Lymphatic imaging: focus on imaging probes.

In view of the importance of sentinel lymph nodes (SLNs) in tumor staging and patient management, sensitive and accurate imaging of SLNs has been intensively explored. Along with the advance of the imaging technology, various contrast agents have been developed for lymphatic imaging. In this review, the lymph node imaging agents were summarized into three groups: tumor targeting agents, lymphatic targeting agents and lymphatic mapping agents. Tumor targeting agents are used to detect metastatic tumor tissue within LNs, lymphatic targeting agents aim to visualize lymphatic vessels and lymphangionesis, while lymphatic mapping agents are mainly for SLN detection during surgery after local administration. Coupled with various signal emitters, these imaging agents work with single or multiple imaging modalities to provide a valuable way to evaluate the location and metastatic status of SLNs.

Introduction

Besides removing interstitial fluid from tissues to maintain tissue interstitial pressure, the lymphatic system plays a very important role in immune response by providing a transport route for antigen-presenting cells (APCs) and white blood cells 1. At the same time, it serves as a transport route for disseminating tumor cells, resulting in metastases. Although the exact mechanism is not clear, it is well taken that the lymphatics has advantages over the blood circulation for tumor metastasis, possibly due to the large cavity of lymph vessels and slow velocity of lymph 2. Consequently, many types of malignant tumors such as breast cancer, melanoma, and prostate cancer are prone to metastasize first to regional lymph nodes (LNs), through tumor associated lymphatic channels 3, 4.

The amount of spread to nearby lymph nodes is one of the components for TNM staging system, which has been accepted by the Union for International Cancer Control (UICC) and the American Joint Committee on Cancer (AJCC). In addition, the status of the tumor draining LNs or sentinel LNs (SLNs) serves as an indicator of prognosis and therapeutic decision-making 5, 6. So far, the gold standard to stage the LNs is lymphadenectomy and histologic evaluation, which is invasive and limited by surgical field for nodal sampling and lack of accuracy 7. With the development of imaging techniques, currently, pre-surgical diagnosis of SLNs is often based on their morphologic change observed by magnetic resonance imaging (MRI) or x-ray computed tomography (CT). The application and limitation of these imaging modalities have been summarized in details elsewhere 8.

Since the current assessment of lymph nodes relies on morphology and anatomy rather than function and physiology, tumor metastasis is mainly evaluated based on the size and the shape of the involved lymph node 9. However, nodal metastases are often microscopic, so neither CT nor MRI can rule them out reliably 10. Besides, it is very challenging for CT and MRI to visualize SLNs when they are small or have similar signal intensities with surrounding healthy soft tissues 11. Consequently, various imaging probes have been developed with the aim to better visualize and characterize the lymphatics 12. Based on imaging purpose and underlying mechanisms, these probes can be categorized into three classes, including tumor targeting agents, lymphatic targeting agents and lymphatic mapping agents.

Tumor targeting agents

As the name indicates, tumor targeting agents recognize specific biomarkers, pathways on/within tumor cells or tumor microenvironment to achieve tumor/non-tumor signal contrast for tumor visualization. Majority of tumor targeting agents are labeled with radionuclides for either single photon emission computed tomography (SPECT) or positron emission tomography (PET). For example, fluorine-18 labeled fluorodeoxyglucose (18F-FDG), a glucose analog, usually shows high tumor accumulation due to the increased rate of glycolysis in various malignant cells 13-15. So far, 18F-FDG is the most valuable PET imaging tracer in clinical oncology and it is well accepted that FDG-PET is superior to morphologic imaging procedures for staging LNs adjacent to tumors (Figure 1) 16-18. The high uptake of FDG in tumor metastasized LNs also allows Cerenkov luminescence imaging with a specially designed dark box 17, which may be used to provide intraoperative guidance in the detection of positive lymph nodes. Due to the extremely low yield of Cerenkov photon, clinical value of this method will need further validation.

Figure 1

A, A representative PET/CT images of 18F-FDG-positive axillary lymph node. B & C, Results for discordant false-negative MR imaging and true-positive 11C-choline PET/CT with a score for T1-weighted MR imaging of 3 (B) and a score for 11C-choline PET/CT of 5 (C) for LN metastasis are shown. The right internal iliac LN was indicated by arrow. (Reprinted and modified with the permission from references 17 and 24).

However, increased 18F-FDG uptake is not only observed in malignant tumors but also in inflammation and infection. Especially, the lack of specificity results in inaccurate identification of malignant lymph nodes in the mediastinum, which have been confirmed by numerous clinical studies 8. Low FDG uptake in certain cancer types also encourages looking into alternative imaging tracers. Prostate cancer is characterized by an increased uptake of choline into the cell to meet increased synthesis of phosphatidylcholine, an important cell membrane phospholipid. Therefore, choline, labeled either with 18F or 11C, has been used for PET imaging of prostate cancer 19-22. 11C-choline could detect LN metastasis from prostate cancer with a sensitivity of 80%, specificity of 96%, and accuracy of 93% 23. In a recent clinical study in patients with prostate cancer, 11C-choline PET/CT was found to be superior for pelvic LN metastasis than multi-parametric MR imaging (Figure ) 24. Increased lipid synthesis in prostate cancer also results in high retention of 11C-acetate or 18F-acetate. It has been demonstrated that 11C-acetate is better than 18F-FDG in detecting local recurrences and regional lymph node metastases of prostate cancer 25. Prostate-specific membrane antigen (PSMA) is significantly overexpressed on the surface of prostate cancer cells 26. It has also been reported that PSMA PET/CT using a 68Ga-labeled PSMA ligand can detect lesions characteristic of prostate cancer with improved contrast when compared to 18F-fluoromethylcholine PET/CT, especially at low PSA levels 27. These results suggest that PSMA targeting imaging may be useful in detection of lymph node metastases of prostate cancer.

Epithelial cell adhesion molecule (EpCAM) is a 40-kDa type I transmembrane protein found on epithelial cells. Overexpression of EpCAM was found in many metastasizing epithelial cancers 28, 29 and has been shown to be associated with the recurrence of prostate cancer 30. In view of these facts, Hall et al. 31 developed and labeled monoclonal antibodies (mAbs) against EpCAM with a positron emitter, 64Cu and a near-infrared fluorophore, IRDye800 for both noninvasive and intraoperative detection of metastatic LNs in a prostate cancer model. Between 18 and 24 h post intravenous injection, tumor metastases in LNs can be clearly visualized with both PET and optical imaging (Figure ).

Integrins are a family of 24 trans-membrane proteins which mediate cell-cell adhesion and attachment of cells to extracellular matrix (ECM) 32. Among them, αvβ3 integrin has been intensively investigated as a target for angiogenesis imaging and therapy of various types of tumors, owing to its positive role in regulating the survival of endothelial cells and promoting angiogenesis in malignant diseases 33-36. One dominant category of imaging probes were based on the peptide ligand of integrin αvβ3 with the sequence of arginine-glycine-aspartic acid (RGD) 37-40. Several RGD based tracers are in different phases of clinical trials 41. In one small scale clinical study using 18F-galacto-RGD, lymph-node metastases were detected in 3 of 8 patients 42. In another study using 99mTc-3PRGD2 SPECT, the primary lesions within lung and mediastinum could be detected along with most of the lymph node metastases 43.

Theoretically, any tumor targeting agent can be used to detect both primary tumors and metastases within LNs. Although with high sensitivity, the relatively low resolution (approximately 4-8 mm in clinical and 1-2 mm in small animal imaging systems) of PET limits its detection of micrometastases within LNs 44. The combination of PET and CT has matured into an important clinical diagnostic tool by providing anatomical and functional data sets in a single session with accurate image co-registration 45. A number of clinical studies demonstrated that sensitivity, specificity, positive predictive value, negative predictive value and accuracy of lymph node staging were all significantly improved with FDG-PET/CT compared with CT alone 46, 47. Recently, with the availability of PET/MRI, FDG PET/MR has been applied to lymphoma staging and showed high sensitivity and specificity for nodal involvement in lymphoma 48, 49. Even with these hybrid systems, novel tumor targeting probes with high sensitivity, specificity and signal contrast are still needed to visualize microscopic metastasis within LNs.

Lymphatic targeting agents

Tumor-induced lymph-angiogenesis (expansion of the lymphatic vasculature) in the tumor draining LNs usually precedes metastasis and leads to increased tumor spread to distal LNs and further to distal organs 50, 51. A number of lymphatic specific markers such as podoplainin, Prox-1, LYVE-1, and VEGFR-3 have been identified 52-54. Lymphatic targeting agents usually have been developed by labeling antibodies, or peptidic ligands against these lymphatic specific markers. For example, an IgM monoclonal antibody against the glycoproteins, which is responsible for recruiting lymphocytes into peripheral LNs 55, has been labeled with Cy7 dye for LN imaging. The dye conjugated antibody showed surprisingly high accumulation in peripheral LNs as early as 1 h after tail vein injection 56. The same antibody has been conjugated onto polymer shell microbubbles for LN detection using ultrasound imaging after intravenous administration 57.

The lymphatic vessel endothelial hyaluronan receptor (LYVE-1) is expressed predominantly on lymphatic endothelium. As an ortholog of CD44, the function of LYVE-1 is to bind HA and regulate cell migration within the lymphatic system 58. Using an 124I-labeled antibody against LYVE-1, Mumprecht et al. 59 performed PET with mouse lymph-angiogenesis models and found that the LNs bearing metastases could be visualized by PET, even though the metastases were not detected by 18F-FDG PET (Figure ).

Compared with antibodies, peptide-based imaging probes allow faster clearance due to much smaller molecular size. Lyp-1 is a cyclic 9-amino-acid cyclic peptide identified by in vivo phage display technology, which homes to lymphatic endothelial cells 54, 60. Intravenous administration of FITC-LyP-1 led to prominent accumulation in the tumor tissue 16-20 h after intravenous injection 61. The LyP-1 peptide has also been labeled with a near-infrared fluorophore Cy5.5 for optical imaging. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla, indicating increased lymphangiogesis within these LNs 62.

Most approaches for cancer metastasis imaging in patients have focused on the detection of the cancer cells themselves 63, 64. As mentioned before, nodal metastases are often microscopic. It is very challenging to visualize the tumor tissue within the LNs either by anatomical imaging or molecular imaging using tumor targeting probes 65. Thus, the ability to detect LN lymphangiogenesis may serve as an alternative way to predict LN metastasis. However, lymphangiogenesis also happens under inflammatory stimulation since high levels of lymphangiogenic factors are produced by macrophages and granulocytes in inflamed tissue 66. One should be cautious about image interpretation since these lymphatic targeting agents would not differentiate tumor induced lymphangiogenesis from inflammatory reaction. This may be one of the main reasons why these lymphatic targeting agents have not been used in the clinic.

Lymphatic mapping agents

Axillary lymph node dissection (ALND) is a surgical procedure to remove the lymph nodes from axilla for diagnosis and staging of breast cancer. Although it is the most accurate method to assess nodal status, ALND is associated with several adverse long-term side effects due to the extensive surgery. As an alternative, lymphatic mapping with sentinel lymph node biopsy (SLNB) has emerged as an effective method to detect axillary metastases. Although still debatable, the clinical advantages of SLNB over ALND are apparent, and the procedure is becoming the preferred standard in patients with breast cancer or melanoma 67. Moreover, SLNB has become established clinical practice in patients with other types of cancer including penile, anal, colorectal and prostate cancer 68.

Different from the aforementioned imaging agents, lymphatic mapping agents are developed to meet the requirement of SLNB, i.e. to detect SLNs. Consequently, most of the imaging agents in this category are administered locally, which then migrate to and are trapped inside the SLNs. So far, the most commonly used lymphatic mapping method in the clinic is a combined injection of 99mTc-labeled colloids first and vital dyes (patent blue, isosulfan blue or indocyanine green (ICG)) several hours later. SLNs can be visualized pre-operationally either by gamma scintigraphy or SPECT. The SLNs during surgery could be located with a hand-held gamma ray counter and visual contrast of the blue dye. The value of this procedure has been substantiated in numerous clinical studies 69, 70.

However, this method has several drawbacks. Firstly, it requires separate administration of 99mTc-labeled colloids and dyes because of different rate of local migration 71. Secondly, scintigraphy and SPECT show relatively low sensitivity and spatial resolution. In addition, blue dye injections may stain the surgical field blue, which can be a hindrance during surgery 72. With the advancement of imaging instruments and material sciences, numerous lymphatic mapping probes have been developed, aiming to improve identification and mapping of lymph nodes, especially sentinel lymph nodes during surgery 73, 74.

To avoid injection of 99mTc-labeled colloid and blue colored vital dye separately, Evans blue (EB), a dye molecule binding with plasma proteins, has been labeled with 99mTc for SLNB. 99mTc-EB combines both radioactive and colored signals and can be administered as a single dose for SLN identification 75. To increase the migration rate and LN retention, 99mTc-tilmanocept has been developed, which consists of a dextran frame linked with multiple diethylenetriaminepentaacetic acid (DTPA) for 99mTc labeling and mannose residues for CD206 binding. CD206 is a mannose receptor, primarily presented on the surface of macrophages and dendritic cells in lymph nodes 76. Because of its small size, 99mTc-tilmanocept can migrate quickly through the afferent lymph vessels and reside within SLNs due to the specific binding. Several clinical studies have confirmed that 99mTc-tilmanocept does not escape from the SLN to the second echelon lymph nodes, and has superior identification rates and sensitivity over blue dyes 68, 77. A hybrid fluorescent-radioactive tracer has also been applied for sentinel node identification by mixing ICG with 99mTc-labeled albumin nanocolloid 78. The lymphatic drainage pattern of ICG/99mTc-nanocolloid is identical to that of 99mTc-nanocolloid in clinical setting and all preoperatively identified sentinel nodes could be localized using combined radio- and fluorescence guidance intraoperatively.

Compared with SPECT, PET has higher sensitivity and temporal resolution. PET lymphography has been investigated with intradermal administration of 18F-FDG for combined diagnostic and intraoperative visualization of LNs 79. Within 30 min after tracer injection, lymphatic vessels and LNs can be clearly revealed by PET in an animal modal. However, the clinical application of 18F-FDG PET lymphography may be challenged by the fast migration of the small molecules into blood circulation. Recently, we synthesized a NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid) conjugated truncated Evans blue (NEB). 18F-labeling was achieved through the formation of 18F-aluminum fluoride complex 80. After intravenous injection, 18F-AlF-NEB complexes with serum albumin very quickly and thus most of the radioactivity is retained in the blood circulation 80. After local injection, 18F-AlF-NEB also forms complexes with endogenous albumin in the interstitial fluid and allows for visualizing the lymphatic system. The LNs can be distinguished clearly by high intensity PET signal from 18F-AlF-NEB (Figure ) 81.

Superb spatial resolution endows MRI the ability to accurately reflect the anatomical location and resolve heterogeneity within LNs. After local administration of superparamagnetic iron oxide (SPIO), the adjacent LNs can be visualized on T2-weighted MRI since significant amount of particles is accumulated within the LNs, mainly through macrophage endocytosis 82, 83. More importantly, tumor metastasis can be distinguished by heterogeneous signal enhancement because metastatic tumor tissue takes up SPIO much less efficiently than the lymphatic tissue (Figure ) 84, 85.

In some special cases when whole body LNs need be evaluated, intravenous administration may be preferred. After intravenous injection, some small sized (30-50 nm) lymphotropic nanoparticles such as ultrasmall SPIO (USPIO) are slowly extravasated from the vasculature into the interstitial space, from which they are transported to lymph nodes by way of lymphatic vessels 86. Accumulation of nanoparticles in benign nodes causes a decrease in signal intensity on T2-weighted and T2*-weighted MRI scans 86. The metastasized tumor tissue in malignant lymph nodes lacking normal macrophages cannot phagocytose USPIO and thus retain the bright signals in MRI scans 87. Consequently, in patients with prostate cancer, nodal metastases could be correctly identified in all patients with a significantly higher sensitivity than conventional MRI or nomograms (Figure ) 88. One disadvantage of this imaging strategy is the slow transport of USPIO particles to the lymphatic system so delayed imaging at 24-36 h after contrast agents injection is necessary 89. In addition, low sensitivity of MRI requires relatively large amount of imaging contrast agents 90. Unpredictability of iron-induced susceptibility artifacts, and the heterogeneous enhancement profile in normal lymph nodes also increase the difficulty of image interpretation 91.

MR lymphangiography in mice and monkeys has also been performed with T1 contrast agents. Herborn et al. 92 used a blood-pool contrast agent, MS-325 (Gadofosveset) to image regional lymph nodes. MS-325 is albumin-binding Gd-based contrast agent and the protein-binding properties may make this agent large enough to be phagocytosed and lymphotropically cleared. After interstitial injection, lymphatic vessels and tumor-bearing lymph nodes can be detected. The same contrast agent has also been premixed with 10% human serum albumin (HSA) for intradermal injection. Lymphatic drainage was visualized clearly by T1-weighted MRI 93. Kobayashi et al. used different dendrimer-based MRI contrast agents to visualize the anatomy and physiology of deep lymphatic vessels and lymph nodes in mouse models 94-96.

Optical imaging guided surgery has been intensively studied due to its low cost, simplicity, and adaptability. Besides, the limited tissue penetration is less critical because of open field of view during surgery 97-101. For example, NIR fluorescent dyes, such as indocyanine green (ICG), have been investigated for sentinel node navigation during surgery either alone or in combination with nanoformulations 26, 27, 102, 103. New imaging systems which integrate invisible light and color video have also been developed to provide intraoperative guidance using NIR lymphatic mapping agents such as indocyanine green (ICG) diluted in human serum albumin (HSA). The NIR fluorescence detection of SLNs was very promising in a small scale of patients with breast cancer 104. Besides small molecular dyes, various nano-scale sized fluorophores have also been applied for SLN imaging and showed promising results in preclinical models 105-109. Kim et al. 106 demonstrated that injection of only 400 pmol of near-infrared quantum dots (a hydrodynamic diameter of 15-20 nm and emission at 840-860 nm) permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using very low excitation fluence rates (5 mW/cm2). With the combination of NIR dyes and microscopic techniques, in vivo functional lymphatic imaging with high spatial and temporal resolution can be achieved 110.

Contrast-enhanced ultrasound imaging (CEUS) using mcirobubbles has been widely used in both preclinical experiments and clinical diagnosis 108, 111-118. In preclinical studies, microbubbles have been shown to accumulate in sentinel lymph nodes but not second-order lymph nodes, probably due to the avidity of the shell material for macrophages 119, 120. In a pilot clinical trial, before surgery, patients with breast cancer received a periareolar intradermal injection of microbubbles, lymphatic channels were visualized immediately by ultrasonography and putative axillary SLNs were identified. The sensitivity of SLN detection in this study was 89% 121. Similar to MRI, differentiation of benign and malignant lymph nodes can be achieved with CEUS because of the different accumulation of microbubbles in normal and metastasized LNs 122. Several limitations of CEUS prevent broad application of this technique in SLN mapping, such as poor spatial resolution, slow migration of the microbubble, inaccessibility to the thorax and deep retroperitoneum, as well as its dependence on operator experience 123. Photoacoustic imaging (PAI) is a hybrid biomedical imaging modality to detect the ultrasonic waves generated by pulse laser induced transient thermoelastic expansion within biological tissues 124-127. In combination with different contrast agents including methylene blue, carbon nanotubes, gold nanocages, gold nanorods and gold nanobeacons 128-131, PAI showed potential in improved detection of metastases in preclinical models. However, no clinical application has been reported so far, possibly due to the lack of bedside imaging system. In addition, the still limited signal penetration and challenges in control of surgery field with conductive gel may also be the hindrance.

With regard to lymphatic imaging, in order to meet the requirement for both pre-operational evaluation and intra-operational guidance, the combination of multiple imaging techniques is often needed 132. Like the conventional lymphatic mapping with 99mTc-labeled colloids/blue dye, SLNs can be visualized pre-operationally either by SPECT and located with a gamma ray counter and visual contrast of the blue dye during surgery 69, 70. With the development and maturation of hybrid systems including PET/CT, SPECT/CT 34, PET/MRI 133, and bed side optical imaging systems 98, 104, various multifunctional lymphatic imaging probes have been investigated to offer the synergistic advantages, especially those combining radionuclide and fluorescence 134. By taking advantage of lymphatic binding property of tilmanocept, Tsien group 135 conjugated an 18F-labeled NIR fluorophore to the dextran backbone. This dual-labeled compound permits PET or scintigraphic imaging of SLN, and enables NIRF-guided excision for multimodality-guided sentinel node visualization and excision (Figure ). By mixing 18F-AlF-NEB with Evans blue, our lab also investigated multimodal imaging of LNs. In several animal models, the LNs can be distinguished clearly by the apparent blue color and strong fluorescence signal from EB as well as high intensity PET signal from 18F-AlF-NEB 136. Kobayashi and co-workers 132 synthesized 111In-labeled radionuclide/five-color NIR optical dual-modal imaging probes using a polyamidoamine dendrimer (generation-6 PAMAM dendrimer) with an ethylenediamine core as the platform component. Radionuclide imaging of this dual-modal imaging probe allows increased depth penetration and absolute quantification whereas multi-color NIR optical imaging offers real time spatial resolution and the ability to distinguish multiple lymphatic drainages 132.

In a relatively short period, PET/MRI system achieved transition from small PET/MRI prototypes for small-animal studies 137, 138 to clinical arena 139. Consequently, multi-modality imaging agents have been investigated for lymphatic mapping with the hope to detect sites of disease with higher sensitivity and accuracy. For example, a multimodal nanoparticle, 89Zr-ferumoxytol, has been tested in preclinical disease models and the results demonstrated that the particles can be used for high-resolution tomographic studies of lymphatic drainage 140. Our group also developed a mesoporous silica-based triple-modal imaging nanoprobe (MSN-probe) that possesses the long-term imaging ability to track tumor metastatic SLNs. In this system, three imaging tags including NIR dye ZW800, T1 contrast agent Gd3+ and positron emitting radionuclide 64Cu were integrated into MSNs by different conjugation strategies. Due to their high stability and long intracellular retention time, signals from tumor draining SLNs are detectable up to 3 weeks (Figure ) 105.

Summary and perspectives

An ideal lymphatic imaging agent should have high signal-to-background ratio for clear SLN detection, be able to differentiate tumor metastasis and provide real-time intraoperative guidance. However, it is very challenging to fulfill all the requirements with a single imaging agent and a single imaging modality. For preclinical studies, the emphasis of imaging is on how to evaluate lymphangiogenesis during pathological processes, especially during the development and metastasis of malignant lesions. Clinically, the focus is still on intraoperative detection of SLN for biopsy and accurate pathological evaluation. This application is mainly for patients with breast cancer and melanoma, but shows potential in other types of cancer including prostate cancer and head and neck cancers. The combination of 99mTc-colloid and vital dyes is still the main stream in clinical practice while other combinations are emerging. With the prevalence of PET and availability of clinically applicable optical imaging systems, more probes with positron emitter and fluorophore labeling are under intensive investigation to provide better pre-operational imaging and intra-operative guidance for SLNB 134.

Various nanoparticles have been investigated for lymphatic imaging, as they have some preferred features including strong signal intensity, tunable size, and modularized modification for multiple modality imaging. For example, based on experience from 99mTc-labeled colloid, the optimal particle size is from 50 to 200 nm since the radioactive colloids are cleared by lymphatic drainage with a speed that is inversely proportional to the particle size after interstitial injection 69. The size of most nanoparticles can be easily tuned to fall in this range. Despite the fact that clinical translation of many nanoparticle formula face formidable obstacles, perceived acute and chronic toxicities, and regulatory hurdles 141, local administration of lymph node mapping will overcome the suboptimal biocompatibility of these NPs.

It is envisioned that PET/optical dual functional imaging probes may be the best combination for clinical SLNB. Pre-operational evaluation could be performed with PET/CT or PET/MRI hybrid systems to provide both LN location and surrounding anatomical reference. While fluorescence optical imaging provides direct visualization of the SLNs and the field of view can be overlaid with bright field images. The localized radioactive signal can substantiate the accuracy of optical imaging. Ex vivo microscopic imaging can be added to provide fast evaluation of tumor micrometastasis in the resected LNs.