J Acupunct Meridian Stud 2012;5(5):206e209

Available online at www.sciencedirect.com

Journal of Acupuncture and Meridian Studies

journal homepage: www.jams-kpi.com

- RESEARCH ARTICLE -

Primo Vessel Inside a Lymph Vessel Emerging From

a Cancer Tissue
Sungwoo Lee 1, Yeonhee Ryu 2, Jinmyung Cha 3, Jin-Kyu Lee 3,
Kwang-Sup Soh 1, Sungchul Kim 4, Jaekwan Lim 1,*

1
Nano Primo Research Center, Advanced Institutes of Convergence Technology, Seoul National University,
Suwon, Republic of Korea
2
Department of Acupuncture, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea
3
Materials Chemistry Laboratory, Department of Chemistry, Seoul National University, Seoul,
Republic of Korea
4
Department of Acupuncture and Moxibustion, Wonkwang University Hospital, Gwangju, Republic of Korea

Available online Aug 15, 2012

Received: Nov 29, 2011 Abstract

Revised: Mar 25, 2012 Primo vessels were observed inside the lymph vessels near the caudal vena cava of
Accepted: Mar 28, 2012 a rabbit and a rat and in the thoracic lymph duct of a mouse. In the current work we
found a primo vessel inside the lymph vessel that came out from the tumor tissue of
KEYWORDS a mouse. A cancer model of a nude mouse was made with human lung cancer cell line
cancer; NCI-H460. We injected fluorescent nanoparticles into the xenografted tumor tissue and
fluorescence; studied their flow in blood, lymph, and primo vessels. Fluorescent nanoparticles flowed
lymph; through the blood vessels quickly in few minutes, and but slowly in the lymph vessels.
nanoparticle; The bright fluorescent signals of nanoparticles disappeared within one hour in the blood
primo vascular system vessels but remained much longer up to several hours in the case of lymph vessels. We
found an exceptional case of lymph vessels that remained bright with fluorescence up
to 24 hours. After detailed examination we found that the bright fluorescence was due
to a putative primo vessel inside the lymph vessel. This rare observation is consistent with
Bong-Han Kim’s claim on the presence of a primo vascular system in lymph vessels. It
provides a significant suggestion on the cancer metastasis through primo vessels and
lymph vessels.

* Corresponding author. Nano Primo Research Center, Advanced Institutes of Convergence Technology, Seoul National University, 864-1,
lui-dong, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-270, Republic of Korea.
E-mail: jakelim09@gmail.com

Copyright ª 2012, International Pharmacopuncture Institute

http://dx.doi.org/10.1016/j.jams.2012.07.003
Primo vessel in cancer tissue
1. Introduction
The role of primo vascular system (PVS) as an additional
metastatic path of cancer cells besides well-known blood
vascular and lymphatic paths was observed [1] after the
discovery of the PVS around cancer issues which were
induced in nude mice by xenografts of human lung cancer
cell line NCI-H460 [2,3]. Further observations of the cancer-
PVS were reported with melanoma in female nude mice [4],
male C57BL6 mice [5], GFP-expressing transgenic mice [6],
and female Swiss Webster mice [7].
In this work we report another observation of a primo
vessel that came out of a cancer tissue in a nude mouse. It
was a very rare case that the primo vessel ran afloat inside
a lymph vessel emerging from the cancer tissue. Several
years ago there were reports that PVS was observed as
a floating threadlike structure inside lymph vessels of
rabbits [8,9], and rats [10e12] as well as the original
discovery by Bong-Han Kim [13]. Therefore, it is not
surprising that a primo vessel was detected in the lymph
vessel. Nevertheless, there are significant points to be
noted in the current work.
First of all, it was the first observation of the primo
vessel that came out of the cancer tissue whereas the
previous observations [2e5] were the PVS lying outside the
surrounding membrane of the cancer tissue, and therefore
it was not clear whether they were directly connected to
the cancer tissue. In the previous cases [1e7] the PVS was
physically separated from and independent of blood or
lymph vessels. In an exceptional case of green fluorescent
protein (GFP)-expressing mouse, a primo vessel came out of
a blood vessel inside a tumor tissue [6].
Second, the presence of the primo vessel inside the
lymph vessel emerging from the cancer tissue raises the
role of the PVS as a mediator of metastasis even in lymph
vessels. If the PVS somehow facilitates the migration of
cancer cells this particular kind of lymph vessel with PVS
inside could become a main path of metastasis.
Finally the observation technique was particularly
interesting. After injecting fluorescent nanoparticles into
a cancer tissue we observed the fluorescence images of
nanoparticles flowing in the blood and lymph vessels that
emanated from the cancer tissue. The flow of nanoparticles
in blood vessels were fast and disappeared in a few
minutes, while the flow in lymph vessels was relatively slow
and lasted for up to 24 hours. In 24 hours there was no
detectable fluorescence of nanoparticles in the lymph
vessels. Surprisingly there was one lymph vessel, which
remained bright with fluorescence even after 24 hours. It
turned out that the bright fluorescence was due to nano-
particles in the fibrous extracellular matrices of the primo
vessel in the lymph vessel. Thus, flowing time characteris-
tics alone can differentiate blood, lymph, or primo vessel.
2. Materials and methods
2.1. Culture of human lung cancer cells
The cell line NCI-H460 was obtained from the Korean Cell
Line Bank. Human lung cancer cells were cultured in an
RPMI-1640 medium supplemented with 1% penicillin-
207
streptomycin and 10% fetal bovine serum (FBS; Invitrogen,
Carlsbad, California, USA). Cells were incubated at 37 C in
a humidified atmosphere containing 5% CO2.
2.2. Animals and cancer model
Female athymic nude mice (aged 5 weeks old, weighing
15e20 g, Charles River Laboratories) were used in accor-
dance with institutional guidelines under approved proto-
cols. For subcutaneous xenografts of human cancer,
animals were anesthetized with intraperitoneal (i.p.)
Zoletil (Verbac, Carros, France)/Rompun (Bayer, Leverku-
sen, Germany) and subcutaneously inoculated with
2  106 cells (in 0.2 mL RPMI-1640 medium).
2.3. Fluorescent nanoparticles
Cobalt-ferrite magnetic nanoparticles embedded in amor-
phous silica shells were synthesized. The shell contained
a fluorescent dye, rhodamine B isothiocyanate (orange l
max Z 555 nm), in the middle and a biocompatible poly-
ethylene glycol on the outside. The average core size of the
fluorescent magnetic nanoparticles was 9 nm, while the
average size of the enclosing shell was 50 nm [14]. The
suspension was composed of fluorescent magnetic namo-
particles and  99.9 % ethyl alcohol in a ratio 1:1.
2.4. In vivo visualization of blood, lymph, and
primo vessels
Three to four weeks after inoculation, the mice were
anesthetized with Zoletil/Rompun intraperitoneally. The
0.2 ml of fluorescent nanopartcles was directly injected
into cancer tissue with syringe. Immediately after injec-
tion, fluorescence images of fluorescent nanopartcles
flowed in lymph and primo vessels were taken by fluores-
cence stereomicroscope (MVX10, Olympus, Tokyo, Japan)
and monochrome CCD camera (DP30BW, Olympus, Tokyo,
Japan) without any surgery. Fluorescence images of blood
vessels were taken after incising the small area of skin
around cancer tissue.
2.5. Histological analysis
Samples were fixed in 10% neutral buffered formalin and
embedded in paraffin using routine procedures. Transverse
sections of 5 mm thickness were cut with a microtome,
stained with hematoxylin and eosin (H&E), and the samples
observed and photographed under a light microscope
(BX51, Olympus, Japan) and CCD camera (Infinity 2,
Lumenera, Ottawa, Canada).
3. Results
Fortuitously, we were able to find the primo vessel in
a lymph vessel that accompanied a blood vessel, which
came out of the tumor tissue as shown in Fig. 1. Nano-
particles flowed away in the blood vessel very fast. Within
several minutes after injection of nanopartcles into the
tumor tissue the blood vessel returned to its normal
208 S. Lee et al.

Figure 1 (A) Ventral view of the mouse with a xenografted tumor tissue (Figure taken from outside of the skin). The bright part is
the tumor tissue (arrow) and the bright curve is the lymph vessel (double arrows). The brightness is due to the fluorescence signal
of nanoparticles injected into the tumor tissue. Black rectangular region was studied after turning over the skin to observe the area
from the abdominal wall side in panel (B); (B) fluorescence signal of nanoparticles flowed through the lymph vessel (arrows) along
the blood vessel (dark wide curve). Image was obtained after incision and turning over the skin to expose the inner side of the skin.
The bright curve inside the lymph vessel (solid arrows) was taken after 24 hours from injection of nanopartiles.

appearance (dark in fluorescence view). The lymph vessel
kept the flow of nanoparticles much longer up to 24 hours.
After 24 hours the lymph did not show the fluorescence of
nanoparticles because they flowed away. After turning over
the skin and observing from the abdominal side we found
and an apparent lymph vessel remained bright in fluores-
cence even after 24 hours. It was a rare case (Fig. 1B).
The H&E image of the cross section of the blood and
accompanying lymph vessel showed indeed a thin thread-
like primo vessel bearing nanoparticles. Apparently they
flowed in the primo vessel whose diameter was about
20 mm, and had one nucleus, and fibrous materials in the
vessel (Fig. 2). The nanoparticles were not uniformly
distributed in the cross section of the primo vessel. They
were trapped in the fibrous extra cellular matrices of the
primo vessel. There were no nanoparticles in the lymph
vessel or on the lymph wall at all.
4. Discussion
The flow time of the nanoparticles was distinctively different
for the three cases of blood, lymph and primo vessels. In the
blood vessels the signals of nanoparticles remained in the
order of 10 minutes, while in the lymph vessels in the order of
several hours but less than 24 hours. Only the primo vessel
showed long lasting signals beyond 24 hours.
There were many reports on the threadlike primo vessel
inside lymph vessel of rats and rabbits [8e13], and the one
in a mouse was first reported by Choi et al. [15]. The
presence of a primo vessel inside a lymph vessel emerging
from a cancer tissue in a nude mouse was observed for the
first time. Although intralymph vascular PVS was observed
in many occasions the one in connection with cancer tissue
calls for special attention. Cancer metastasis is well known
to occur through lymph flow, and in addition through
a primo vessel [1]. It is a natural conjecture that metastasis
may occur more frequently through the double route of the
combined system of primo vessel inside a lymph vessel. It
would be worth to examine whether a more vicious
metastasis has such combined routes.
It raises an open question how many of the currently
known lymphatic metastases were, in fact, the combined
path of a PVS inside a lymph vessel. The finding of this
combined system was by chance, and very rare. No

Figure 2 (A) Cross section hematoxylin and eosin (H&E) image of the isolated sample shown Figure 1B. The primo vessel (arrow)
is located inside the lymph vessel (dotted arrows). The large blood vessel is indicated with double arrow; (B) fluorescence signal of
nanoparticles in the primo vessel (arrow) located inside the lymph vessel. Fluorescent nanoparticles were only in the primo vessel.
The diameter of the primo vessel is about 20 mm as expected for a primo vessel; (B) fluorescent image of (A).
Primo vessel in cancer tissue
thorough histological analysis was performed due to lack of
samples. We do not currently know the way to observe such
an intra-lymph PVS systematically. Further investigation in
this purpose is necessary. In addition, we did not study
other types of cancer models than the xenograft one which
is worth more studies. One last remark on the limit of the
current work is that no direct proof of flow of nanoparticles
was given. It is not ruled out that nanoparticles flowed in
the lymphatic fluid and were absorbed by the primo vessel.
Further systematic studies are necessary to resolve this
criticism.
Acknowledgments
This work was supported by the grant (#2011-P3-10) of
Advanced Institutes of Convergence Technology(AICT), and
was also supported in part by the grant of the Traditional
Korean Medicine R&D Project, Ministry for Health &
Welfare, Republic of Korea (B110076).
References
1. Yoo JS, Kim HB, Won N, Bang J, Kim S, Ahn S, et al. Evidence for
an additional metastatic route: in vivo imaging of cancer cells
in the primo-vascular system around tumors and organs. Mol
Imaging Biol. 2010;13:471e480.
2. Yoo JS, Kim HB, Ogay V, Lee BC, Ahn S, Soh KS. Bonghan ducts
as possible pathways for cancer Metastasis. J Acupuncture
Merid Stud. 2009;2(2):118e123.
3. Yoo JS, Hossein Ayati M, Kim HB, Zhang W, Soh KS. Charac-
terization of the primo-vascular system in the abdominal cavity
of lung cancer mouse model and its differences from the
lymphatic system. PLos One. 2010;5:e9940.
4. Yoo JS, Oyungerel B, Han IY, Kim JY, Lee CH, Choi KD, et al.
Molecular compositional differences of the primo and the
lymphatic vascular systems in murine melanoma models. In:
Soh K-S, Kang KA, Harrison DK, eds. The Primo Vascular
System. New York, USA: Springer; 2010:185e191.
209
5. Hong M, Park SS, Do H, Jhon GJ, Suh M, Lee Y. Primo vascular
system of murine melanoma and heterogeneity of tissue
oxygenation of the melanoma. J Acupunct Meridian Stud.
2011;4:159e163.
6. Heo CJ, Hong MY, Jo A, Lee YH, Suh MA. Study of the primo
vascular system utilizing a melanoma tumor model in a green
fluorescence protein expressing mouse. J Acupunct Meridian
Stud. 2011;4:198e202.
7. Akers W, Liu Y, Sudlow G, Lee J, Yoo JS, Lee B-C, et al. Iden-
tification of primo-vascular system in murine tumors and
viscera. In: Soh K-S, Kang KA, Harrison DK, eds. The Primo
Vascular System. New York, USA: Springer; 2010:179e183.
8. Lee BC, Yoo JS, Baik KY, Kim KW, Soh KS. Novel threadlike
structures (Bonghan ducts) inside lymphatic vessels of rabbits
visualized with a Janus Green B staining method. Anat Rec
(Part B: New Anat). 2005;286(1):1e7.
9. Lee BC, Soh KS. Contrast-enhancing optical method to observe
a Bonghan duct floating inside a lymph vessel of a rabbit.
Lymphology. 2008;41:178e185.
10. Johng HM, Yoo JS, Yoon TJ, Shin HS, Lee BC, Lee C, et al. Use of
magnetic nanoparticles to visualize threadlike structures
inside lymphatic vessels of rats. Evid Complement Altern Med.
2007;4:77e81.
11. Yoo JS, Johng HM, Yoon TJ, Shin HS, Lee BC, Lee C, et al.
In vivo fluorescence imaging of threadlike tissues (Bonghan
Ducts) inside lymphatic vessels with nanoparticles. Current
Applied Physics. 2007;7:342e348.
12. Lee C, Seol SK, Lee BC, Hong YK, Je JH, Soh KS. Alcian blue
staining method to visualize Bonghan threads inside large
caliber lymphatic vessels And X-ray microtomography to reveal
their microchannels. Lymph Res Biol. 2006;4:181e190.
13. Kim BH. The Kyungrak system. J Jo Sun Med. 1965;108:1e38 [in
Korean].
14. Yoon TJ, Kim JS, Kim BG, Yu KN, Cho MH, Lee JK. Multifunc-
tional nanoparticles possessing a “magnetic motor effect” for
drug or gene delivery. Angew Chem Int Ed Engl. 2005;44:
1068e1071.
15. Choi IH, Jeong HK, Hong Y- K. Detection of the primo vessels in
the rodent thoracic lymphatic ducts. In: Soh Kwang-Sup, Kang,
Kyung A, Harrison, David K, eds. The Primo Vascular System.
New York, USA: Springer; 2010:121e125.