling it to clear from the lymph nodes within a few hours. Antibodies requires a few days (data not shown), which can be a problem for radioimmunoscintigraphy of cancer-positive lymph nodes. Figure 7(c) shows the rate of clearance of (In-DTPA)n-trastuzumab-(IRDye800)m averaged over time in five animals. We found variability in the uptake of the imaging agent into the axillary node among the mice, which is represented by a large error bar at t=0 h [Fig. 7(c)], but all mice attained maximum fluorescence in nodes within t=1 h. The half-life of clearance was between 10 to 12 h, which is consistent with previous reports using radio-labeled antibodies. As seen in the i.v. administration of (In-DTPA)n-trastuzumab-(IRDye800)m, we also observed a fluorescence signal emanating from the liver region after an i.d. injection (albeit at later times), which is consistent with antibody degradation and clearance.
Fig. 7
Fig. 7
Representative white light (a) and optical imaging (b) of accumulation and clearance of (In-DTPA)n-trastuzumab-(IRDye800)m (1 μg, 5.4 pmole) in the axillary node after administration into the forepaw footpad. Quantitative analysis of lymph node (more ...)
This study demonstrates the potential of using NIR fluorescence imaging for impacting tumor nodal staging. By enhancing the accuracy of nodal staging with molecular-specific imaging agents, we can offer novel approaches to diagnose sites that are often harder to access for ex vivo pathology, e.g., internal mammary nodes and supraclavicular nodes in breast and pelvic lymph nodes in prostate cancer patients. By injecting the agent directly into lymphatics through i.d. or s.c. routes, we can considerably reduce the dose administered and the amount of nonspecific binding in other organs that is characteristic of an i.v. route of administration. Since antibodies are cleared through the liver and kidneys, which are major organs surrounding the lymph nodes draining the prostate, i.d. delivery is especially advantageous in assessing metastases in prostate cancer.
Despite nuclear imaging being the clinical “gold standard” of molecular imaging, optical imaging provides superior signal-to-noise ratios with low camera integration times due to high photon count rate. These advantages have recently provided investigators with an opportunity to analyze lymph function and quantify pulsatility in humans, swine, and mice.1921 Furthermore, they have enabled the synthesis of novel optical-based targeting agents to address other lymph-based disorders, as demonstrated by HA-NIR, a molecular target of the lymph vascular endothelial receptor (LYVE-1).19 In the present study, we have shown the feasibility of performing optical imaging in detecting lymph nodes in a preclinical animal model using molecular targets such as antibodies. (111In-DTPA)n-trastuzumab-(IRDye800)m is a dual-labeled antibody that exhibits high molecular specificity to HER2. We have demonstrated that picomole amounts are sufficient to visualize the axillary and popliteal lymph nodes in mice (Fig. 6). We observed that the fluorescent intensity due to dual-labeled (In-DTPA)n-trastuzumab-(IRDye800)m reduced to half of its highest value at 12 h after administration [Fig. 7(c)]. But further studies addressing questions of specificity and sensitivity will have to be conducted to prove efficacy for cancer detection. Nonetheless, the combination of NIR and nuclear imaging of lymph node metastases in cancer patients with dual-labeled molecular agents that are administered through i.d. or s.c. routes shows promise for improving the accuracy of tumor node metastasis staging.
Acknowledgments
This work was supported by the National Institutes of Health, Grant Nos. P50 CA58183, R01 EB003132, and R01 CA112679.
Contributor Information
Lakshmi Sampath, Baylor College of Medicine, Division of Molecular Imaging, Department of Radiology and Molecular Physiology and Biophysics and The Baylor Breast Center, Houston, Texas 77030.
Wei Wang, Baylor College of Medicine, Division of Molecular Imaging, Department of Radiology, Houston, Texas 77030.
Eva M. Sevick-Muraca, Baylor College of Medicine, Division of Molecular Imaging, Department of Radiology and Molecular Physiology and Biophysics and The Baylor Breast Center, Houston, Texas 77030.
1. Weinstein JN, Parker RJ, Keenan AM, Dower SK, Morse HC, III, Sieber SM. Monoclonal anitbodies in the lymphatics: toward the diagnosis and therapy of tumor metastases. Science. 1982;218(4579):1334–1337. [PubMed]
2. Weinstein JN, Steller MA, Keenan AM, Covell DG, Key ME, Sieber SM, Oldham RK, Hwang KM, Parker RJ. Monoclonal antibodies in the lymphatics: selective delivery to lymph node metastases of a solid tumor. Science. 1983;222(4622):423–426. [PubMed]
3. Steller MA, Parker RJ, Covell DG, Holton OD, III, Keenan AM, Sieber SM, Weinstein JN. Optimization of monoclonal antibody delivery via the lymphatics: the dose dependence. Cancer Res. 1986;46(4 Pt 1):1830–1834. [PubMed]
4. Wahl RL, Geatti O, Liebert M, Wilson B, Shreve P, Beers BA. Kinetics of interstitially administered monoclonal antibodies for purposes of lymphoscintigraphy. J Nucl Med. 1987;28(11):1736–1744. [PubMed]
5. Mulshine JL, Keenan AM, Carrasquillo JA, Walsh T, Linnoila RI, Holton OD, Harwell J, Larson SM, Bunn PA, Weinstein JN. Immunolymphoscintigraphy of pulmonary and mediastinal lymph nodes in dogs: a new approach to lung cancer imaging. Cancer Res. 1987;47(13):3572–3576. [PubMed]
6. Porter CJ, Charman SA. Lymphatic transport of proteins after subcutaneous administration. J Pharm Sci. 2000;89(3):297–310. [PubMed]
7. Order SE, Bloomer WD, Jones AG, Kaplan WD, Davis MA, Adelstein SJ, Hellman S. Radionuclide immunoglobulin lymphangiography: a case report. Cancer. 1975;35(6):1487–1492. [PubMed]
8. DeLand FH, Kim EE, Goldenberg DM. Lymphoscintigraphy with radionuclide-labeled antibodies to carcinoembryonic antigen. Cancer Res. 1980;40(8 Pt 2):2997–3000. [PubMed]
9. Tjandra JJ, Russell IS, Collins JP, Andrews JT, Lichtenstein M, Binns D, McKenzie IF. Immunolymphoscintigraphy for the detection of lymph node metastases from breast cancer. Cancer Res. 1989;49(6):1600–1608. [PubMed]
10. Kairemo KJ. Immunolymphoscintigraphy with 99mTc-labeled monoclonal antibody (BW 431/26) reacting with carcinoembryonic antigen in breast cancer. Cancer Res. 1990;50(3 Suppl):949s–954s. [PubMed]
11. Keenan AM, Weinstein JN, Mulshine JL, Carrasquillo JA, Bunn PA, Jr, Reynolds JC, Larson SM. Immunolymphoscintigraphy in patients with lymphoma after subcutaneous injection of indium-111-labeled T101 monoclonal antibody. J Nucl Med. 1987;28(1):42–46. [PubMed]
12. Keenan AM, Weinstein JN, Carrasquillo JA, Bunn PA, Jr, Reynolds JC, Foon KA, Smarte NC, Ghosh B, Fejka RM, Larson SM, et al. Immunolymphoscintigraphy and the dose dependence of 111In-labeled T101 monoclonal antibody in patients with cutaneous T-cell lymphoma. Cancer Res. 1987;47(22):6093–6099. [PubMed]
13. Abdel-Nabi HH, Ortman-Nabi JA, See W, Lee J, Ireton R, Boileau M, Unger MW, Halverson C. Clinical experience with intralymphatic administration of 111In-labeled monoclonal antibody PAY 276 for the detection of pelvic nodal metastases in prostatic carcinoma. Eur J Nucl Med. 1990;16(3):149–156. [PubMed]
14. Lotze MT, Carrasquillo JA, Weinstein JN, Bryant GJ, Perentesis P, Reynolds JC, Matis LA, Eger RR, Keenan AM, Hellstrom I, et al. Monoclonal antibody imaging of human melanoma. Radioimmunodetection by subcutaneous or systemic injection. Ann Surg. 1986;204(3):223–235. [PubMed]
15. Nelp WB, Eary JF, Jones RF, Hellstrom KE, Hellstrom I, Beaumier PL, Krohn KA. Preliminary studies of monoclonal antibody lymphoscintigraphy in malignant melanoma. J Nucl Med. 1987;28(1):34–41. [PubMed]
16. Engelstad BL, Spitler LE, Del Rio MJ, Ramos EC, Rosendorf LL, Reinhold CE, Khentigan A, Huberty JP, Corpuz SW, Lee HM, et al. Phase 1 immunolymphoscintigraphy with an In-111-labeled antimelanoma monoclonal antibody. Radiology. 1986;161(2):419–422. [PubMed]
17. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244(4905):707–712. [PubMed]
18. Sampath L, Kwon S, Ke S, Wang W, Schiff R, Mawad ME, Sevick EM. Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor-2 (HER2) over-expression in breast cancer. J Nucl Med. 2007;48(9):1501–1510. [PubMed]
19. Sharma R, Wang W, Rasmussen JC, Joshi A, Houston JP, Adams KE, Cameron AG, Ke S, Kwon S, Mawad ME, Sevick EM. Quantitative imaging of lymph function. Am J Physiol Heart Circ Physiol. 2007;292:H3109–H3118. [PubMed]
20. Sevick-Muraca EM, Sharma R, Rasmussen JC, Marshall MV, Wendt JA, Pham HQ, Bonefas E, Houston JP, Sampath L, Adams KE, Blanchard DK, Fisher RE, Chiang SB, Elledge R, Mawad ME. Imaging of lymph flow in breast cancer patients after microdose administration of a near-infrared fluorophore: feasibility study. Radiology. 2008;246(3):734–741. [PMC free article] [PubMed]
21. Kwon S, Sevick-Muraca EM. Noninvasive quantitative imaging of lymph function in mice. Lymphatic Res Biol. 2007;5(4):219–231. [PubMed]
22. Knapp DW, Adams LG, Degrand AM, Niles JD, Ramos-Vara JA, Weil AB, O’Donnell MA, Lucroy MD, Frangioni JV. Sentinel lymph node mapping of invasive urinary bladder cancer in animal models using invisible light. Eur Urol. 2007;52(6):1700–1708. [PMC free article] [PubMed]
23. Parungo CP, Soybel DI, Colson YL, Kim SW, Ohnishi S, DeGrand AM, Laurence RG, Soltesz EG, Chen FY, Cohn LH, Bawendi MG, Frangioni JV. Lymphatic drainage of the peritoneal space: a pattern dependent on bowel lymphatics. Ann Surg Oncol. 2007;14(2):286–298. [PMC free article] [PubMed]
24. Tanaka E, Ohnishi S, Laurence RG, Choi HS, Humblet V, Frangioni JV. Real-time intraoperative ureteral guidance using invisible near-infrared fluorescence. J Urol (Baltimore) 2007;178(5):2197–2202. [PMC free article] [PubMed]
25. Corlu A, Choe R, Durduran T, Rosen MA, Schweiger M, Arridge SR, Yodh AG. Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans. Opt Express. 2007;15(11):6696–6716. [PubMed]
26. Sevick-Muraca EM, Rasmussen JC. Molecular imaging with optics: a primer and a case for near-infrared fluorescence techniques in personalized medicine. J Biomed Opt. 2008;13(4):041305. [PMC free article] [PubMed]
27. Godavarty A, Eppstein MJ, Zhang C, Sevick-Muraca EM. Detection of single and multiple targets in tissue phantoms with fluorescence-enhanced optical imaging: feasibility study. Radiology. 2005;235(1):148–154. [PubMed]
28. Godavarty A, Zhang C, Eppstein MJ, Sevick-Muraca EM. Fluorescence-enhanced optical imaging of large phantoms using single and simultaneous dual point illumination geometries. Med Phys. 2004;31(2):183–190. [PubMed]
29. Roy R, Godavarty A, Sevick-Muraca EM. Fluorescence-enhanced optical tomography of a large tissue phantom using point illumination geometries. J Biomed Opt. 2006;11(4):044007. [PubMed]
30. Joshi A, Bangerth W, Hwang K, Rasmussen JC, Sevick-Muraca EM. Fully adaptive FEM based fluorescence optical tomography from time-dependent measurements with area illumination and detection. Med Phys. 2006;33(5):1299–1310. [PubMed]
31. Li C, Wang W, Wu Q, Ke S, Houston J, Sevick-Muraca E, Dong L, Chow D, Charnsangavej C, Gelovani JG. Dual optical and nuclear imaging in human melanoma xenografts using a single targeted imaging probe. Nucl Med Biol. 2006;33(3):349–358. [PubMed]
32. Houston JP, Ke S, Wang W, Li C, Sevick-Muraca EM. Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe. J Biomed Opt. 2005;10(5):054010. [PubMed]
33. Wang W, Ke S, Kwon S, Yallampalli S, Cameron AG, Adams KE, Mawad ME, Sevick-Muraca EM. A new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chain. Bioconjugate Chem. 2007;18(2):397–402. [PubMed]
34. Yarden Y. Biology of HER2 and its importance in breast cancer. J Surg Oncol, Suppl. 2001;61(Suppl 2):1–13. [PubMed]
35. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA. 1992;89(10):4285–4289. [PubMed]
36. Tokunaga E, Oki E, Nishida K, Koga T, Egashira A, Morita M, Kakeji Y, Maehara Y. Trastuzumab and breast cancer: developments and current status. Int J Clin Oncol. 2006;11(3):199–208. [PubMed]
37. Yeon CH, Pegram MD. Anti-erbB-2 antibody trastuzumab in the treatment of HER2-amplified breast cancer. Invest New Drugs. 2005;23(5):391–409. [PubMed]
38. Simon R, Nocito A, Hubscher T, Bucher C, Torhorst J, Schraml P, Bubendorf L, Mihatsch MM, Moch H, Wilber K, Schotzau A, Kononen J, Sauter G. Patterns of HER-2/neu amplification and overexpression in primary and metastatic breast cancer. J Natl Cancer Inst. 2001;93(15):1141–1146. [PubMed]
39. Regitnig P, Schippinger W, Lindbauer M, Samonigg H, Lax SF. Change of HER-2/neu status in a subset of distant metastases from breast carcinomas. J Pathol. 2004;203(4):918–926. [PubMed]
40. Smith-Jones PM, Solit DB, Akhurst T, Afroze F, Rosen N, Larson SM. Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors. Nat Biotechnol. 2004;22(6):701–706. [PubMed]
41. Blend MJ, Stastny JJ, Swanson SM, Brechbiel MW. Labeling anti-HER2/neu monoclonal antibodies with 111In and 90Y using a bifunctional DTPA chelating agent. Cancer Biother Radiopharm. 2003;18(3):355–363. [PubMed]
42. Dennis MS, Jin H, Dugger D, Yang R, McFarland L, Ogasawara A, Williams S, Cole MJ, Ross S, Schwall R. Imaging tumors with an albumin-binding Fab, a novel tumor-targeting agent. Cancer Res. 2007;67(1):254–261. [PubMed]
43. Tang Y, Wang J, Scollard DA, Mondal H, Holloway C, Kahn HJ, Reilly RM. Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using (111)In-trastuzumab (Herceptin) Fab fragments. Nucl Med Biol. 2005;32(1):51–58. [PubMed]
44. Tang Y, Scollard D, Chen P, Wang J, Holloway C, Reilly RM. Imaging of HER2/neu expression in BT-474 human breast cancer xenografts in athymic mice using [(99m)Tc]-HYNIC-trastuzumab (Herceptin) Fab fragments. Nucl Med Commun. 2005;26(5):427–432. [PubMed]
45. Lub-de Hooge MN, Kosterink JG, Perik PJ, Nijnuis H, Tran L, Bart J, Suurmeijer AJ, de Jong S, Jager PL, de Vries EG. Preclinical characterisation of 111In-DTPA-trastuzumab. Br J Pharmacol. 2004;143(1):99–106. [PubMed]
46. Huh YM, Jun YW, Song HT, Kim S, Choi JS, Lee JH, Yoon S, Kim KS, Shin JS, Suh JS, Cheon J. In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals. J Am Chem Soc. 2005;127(35):12387–12391. [PubMed]
47. Artemov D, Mori N, Okollie B, Bhujwalla ZM. MR molecular imaging of the HER-2/neu receptor in breast cancer cells using targeted iron oxide nanoparticles. Magn Reson Med. 2003;49(3):403–408. [PubMed]
48. Copland JA, Eghtedari M, Popov VL, Kotov N, Mamedova N, Motamedi M, Oraevsky AA. Bioconjugated gold nanoparticles as a molecular based contrast agent: implications for imaging of deep tumors using optoacoustic tomography. Mol Imaging Biol. 2004;6(5):341–349. [PubMed]
49. Hilger I, Leistner Y, Berndt A, Fritsche C, Haas KM, Kosmehl H, Kaiser WA. Near-infrared fluorescence imaging of HER-2 protein over-expression in tumour cells. Eur Radiol. 2004;14(6):1124–1129. [PubMed]
50. Koyama Y, Hama Y, Urano Y, Nguyen DM, Choyke PL, Kobayashi H. Spectral fluorescence molecular imaging of lung metastases targeting HER2/neu. Clin Cancer Res. 2007;13(10):2936–2945. [PubMed]
51. Tada H, Higuchi H, Wanatabe TM, Ohuchi N. In vivo real-time tracking of single quantum dots conjugated with monoclonal anti-HER2 antibody in tumors of mice. Cancer Res. 2007;67(3):1138–1144. [PubMed]
52. Li-Shishido S, Watanabe TM, Tada H, Higuchi H, Ohuchi N. Reduction in nonfluorescence state of quantum dots on an immunofluorescence staining. Biochem Biophys Res Commun. 2006;351(1):7–13. [PubMed]
53. Behr TM, Behe M, Wormann B. Trastuzumab and breast cancer. N Engl J Med. 2001;345(13):995–996. [PubMed]
54. Perik PJ, Lub-De Hooge MN, Gietema JA, van der Graaf WT, de Korte MA, Jonkman S, Kosterink JG, van Veldhuisen DJ, Sleijfer DT, Jager PL, de Vries EG. Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol. 2006;24(15):2276–2282. [PubMed]