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Sentinel Lymph Node Imaging Using Quantum Dots in Mouse Tumor Models Byron Ballou,*,†,‡ Lauren A. Ernst,† Susan Andreko,† Theresa Harper,| James A. J. Fitzpatrick,†,§ Alan S. Waggoner,†,‡ and Marcel P. Bruchez†,§,⊥ Molecular Biosensor and Imaging Center, Department of Biological Sciences, and Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, and Aviir, 2463 Faber Place, Palo Alto, California 94303 . Received August 22, 2006; Revised Manuscript Received November 21, 2006
We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined. Imaging during necropsy confirmed confinement of the quantum dots to the lymphatic system and demonstrated easy tagging of sentinel lymph nodes for pathology. Examination of the sentinel nodes identified by quantum dot localization showed that at least some contained metastatic tumor foci.
INTRODUCTION
Table 1. Quantum Dot Surfaces Used in this Report
Mapping the reticuloendothelial system (RES) and locating draining lymph nodes were among the first uses of quantum dots in ViVo (1-3). Fluorescence imaging should allow a simple optical readout, as well as higher resolution and a wider dynamic range than dye absorption or scintigraphy. For exposed tissues, the localization capability and sensitivity are potentially better than given by either visible dyes or radioactives, especially when using near-infrared emission, because of good tissue penetration and lowered background in the infrared (3-5). Frangioni and colleagues (3, 6-9) as well as others (1016) have published studies showing mapping of draining lymph nodes by injection of fluorophores or fluorescently labeled macromolecules and particulates into various normal tissues. However, to our knowledge this is the first study that uses quantum dots injected into tumors to map sentinel nodes. In this paper, we also investigate whether changing the surface of PEG-conjugated quantum dots influences migration to sentinel nodes. We would expect binding and retention in lymph nodes to be influenced by surface charge, but simple drainage from the tumor volume would not.
MATERIALS AND METHODS Quantum Dots were provided by Quantum Dot Corporation (now a division of Invitrogen). For the experiments reported here, we used 655 nm emitting ZnS-CdSe and 800 nm emitting ZnS-CdSe-CdTe core-shell quantum dots having a primary amphiphilic polymer coating (amp (17)), which yields highly fluorescent quantum dots with a high density of carboxyl groups on the surface. These amp-coated QDs were further substituted using poly(ethylene glycol) polymers having terminal methoxy, * Corresponding author. Byron Ballou, MBIC, CMU, 4400 Fifth Avenue, Pittsburgh, PA 15213, e-mail
[email protected]. Tel: 412-268-4779. Fax: 412-268-6571. † Molecular Biosensor and Imaging Center, Carnegie Mellon University. ‡ Department of Biological Sciences, Carnegie Mellon University. § Department of Chemistry, Carnegie Mellon University. | Aviir. ⊥ T.H. and M.P.B. were at Quantum Dot Corporation (now a part of Invitrogen, 29851 Willow Creek Road, Eugene OR 97402) at the time this research was done.
*Measured in 0.01 M sodium borate buffer, pH 8.5. It was not possible to perform fluorescence correlation spectroscopy on the 800 nm quantum dots using our apparatus, which uses an 800 nm emitting laser for twophoton excitation. We expect the hydrodynamic diameters to be roughly similar to those of the 655 nm quantum dots.
carboxy, or amino termini (2) (see Table 1). Thus, PEGconjugated quantum dots having terminal neutral, positively charged, or negatively charged PEG surfaces could be compared as to their ability to access sentinel nodes after injection into a tumor. Properties of the quantum dots are summarized in Table 1. Quantum yields were determined as described (2), using Rhodamine 101 as a standard, and with excitation at 530 nm. Hydrodynamic diameters were determined in 0.01 M sodium borate buffer, pH 8.5, using two-photon fluorescence correlation spectroscopy (18, 19). To minimize aggregation, all quantum dots were kept in 0.01 M sodiuim borate, pH 8.5. Just before use, the quantum dots were filtered through a 0.2 µm filter and made up to normal tonicity by addition of 1/9 volume of 1.5 M NaCl, then adjusted to the desired volume by adding Dulbecco’s phosphate-buffered saline (PBS).
10.1021/bc060261j CCC: $37.00 © 2007 American Chemical Society Published on Web 01/31/2007
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Figure 1. Nude mouse bearing M21 melanoma, dorsal view 3 min after injection into the tumor using 655 nm PEG 5k-COOH quantum dots. Left, visible light; right, fluorescence at 655 nm. Migration toward the inguinal node is already apparent.
Figure 2. Same mouse as Figure 1, dissected at 40 min postinjection; ventral view. (A) Visible light. (B) Fluorescence at 655 nm. (C) Same as B, but with tumor masked. (D) Same as B, log-transformed image. Both C and D allow better display of the less bright lymphatics and smaller lymph nodes. Note drainage from tumor into lymphatics, accumulation in right inguinal, axillary, lumbar, and renal lymph nodes, but no fluorescence in the general circulation or in lymph nodes on the left side of the body. Arrows in C and D as in B; captions are omitted for clarity. We use the term “axillary” to describe lymph nodes often termed brachial (or “proper axillary”) nodes as well as axillary (or “accessory axillary”) nodes; in our preparations, these nodes are often not well separated (compare ref 48). Table 2. Quantum Dot Combinations Used in This Reporta
a
study
Qdot 1 surface
Qdot 1 emission
Qdot 2 surface
Qdot 2 emission
1 2 3 4
PEG 5k-OMe PEG 6k-NH2 PEG 5k-OMe PEG 5k-COOH
800 nm 655 nm 800 nm 655 nm
PEG 6k-NH2 PEG 5k-OMe PEG 5k-COOH PEG 5k-OMe
655 nm 800 nm 655 nm 800 nm
Three mice were injected using each mixture.
Animals. Athymic nude mice were obtained from Harlan Sprague Dawley and were used at ages from 6 weeks to 3 months. All operations involving animals were performed in accord with institutional animal use and care regulations. Tumors. Two tumor models were used, the M21 human melanoma (20, 21) and the MH-15 mouse teratocarcinoma (22). For each tumor model, the tumor was grown subcutaneously in the right thigh; this location drains to the inguinal lymph node. Because the inguinal node is large and superficial, a rapid, reproducible assessment of quantum dot migration by noninvasive imaging was possible. After tumor growth to a suitable size (usually 2-4 mm diameter, 1-2 mm thick), mice were
anesthetized using pentobarbital ip to effect (typically 62.5 mg/ kg). Quantum dots were injected into tumors using a volume of 5-25 µL of a quantum dot preparation containing 25100 pmol of quantum dots. The volume was adjusted to avoid noticeably inflating the tumors. In all cases, we attempted to inject directly into tumors and not into surrounding tissue. In two cases, we removed the skin over the tumor, then injected the visible tumor; no significant leakage was observed. Migration of fluorescence to the lymph nodes was identical to that reported in this paper (data not shown). We cannot exclude the possibility that a small amount of material may have leaked into subcutaneous space in some instances, but any significant
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Figure 3. Mouse bearing M21 melanoma, injected sequentially in the tail vein first using 655 nm PEG 5k-COOH quantum dots, followed 20 min later by injection into the tumor using 800 nm PEG 5k-OMe quantum dots. Necropsy 90 min after tail vein injection. (A) Visible light image. (B) General labeling by IV-injected quantum dots, 655 nm window. Bone marrow, lymph nodes, and liver are prominent in the 655 window.
amount would have been detected by imaging immediately after injection. Tail vein injection was as described (2). Quantum dot doses given by tail vein were typically 300 pmol in 200 µL of normal saline. Imaging. Tumors were imaged using an Andor DU 434-BRDD cooled CCD camera (Andor Technology, South Windsor, CT 06074) equipped with a 50 mm AF Nikkor lens and any of several interference filters fitted between the lens and the CCD (filters were supplied by Chroma Technologies, Brattleboro, VT 05301). Mice were illuminated using four 450 nm emitting 5 W LEDs (Luxeon Royal Blue LXHL-MRRC, Lumileds Lighting, San Jose, CA 95131); 655 nm emitting quantum dots were imaged using a 654 nm × 24 nm interference filter. 800 nm emitting quantum dots were normally imaged using an 820 nm × 40 nm interference filter. Where the two different emitters were used together, to eliminate any residual spillover from 655 nm emitting quantum dots, 800 nm emitters were illuminated using a 250 W fiberoptic equipped halogen illuminator (Cuda Products, Jacksonville, FL 32217) filtered through a 750 nm × 30 nm interference filter, and emission was observed using an 840 nm × 20 nm filter. This resulted in much less observed emission from the 800 nm quantum dots and a higher background, but eliminated completely a small spillover (∼2%) from the 655 nm emitting quantum dots seen using the 450 nm LED illuminators and the 820 nm × 40 nm interference filter. There was no spillover from 800 nm quantum dots into the 655 nm channel. A difficulty in displaying lymph nodes in the same image as the tumor is caused by the much greater brightness of the tumor. Either a logarithmic transform of the image or masking the tumor permits the display of small lymph nodes or faint lymphatics in the same image as the tumor. Where a logtransformed image is used, we display the original and transformed images together. To demonstrate that transfer of quantum dots from tumors occurred through the lymphatics, lymph nodes were labeled generally by injecting animals in the tail vein, followed by injection in the tumor using a different wavelength quantum dot. This allowed us to assess whether any trapping of quantum dots had occurred by leakage from the tumor into the vascular compartment, followed by uptake into lymph nodes. In only 2 cases of a total of 27 mice examined, including the 12 mice reported in this paper, was there any indication of such leakage. Both cases involved large tumors having significant necrotic areas.
To compare the effects of surface charge on migration to sentinel nodes, a mixture of quantum dots having a methoxyPEG surface and either a carboxy-PEG or amino-PEG surface was injected, and images were made in two different spectral windows corresponding to the neutral or charged quantum dots; the reciprocal combination was also injected. The tested combinations of emission wavelengths and surfaces are summarized in Table 2. For the results given in this paper, three mice were labeled using each combination. Microscopy. Lymph nodes were removed at necropsy and subjected to normal paraffin embedding and staining procedures. Examination by a veterinary pathologist confirmed the presence or absence of tumor in lymph nodes. Where necessary, paraffin sections were stained using tumor-binding monoclonal antibodies: anti-SSEA-1 for the MH-15 teratocarcinoma (22) or 9.2.27 for the M21 melanoma (20).
RESULTS In most cases, injection of quantum dots into the tumor yielded rapid transfer of fluorescence into adjacent lymph nodes, visible through the skin. Figure 1 shows that movement out of the tumor to adjacent lymphatics is rapid. Drainage toward the inguinal node can be seen through the skin almost immediately after injection. Figure 2 shows the same animal as in Figure 1, necropsied 40 min after injection. Drainage through the lymphatics to the right inguinal and axillary lymph nodes is clearly apparent. Migration of quantum dots to the right lumbar and renal nodes is also detectable. There is no labeling of the lymph nodes on the left side of the mouse. Note that Figure 1 shows a dorsal view, while all the other figures show ventral views. The tumors were always on the dorsal side of the thigh, so the tumor in the ventral images is partly obscured by the mouse thigh. Figures 3 and 4 show that drainage to adjacent lymph nodes is not caused by any difference in ability to trap quantum dots between nodes adjacent to the tumor and more distant lymph nodes. All lymph nodes were labeled by the 655 nm PEG 5kCOOH quantum dots that were tail-vein injected; only nodes close to the tumor were labeled by the 800 nm PEG 5k-OMe quantum dots injected into the tumor. (Carboxy-terminal quantum dots were used for general labeling to ensure rapid uptake by lymph nodes (1, 2).) To determine the effect of terminal charged groups on migration from tumors to lymph nodes, neutral PEG 5k-OMe
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Figure 4. Same mouse as Figure 3, visualized in 655 nm window (left; A,C,E; tail-vein injection, 655 nm PEG 5k-COOH) and 800 nm window (right; B,D,F; intratumoral injection, 800 nm PEG 5k-OMe). Internal organs are removed to display all lymph nodes. (A,B) Superficial skin removed, but peritoneum intact. (A) Fluorescence of peripheral lymph nodes and liver. (B) Unilateral fluorescence of lymph nodes adjacent to tumor, no staining of liver. (C,D) Same as A,B, but with tumor masked. The lymphatic drainage from the tumor is more easily seen in D. (E,F) Most internal organs removed. Bilateral staining of lumbar and renal nodes visible in E, but only unilateral staining is visible in F.
quantum dots were injected together with either PEG 5k-COOH or PEG 6k-NH2 quantum dots (Table 2; coprecipitation rules out injecting amino- and carboxy-PEG mixtures). Figures 5
and 6 (reciprocal combination) show that migration to sentinel nodes was similar when mixed methoxy- and carboxyterminated PEG substituents were used. Results were also similar
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Figure 5. Quantum dots having either carboxy or methoxy surfaces migrate similarly. A mixture of 655 nm PEG 5k-OMe and 800 nm PEG 5k-COOH was injected into the tumor. Necropsy at 40 min after injection. (A) 655 nm window, tumor visible. (B) 800 nm window, tumor visible. (C) 655 nm window, tumor masked. (D) 800 nm window, tumor masked. Visibility of tumor through the leg is better using 800 nm quantum dots, but signal is lower and background is higher in B,D due to long-wavelength illumination (750 nm); however, no spillover from 655 nm quantum dots is possible. (E-G) Lymph node from mouse in Figure 5. The node was removed, fixed in buffered formalin, paraffin-embedded, and then stained using conventional hematoxylin and eosin. (E) Bright-field color image; note germinal centers (asterisks). (F) 655 nm fluorescence image (PEG 5k-OMe quantum dots). (G) 800 nm fluorescence image (PEG 5k-COOH quantum dots.) Fluorescence is confined to lymphatic vessels and sinuses.
Figure 6. A mixture of 655 nm PEG 5k-COOH and 800 nm PEG 5k-OMe quantum dots was used, reciprocal to that of Figure 5. Necropsy and imaging were similar to those of Figure 5. (A) 655 nm window, tumor visible. (B) 800 nm window, tumor visible. (C) 655 nm window, tumor masked. (D) 800 nm window, tumor masked.
when injections of mixed amino- and methoxy-PEG-terminated quantum dots were used (Figure 7). Reciprocal combinations
of emission wavelength and surface (Table 2) all gave similar results, so the nature of the core-shell material was unimportant.
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Figure 7. A mixture of 655 nm PEG 6k-NH2 and 800 nm PEG 5k-OMe quantum dots was used. Necropsy 40 min after intratumoral injection into M21 melanoma. (A) 655 nm window, tumor visible. (B) 800 nm window, tumor visible. (C) 655 nm window, tumor masked. (D) 800 nm window, tumor masked. Quantum dots having either surface migrate similarly to adjacent inguinal node (arrows). T, tumor.
Figure 8. Nude mouse having large (∼0.5 g) MH-15 teratocarcinoma, main growth dorsal to the right thigh, injected using 655 nm PEG 5k-OMe quantum dots. Ventral view. Left, visible light; right, 655 nm fluorescence. The thigh muscle has been infiltrated by the tumor, and there are local metastases to the adjacent tissue, including the right inguinal lymph node. Image taken 24 h postinjection.
Figure 8 shows the result of injecting a large MH-15 teratocarcinoma with neutral PEG-surfaced quantum dots. In this case, only the adjacent inguinal lymph node showed uptake of the quantum dots.
DISCUSSION We have demonstrated that quantum dots define sentinel lymph nodes in mice after injection into tumors. Our results indicate that, for tumors in mice, terminal charged groups on PEG-conjugated quantum dots have little or no effect on drainage to surrounding lymph nodes. These results were surprising, since we expected that, as with whole-body imaging after intravenous injection, charged surfaces on quantum dots would increase lymph node uptake (1, 2). Our results also show that quantum dots having significantly different hydrodynamic diameters migrate similarly to lymph nodes. We would expect significant binding of intersitital tissue fluid components to the quantum dots, including enhanced binding to charged surfaces as compared to neutral PEG; this
makes the results we observed even more remarkable and reinforces our belief that we are following mostly fluid flow from the tumors. On the time scale of these experiments, it is unlikely that any significant selective uptake by the lymph node occurs. Figure 5 shows that quantum dots in sentinel lymph nodes are found only on vessel boundaries and not in monocytes surrounding the germinal centers. For visualization of superficial lymph nodes and lymphatics, or for parts of the lymphatic system exposed during necropsy, there was little difference in visibility between 655 nm emitting and 800 nm emitting quantum dots. For imaging deeper nodes or visualizing tumors through the thigh, we found 800 nm emitting quantum dots to be superior, as expected (1, 23). Frangioni and colleagues proposed the use of NIR-emitting quantum dots for sentinel lymph node mapping and demonstrated migration of quantum dots to lymph nodes when injected into normal tissues in mice. They also demonstrated that quantum dots can be used for lymph node mapping in a large animal model (pig) at centimeter depths (3). In subsequent
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papers, this group has shown quantum dot fluorescence imaging of lymph nodes draining the lungs (6, 9) and esophagus (8). Recently, Zimmer et al. (24) demonstrated that sufficiently small InAs-ZnSe quantum dots, coated using PEG-DHLA, localized in sentinel lymph nodes when injected into normal tissues. A sequence of draining lymph nodes could be labeled using these quantum dots; the authors attributed passage through the first draining lymph node to the small size of their quantum dots (>10 nm hydrodynamic diameter). In our hands, a sequence of lymph nodes was labeled using much larger, polymer-coated quantum dots; however, the bypass route through mouse lymph nodes (25) likely accounts for our results. Other fluorophores used for sentinel lymph node imaging include free indocyanine green (11, 26), indocyanine green adsorbed to albumin (26), free IRDye800CW (26), albumin conjugated to IRDye78 (7) or IRDye800CW (8, 26), albumin colloid conjugated to IRDye800CW (26), fluorescent polystyrene microspheres (14, 15), and two targeted conjugates, Cy5vitamin B12 (“Cobalofluor”) (13) and MECA-78, a cyanineconjugated IgM antibody (11). Finally, enzyme-specific dequenching of a Cy5.5 conjugated polymer has been used to define lymph nodes (16, 27). Frangioni and colleagues directly compared quantum dots to human serum albumin conjugates (8, 26) and to isosulfan blue (9). Both quantum dots and organic fluorochromes imaged sentinel lymph nodes successfully; the quantum dots, probably because of their relatively large sizes (10-30 nm), normally targeted only the first draining lymph node, while organic dyes and smaller conjugates continued further to more distant lymph nodes (26) (but see ref 3.) As compared to other methods of sentinel node location, fluorescence imaging offers improved sensitivity and greater speed, at least for lymph nodes that are close enough to the surface to be imaged or are revealed during surgery. Combining quantum dot fluorescence imaging with other noninvasive techniques (e.g., scintigraphy, MRI, or PET) would allow mapping of deep nodes; after exposure during surgery, fluorescence would then give more precise, detailed localization, potentially including microscopic levels (3, 28). Alternatively, fluorescence endoscopic examination could be used during minimally invasive surgery (8, 13). The equipment required for near-infrared imaging is relatively cheap and readily available, and a color video-NIR combined system has been described (9, 29). Easy visualization of transport through lymphatics and accumulation in lymph nodes should be helpful for understanding the pattern of flow in the vicinity of tumors. The ability to use multiple fluorescent emission colors may be useful in tumor diagnosis; ideally, sentinel nodes might be labeled using nonspecific quantum dots, while quantum dots conjugated to appropriate ligands and fluorescing at a different wavelength might be used to localize metastases in lymph nodes. The advantages of quantum dots remain: very high brightness, wide range of fluorescence emission wavelengths, excitation at any wavelength below the emission wavelength, and extraordinary photostability. No organic fluorophore matches this set of advantages. Moreover, the long fluorescence lifetimes of quantum dots potentially allow nearly zero background imaging by time-gating (30). The difficulties remain also: relatively large size caused by the coats required for stability in aqueous solution; and potential toxicity, due either to heavy metal toxicity from dissolution of the quantum dots or to the size and surface characteristics of the nanoparticles. Several recent articles have discussed both actual and potential toxicity of quantum dots (31-39). Both major concerns are being addressed; new quantum dot compositions and coats should yield nanoparticles that are both smaller and less toxic (24, 4047). Molecules or particulates multiply substituted with organic
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dyes are good alternatives that have been demonstrated to function well for sentinel node location (7, 8, 26); however, the NIR organic fluorophores themselves may also be toxic. In our hands, the toxicity of amp-coated CdSe-ZnS quantum dots is minimal or nonexistant, as assessed by pathological examination of animals injected with quantum dots and kept for long periods (now over 2 years.) Even after this time, at least some of the quantum dots retain their fluorescence, suggesting that they remain intact ((1) and in preparation). We therefore believe that the clinical use of existing quantum dots, suitably purified and at low doses relevant to sentinel lymph node detection, would result in minimal toxicity, though less toxic compositions and surfaces will be welcome.
ACKNOWLEDGMENT The authors are grateful for assistance with quantum dot manufacture by Joseph Treadway (present address Invitrogen, 29851 Willow Creek Road, Eugene OR 97402) and to B. Christoffer Lagerholm (present address Memphys, Physics Department, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark) for assistance in infrared imaging. We are grateful to Newell R. Washburn (Department of Chemistry, Carnegie Mellon University) for use of the FCS apparatus. We also thank Lisa McGaw (Department of Biological Sciences, Carnegie Mellon University) for assistance with animal handling. This work was supported by NIH grant no. R01 EB 000364. The work at Quantum Dot Corporation was partly funded by NIST-ATP 70NANB0H3000.
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