Anda di halaman 1dari 6

Breast Cancer Res Treat (2007) 103:2328

DOI 10.1007/s10549-006-9347-0

PRECLINICAL STUDY

Simultaneous two-color spectral fluorescence lymphangiography


with near infrared quantum dots to map two lymphatic flows
from the breast and the upper extremity
Yukihiro Hama Yoshinori Koyama
Yasuteru Urano Peter L. Choyke
Hisataka Kobayashi

Received: 14 July 2006 / Accepted: 17 July 2006 / Published online: 21 September 2006
 Springer Science+Business Media B.V. 2006

Abstract Due to their small size and poor access, the Quantum dots Spectral fluorescence imaging
lymphatic function has been difficult to study in vivo. Near infrared
Especially difficult is the mapping of lymphatic drain-
age from two basins into the same node. Quantum dots
can be used to perform multicolor images with high Introduction
fluorescent intensity and are of a nano-size size suitable
for lymphatic imaging via direct interstitial injection. The lymphatic system is difficult to evaluate because its
Here we show simultaneous two-color in vivo wave- channels are small and not directly accessible. More-
length-resolved spectral fluorescence lymphangiogra- over, when assessing lymphatic drainage from two
phy using two near infrared quantum dots with separate drainage basins, techniques such as X-ray
different emission spectra, which allow non-invasive lymphangiography, MR lymphangiography or radio-
and simultaneous visualization of two separate lym- nuclide radioscintigraphy are inadequate because it is
phatic flows draining the breast and the upper impossible to differentiate the contributions from each
extremity and variations in the drainage patterns and lymphatic basin. Quantum dots (Qdots) are charac-
the water sheds within the axillary node. Two-color terized by sharply defined emission spectra and can be
spectral fluorescence lymphangiography can provide synthesized to emit a variety of wavelengths including
insight into mechanisms of drainage from different in the near infrared (NIR) [1]. Qdots can vary in size
lymphatic basins that may lead to sentinel lymph nodes but some are appropriately sized to conduct lymphatic
detection of the breast cancer as well as prevention of studies based on prior studies using macromolecular
complications such as lymphedema of the arm. dendrimer particles on MRI, which demonstrate opti-
mal lymphatic uptake for particles 912 nm in diame-
Keywords Lymphatic drainage Imaging Breast ter [2, 3]. Kim et al. [4] reported remarkable in vivo
cancer Lymph node Lymphedema Nanotechnology imaging of the lymphatics using a NIR Qdot in order to
detect the sentinel lymph node (SLN) arising from
Yukihiro Hama and Yoshinori Koyama are contributed
breast tissue. However, few in vivo multicolor imaging
equally to this work. studies for visualizing two separate physiological
functions have been reported [5], although the multi-
Y. Hama Y. Koyama P. L. Choyke H. Kobayashi (&) color imaging with two Qdots has been introduced in in
Molecular Imaging Program, Center for Cancer for Cancer
vitro microscopic imaging as a possible application of
Research, National Cancer Institute, NIH, Bldg. 10, Room
1B40, MSC 1088, Bethesda, MD 20892-1088, USA nanotechnology to the bio-imaging. Therefore, we
e-mail: Kobayash@mail.nih.gov chose two NIR Cadmium-Tellurium (CdTe) Qdots
with different emission spectra (705 and 800 nm peak
Y. Urano
emission), based on their appropriate physical nano-
Graduate School of Pharmaceutical Sciences,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, size for lymphatic imaging compared with other kinds
Tokyo 113-0033, Japan of Qdots such as Cadmium Selenium (CdSe) series. We

123
24 Breast Cancer Res Treat (2007) 103:2328

simultaneously injected the two Qdots, into the mam- images captured at each wavelength. Collected images
mary gland and the skin of the middle phalange in the were analyzed by the Maestro software, which uses
upper extremity in order to monitor the lymphatic spectral unmixing algorithms to separate autofluores-
drainage functions from these two basins, which both cence from quantum dot signals, and a composite im-
drain into the axillary lymph nodes. age consisting of Qdot 705 ITKTM and Qdot 800
ITKTM signals and autofluorescence was generated.
Unmixed composite images of in vivo lymphangiog-
Materials and methods raphy as well as ex vivo LN images for both Qdot 705
ITKTM and Qdot 800 ITKTM were compared inde-
Chemicals pendently by two reviewers (Y.K. and H.K.). The
qualitative visibility of the axillary LNs on lymphan-
Carboxyl quantum dots, Qdot 705 ITKTM (peak giograms obtained with each fluorescence signal (705
emission wavelength at 705 nm) and Qdot 800 or 800 nm) was rated on a 5-point scale (, , +, ++,
ITKTM (peak emission wavelength at 800 nm), which and +++). Any discrepancy between the two reviewers
had CdTe core with an additional thin semiconductor was resolved by discussion.
shell (zinc sulfide) of ~6 and ~12 nm in diameter,
respectively, coated by thin polymer (12 nm) con-
taining carboxyl groups, were purchased from Invitro- Two-color spectral fluorescence microscopic
gen Corporation (Carlsbad, CA, USA). A mixture of imaging of the lymph node section
800 nmol/l Qdot 705 ITKTM or Qdot 800 ITKTM
was prepared in PBS. The mice were killed with intravenous injection of
sodium pentobarbital 1 h after Qdots injection. The
axillary, lateral thoracic and cervical LNs were dis-
In vivo wavelength-resolved two-color spectral sected and removed and frozen in Tissutek (Sakura,
fluorescence imaging Torrance, CA, USA) and stored at 80C. Frozen
samples were sectioned by a cryostat microtome
All in vivo procedures were carried out in compliance (CM1800, Leica, Bannockburn, IL, USA) at a thick-
with the Guide for the Care and Use of Laboratory ness of 30 lm. Slides were analyzed under an Olympus
Animal Resources (1996), National Research Council, BX61 microscope (Olympus America Inc., Melville,
and approved by the National Cancer Institute Animal NY, USA) equipped with the following filters: Cy5
Care and Use Committee. Ten-week-old normal fe- cube set for Qdot 705 ITKTM; excitation wavelength
male athymic mice were anesthetized with intraperi- 590650 nm, emission wavelength 665730 nm band
toneal injection of 1.15 mg sodium pentobarbital pass, Cy7 cube set for Qdot 800 ITKTM; excitation
(Dainabot, Osaka, Japan). Then, ten consecutive mice wavelength 675745 nm, emission wavelength 770
were given intracutaneous injections of 10 ll (8 pmol) 850 nm band pass.
of Qdot 800 ITKTM into the middle phalange of the
left upper extremity and subcutaneous injections of
20 ll (16 pmol) of Qdot 705 ITKTM into the left Results
breast. Another five consecutive mice were adminis-
tered the Qdots in the opposite order (i.e. intracuta- In vivo wavelength-resolved two-color spectral
neous injections of 10 ll (8 pmol) of Qdot 705 fluorescence lymphangiography of the axillary
ITKTM into the middle phalange of the left upper region visualized the territory of the lymphatic
extremity and subcutaneous injections of 20 ll (16 drainage from two different basins
pmol) of Qdot 800 ITKTM into the left breast).
Immediately after injection of the Qdots, wavelength- Two-color NIR lymphangiography following injections
resolved spectral imaging was carried out using a of Qdot 705 ITKTM in the breast and Qdot 800
spectral imaging system (Maestro In-Vivo Imaging ITKTM in the upper extremity successfully visualized
System, CRI Inc., Woburn, MA, USA). Animals were the lymphatic vessels as well as demonstrated the
placed in the prone and in the right lateral decubitus lymphatic drainage territory in all ten mice using
position under pentobarbital anesthesia. The excitation spectral fluorescence imaging (Fig. 1). In the two-color
band pass filter 575605 nm was used. The tunable wavelength-resolved spectral fluorescence lymphangi-
filter was automatically stepped in 10-nm increments ography, eight axillary lymph nodes (LNs) had mixed
from 600 to 950 nm with the same exposure time for contributions from both the breast Qdot (705 nm) and

123
Breast Cancer Res Treat (2007) 103:2328 25

tissues had increased lymphatic flow from the breasts


to the cervical LNs indicating that the principal
drainage pattern of the breast had been altered in these
animals (Fig. 3, Table 1). Ex vivo fluorescence imaging
and fluorescence microscopy depicting the mixture of
Qdots within resected LNs validated the lymphatic
drainage found in the in vivo lymphangiography in all
ten mice (Table 1).

Inappropriate use of quantum dots failed to show


two-color lymphangiography

When the Qdot 800 ITKTM was injected in the breast


and the Qdot 705 ITKTM was injected in the upper
extremity, the lymphatic drainage from the breast was
only faintly visualized on in vivo spectral NIR fluo-
rescence images. In these five animals only the Qdot
705 ITKTM was consistently visualized as the Qdot
800 ITKTM signal was too weak (Fig. 4). Visualization
score of the lymphatic drainage from the breast with
Qdot 800 ITKTM was significantly less than that with
Qdot 705 ITKTM (P < 0.005). The in vivo findings
corresponded with the ex vivo fluorescence imaging
and fluorescence microscopy of the resected LNs
(Table 1).

Discussion

Based on prior results with Gadolinium labeled


dendrimer-based nano particles, 910 nm in diameter
Fig. 1 A schematic illustration of two-color NIR fluorescence is an ideal diameter for uptake by the lymphatic system
imaging with a graph of emission spectra of two NIR quantum
dots (Qdot705; red, and Qdot800; green) visualizing the bidirec- from either the mammary gland or the subcutaneous
tional axillary lymphatic flows from the mammary pad (Qdot705; space for identifying a sentinel LN, however a broad
red) and the upper extremity (Qdot800; green). The axillary, range of diameters (530 nm) can still be used [6, 7].
lateral thoracic and superficial neck lymph nodes were the Interestingly, for visualizing the lymphatics of the up-
draining lymph nodes visualized in this experiment
per extremity by intradermal injection, a larger parti-
cle, of approximately 13 nm or larger in diameter was
the upper extremity Qdot (800 nm) (Fig. 2a). These optimal for visualizing the lymphatic flow [2]. There-
findings were clearly depicted in images at the surgery fore, in this experiment we matched the Qdot size to
(Fig. 2b) and validated by the fluorescence microscopic the lymphatic basin. The smaller Carboxyl Qdot705
images of frozen LN sections (Fig. 2c). Among these has a core diameter of ~6 nm and a thin coating of
eight animals, however, there were variable contribu- 12 nm making it ideal for imaging the lymphatic
tions from each basin indicating different flow rates in drainage from the mammary gland, while the larger
each animal, a phenomenon that was illustrated by the Carboxyl Qdot800 has a core diameter of ~12 nm and
varying proportion of the two Qdots in the resected the same thin coating making it ideal for imaging the
axillary node (Table 1, Fig. 2a). Two axillary LNs re- lymphatic drainage from the upper extremity. With this
ceived only one color (800 nm) of Qdot indicating that selection of Qdots, we were able to image the respec-
the lymphatic flow was exclusively arising from the tive lymphatic drainage basins from both the mammary
upper extremity (Fig. 3). Interestingly, the mice whose gland and the upper extremity and determine the
axillary lymph nodes received lymphatic flow exclu- trafficking of lymphatic flow in the axillary node in all
sively from the upper extremities and not the breast 10 mice. However, when the Qdots were reversed and

123
26 Breast Cancer Res Treat (2007) 103:2328

the larger Qdot800 was injected in the mammary tis- The practical implications of this finding are that it
sue, the lymphatic drainage from the mammary gland is now possible to study the drainage patterns and
was barely visualized due to its larger size. In mice, in mixing of adjacent lymphatic basins in vivo using a
which the lymphatic flows from the mammary gland high resolution imaging in a laboratory environment.
were faintly visualized as shown in Fig. 4, fluorescence There are a number of nodes in the lymphatic system
signal of Qdot800 was too weak compared with that of that receive afferent lymphatic vessels from more
Qdot705 to display both colors on a single display. than one body region especially in human yet there is
variability in these drainage patterns that is poorly
understood. The immunologic consequences of these
drainage patterns have not been studied. Two-color
spectral fluorescence imaging could assist in the
understanding of lymphatic flow patterns. This may
also aid in the understanding of the development of
lymphedema following resection of lymph nodes such
as the axillary or inguinal nodes. Lymphedema is a
major complication of radical breast cancer and mel-
anoma surgery [811] and a possible complication for
the SLN biopsy [914]. Since the procedure described
here is similar to that of lymphoscintigraphy without
radioactivity, it may be possible to visualize the ax-
illary LN both from the upper extremity and from the
breast cancer simultaneously and thereby predict the
likelihood of postoperative lymphedema and reduce
the morbidity associated with axillary lymph node
dissection by avoiding the disruption of lymphatic
flow from the upper extremity. However, to be clini-
cally relevant, Qdots composed of non-toxic materials
will need to be developed.
To our knowledge, this is the first demonstration of
simultaneous imaging of two different lymphatic
drainages in vivo and their trafficking to a LN. Since

Fig. 2 a In vivo and ex vivo spectral fluorescence imaging of a


m

mouse injected with Qdot800 (green) intracutaneously into the


middle digit of the left upper extremity and Qdot705 (red) into
the left mammary pad. The axillary lymph node received the two
different lymphatic flows simultaneously from the mammary pad
and the upper extremity. Lymph nodes as well as the lymphatic
vessels (arrows) were clearly visualized through the skin. b In
vivo spectral fluorescence imaging neck and axillary regions at
the surgery of the mouse shown in a. The lymphatic system
especially lymphatic draining ducts from both the breast (red)
and the extremity (green) were more clearly depicted after
removal of the skin and the fat tissue than before the surgery as
shown in a. c Differential interference contrast (DIC) and
fluorescence microscopy of extracted axillary lymph node in the
same mouse shown in a. The two-color fluorescence microscopy
more clearly and reliably demonstrated the intra-lymphatic
drainage territory as well as the water shed of the two different
lymphatic flows (interface between the green and red Qdots).
The fluorescence microscopy findings were compatible with the
in vivo optical lymphangiography in all mice. Eight of ten mice
examined showed similar findings to the mouse shown in figure
but in two mice drainage was exclusively from the upper
extremity (see Fig. 3). The filters used for excitation and
emission were 590650 and 665730 nm for Qdot705 and 675
745 and 770850 nm for Qdot800, respectively

123
Breast Cancer Res Treat (2007) 103:2328 27

Table 1 Summary of in vivo and ex vivo spectral fluorescence imaging of the axillary and the cervical LNs
Animal# Injected Qdot In vivo spectral Ex vivo spectral In vivo spectral
fluorescence imaging fluorescence imaging fluorescence imaging
of the axillary LN of the axillary LN of the cervical LN
Breast Hand 705 nm 800 nm 705 nm 800 nm 705 nm 800 nm

1 705 800 ++ +++ ++ +++ +++


2 705 800 +++ +++ +++ +++
3 705 800 +++ +++ +++
4 705 800 ++ ++ ++ +++ ++
5 705 800 +++ +++ +++ +++ +
6 705 800 +++ +++ +++ +++ ++
7 705 800 + +++ ++ +++ +++
8 705 800 +++ +++ ++
9 705 800 +++ ++ +++ ++ ++
10 705 800 ++ +++ ++ +++
11 800 705 +++ +++
12 800 705 +++ +++
13 800 705 +++ +++
14 800 705 +++ +++
15 800 705 +++ +++ +

Fig. 3 In vivo and ex vivo spectral fluorescence imaging of a Fig. 4 In vivo and ex vivo spectral fluorescence imaging of a
mouse injected with Qdot800 (green) intracutaneously into the mouse injected with Qdot705 (red) intracutaneously into the
middle digit of the left upper extremity and Qdot705 (red) into middle phalange of the left upper extremity and with Qdot800
the left mammary pad. The axillary lymph node received (green) into the left breast (opposite injection). The lymphatic
lymphatic flow exclusively from the upper extremity (green). drainage from the breast (green) was too weak to show up in two-
The lymphatic vessel from the upper extremity to the axillary color NIR in vivo imaging. All five mice examined showed the
lymph node (green arrow) and that from the breast to the similar finding as the mouse shown in figure
superficial neck lymph node (red arrow) were clearly shown

the two colors of each Qdot were related to their size, Acknowledgment This research was supported by the Intra-
it was possible to match the Qdot to its most appro- mural Research Program of the NIH, National Cancer Institute,
Center for Cancer Research.
priate lymphatic basin, based on diameter thus, opti-
mizing imaging for both lymphatic tracts. Qdots hold
promise as an imaging method for the exploration of References
the lymphatic system that may have implications for
1. Goldman ER, Clapp AR, Anderson GP, Uyeda HT, Mauro
the processing of the cancer metastasis and the im- JM, Medintz IL, Mattoussi H (2004) Multiplexed toxin
mune response to cancer cells or vaccines in the lymph analysis using four colors of quantum dot fluororeagents.
nodes. Anal Chem 76:684688

123
28 Breast Cancer Res Treat (2007) 103:2328

2. Kobayashi H, Kawamoto S, Choyke PL, Sato N, Knopp MV, 8. Morrell RM, Halyard MY, Schild SE, Ali MS, Gunderson
Star RA, Waldmann TA, Tagaya Y, Brechbiel MW (2003) LL, Pockaj BA (2005) Breast cancer-related lymphedema.
Comparison of dendrimer-based macromolecular contrast Mayo Clin Proc 80:14801484
agents for dynamic micro-magnetic resonance lymphangi- 9. Petrek JA, Senie RT, Peters M, Rosen PP (2001) Lymphe-
ography. Magn Reson Med 50:758766 dema in a cohort of breast carcinoma survivors 20 years after
3. Kobayashi H, Kawamoto S, Bernardo M, Brechbiel MW, diagnosis. Cancer 92:13681377
Knopp MV, Choyke PL (2006) Delivery of gadolinium-la- 10. Purushotham AD, Upponi S, Klevesath MB, Bobrow L,
beled nanoparticles to the sentinel lymph node: comparison Millar K, Myles JP, Duffy SW (2005) Morbidity after senti-
of the sentinel node visualization and estimations of intra- nel lymph node biopsy in primary breast cancer: results from
nodal gadolinium concentration by the magnetic resonance a randomized controlled trial. J Clin Oncol 23:43124321
imaging. J Control Release 111:343351 11. Erickson VS, Pearson ML, Ganz PA, Adams J, Kahn KL
4. Kim S, Lim YT, Soltesz EG, De Grand AM, Lee J, (2001) Arm edema in breast cancer patients. J Natl Cancer
Nakayama A, Parker JA, Mihaljevic T, Laurence RG, Dor Inst 93:96111
DM, Cohn LH, Bawendi MG, Frangioni JV (2004) Near- 12. McMasters KM, Tuttle TM, Carlson DJ, Brown CM, Noyes
infrared fluorescent type II quantum dots for sentinel lymph RD, Glaser RL, Vennekotter DJ, Turk PS, Tate PS, Sardi A,
node mapping. Nat Biotechnol 22:9397 Cerrito PB, Edwards MJ (2000) Sentinel lymph node biopsy
5. Gao X, Cui Y, Levenson RM, Chung LW, Nie S (2004) In for breast cancer: a suitable alternative to routine axillary
vivo cancer targeting and imaging with semiconductor dissection in multi-institutional practice when optimal tech-
quantum dots. Nat Biotechnol 22:969976 nique is used. J Clin Oncol 18:25602566
6. Kobayashi H, Kawamoto S, Sakai Y, Choyke PL, Star RA, 13. Scoggins CR, Chagpar AB, Martin RC, McMasters KM
Brechbiel MW, Sato N, Tagaya Y, Morris JC, Waldmann TA (2005) Should sentinel lymph-node biopsy be used routinely
(2004) Lymphatic drainage imaging of breast cancer in mice for staging melanoma and breast cancers? Nat Clin Pract
by micro-magnetic resonance lymphangiography using a Oncol 2:448455
nano-size paramagnetic contrast agent. J Natl Cancer Inst 14. Giuliano AE, Haigh PI, Brennan MB, Hansen NM, Kelley
96:703708 MC, Ye W, Glass EC, Turner RR (2000) Prospective
7. Kobayashi H, Brechbiel MW (2005) Nano-sized MRI con- observational study of sentinel lymphadenectomy without
trast agents with dendrimer cores. Adv Drug Deliv Rev further axillary dissection in patients with sentinel node-
57:22712286 negative breast cancer. J Clin Oncol 18:25532559

123