Objective: The present study has been designed to find a possible new route for the
metastasis of cancer cells on the fascia surrounding tumor tissue using a novel technique
of trypan blue staining.
Materials and Methods: Tumor tissues were grown in the skin of nude mice after subcutaneous
inoculation with human lung cancer cells. Trypan blue was recently identified
as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as
blood or lymph vessels or nerves.
Results: We demonstrate that the trypan blue staining technique allows the first
visualization of BHDs which are connected to tumor tissues

Ahn, Saeyoung

Kim, Hong Bae

Lee, Byung-Cheon

Ogay, Vyacheslav

Soh, Kwang-sup

Yoo, Jung Sun

Nazarbayev University Repository

©2009 Korean Pharmacopuncture InstituteJ Acupunct Meridian Stud 2009;2(2):118−123RESEARCH ARTICLE1.  IntroductionMetastases create major clinical problems in han-dling cancer patients because treatments effective against the disease confined to the original site are often ineffective against metastatic cancer. Metas-tasis is currently known to occur via blood vessels, lymphatics, or movement within body cavities and is efficient, in the sense that most human cancers suc-cessfully metastasize. On the other hand, it is ineffi-cient as most cancer cells are destroyed in transit. For example, if melanoma cells are injected into an animal’s bloodstream, over 99% are destroyed within 24 hours. Another indication of this inefficiency is that about one million cells a day are shed from a mammary cancer, while less than 0.1% of these can be detected in the blood, as they are rapidly de-stroyed by the host’s defense mechanisms [1].A satisfactory solution has yet to be offered re-garding conflicting opinions of the efficiency of metastasis. The question can be posed regarding the possible existence of other unknown, more efficient routes of metastasis. If there is such a route, it may not only resolve the efficiency conflict but also pro-vide new insight into the phenomena that, after an apparently successful treatment of a primary tumor, AbstractObjective: The present study has been designed to find a possible new route for the metastasis of cancer cells on the fascia surrounding tumor tissue using a novel tech-nique of trypan blue staining. Materials and Methods: Tumor tissues were grown in the skin of nude mice after sub-cutaneous inoculation with human lung cancer cells. Trypan blue was recently identi-fied as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as blood or lymph vessels or nerves.Results: We demonstrate that the trypan blue staining technique allows the first visualization of BHDs which are connected to tumor tissues.Conclusion: Since BHDs are known to make up a circulatory system corresponding to acu puncture meridians or collaterals, we propose that, in addition to the currently known blood or lymph vessels, BHDs on tumor tissue fascia may be a novel pathway for metastasis.Received: Mar 03, 2009Accepted: Apr 01, 2009KEY WORDS:Bonghan duct;cancer;metastasis;trypan blueBonghan Ducts as Possible Pathways for Cancer MetastasisJung Sun Yoo, Hong Bae Kim, Vyacheslav Ogay, Byung-Cheon Lee, Saeyoung Ahn*, Kwang-Sup Soh*Biomedical Physics Laboratory, Department of Physics and Astronomy, Seoul National University, Seoul, Korea*Corresponding author. Biomedical Physics Laboratory, Department of Physics and Astronomy, Seoul National University, Seoul 151−747, KoreaE-mail: kssoh1@gmail.comBHDs as possible pathways for cancer metastasis 119including extended lymphadenectomy, metastases can sometimes arise after an extended period.In this article, we report the finding of a possible novel path for metastatic cancer. A novel circulatory system, the so-called Bonghan system with Bonghan ducts (BHDs), has been described on the fascia sur-rounding cancer tissue using an in situ trypan blue staining technique. Cancer tissues were grown in the skin of nude mice by subcutaneous injection of human lung cancer cells.BHDs are anatomical structures corresponding to acupuncture meridians and collaterals and were first found in rabbits and other animals by Bonghan Kim in 1963 [2,3]. However, his results were rarely reproduced [4] and his theory given little credence and ignored for a long time [5,6].Since the first rediscovery of intravascular BHDs inside the blood vessels of rabbits and rats [7,8], the presence of BHDs has been successively confirmed in various organs, such as lymphatic vessels [9−12], brain ventricles, central canals in the spine [13], and on internal organ surfaces [14]. The entire circula-tory network has not been investigated, but evidence of liquid flow in BHD has been produced.Multiple ductules in a BHD were observed in mor-phological studies by using hematoxylin and eosin (H&E) staining methods [15] and various types of electron microscopy [16]. Endothelial cells compris-ing the inner boundaries of the ductules in BHD were different from those in blood or lymph vessels [15]. The liquid flow speeds in BHDs on internal organ sur-faces were measured at 0.3 ± 0.1 mm/s [17], with the liquid flow from the skin toward the internal organs observed by injecting chrome-hematoxylin and flu-orescent nanoparticles in the skin at a rat testis [18]. An electrophysiological study of a Bonghan system showed a vascular smooth muscle-like excitability [19] and muscarinic receptors [20] that support the circulatory functioning of the Bonghan system.In this article, we present the trypan blue stain-ing technique recently developed to visualize BHDs [21], without which it would be extremely difficult to visualize their presence on the transparent fas-cia enwrapping tumor tissue. A histological analysis of a BHD with H&E showed sinuses and flowing cells inside sinuses. Thus the Bonghan circulatory system was found to be connected to tumor tissues, which suggests a possible new route for metastasis.2.  Materials and Methods2.1.  Culture of human lung cancer cellsThe cell line NCI-H460 was provided by the Korean Cell Line Bank. Human lung cancer cells were sub-cultured in a RPMI-1640 medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine se rum (FBS) purchased from Invitrogen. Cells were incubated at 37°C in a humidified atmosphere con-taining 5% CO2.2.2.  Animals and cancer modelFemale athymic nude mice (aged 5−7 weeks old, weighing 15−20 g, n = 10; Charles River Laboratories) were used in accordance with institutional guide-lines under approved protocols. For subcutaneous xenografts of human cancer, animals were anes-thetized with intraperitoneal (i.p.) Zoletil/Rompun and subcutaneously inoculated with 1 × 107 cells (in 1 mL RPMI-1640 medium).2.3.  In vivo visualization of Bonghan ductsTwo to 8 weeks after inoculation, the mice were anes thetized with Zoletil/Rompun i.p. and all surgi-cal procedures were performed under general an-esthesia. The lateral sides of the tumor skin were incised and the skin over the tumor removed care-fully to expose the tumor with an intact outer mem-brane. A 0.1% trypan blue solution, previously filtered through 0.22-μm pore-sized filter paper, was applied dropwise on the exposed membrane. After rinsing away the dye with warm saline, the identification of Bonghan corpuscles and ducts was assessed using direct visualization with a surgical microscope (SZX12, Olympus).2.4.  Histological analysisBonghan ducts and corpuscles were fixed in 10% neutral buffered formalin and embedded in par af-fin using routine procedures. Transverse sections of 7 μm thickness were cut with a microtome, stained with H&E, and the samples observed and photo-graphed under a light microscope (BX51, Olympus).3.  ResultsWith simple microscopy of tumor tissue grown under the skin of a nude mouse, we could not observe any novel, threadlike structures on the surface of the solid tumor after sectioning (Figure 1A). However, with trypan blue staining, BHDs and Bonghan cor-puscles (BHCs) were revealed as stained blue. It is remark able that no other tissues, such as blood ves-sels or fascia, were stained, but BHDs and BHCs were prominently stained (Figure 1B, C). The full length of BHDs was sometimes untraceable because they en-tered nearby fatty tissues (Figure 1C). Another nota-ble observation was the detection of a BHD along a blood vessel or nerve bundle connecting the tumor 120 J.S. Yoo et alFigure 1   Visualization of the Bonghan system on the fascia surrounding tumor tissue in the skin of a mouse. (A) Images of tumor tissue, indicated by arrows; left: a mouse with two tumors, grown for two weeks after subcutaneous inoculation; right: tumor tissue surface after skin sectioning; Bonghan system hardly detectable. (B) In situ trypan blue staining of ducts (dotted arrows) and corpuscles (arrow heads) on fascia surrounding tumor tissue; right panel, magnified view of left panel; trypan blue did not stain blood vessels. (C) Multiple ducts (dotted arrows) on fascia surface of tumor tissue; right panel, magnified view of left panel; branching of duct and duct enters nearby fat layer (double arrow). (D) Trypan blue technique revealed Bonghan ducts (dotted arrows) along bundle of blood vessels and nerves; right panel, duct along blood vessel; bundle of blood vessels and nerves connect tumor tissue (an arrow) at lower left corner to outside skin. Samples A, B, C, and D from different mice.ABCD15 mm 2.0 mm2.0 mm1.0 mm2.0 mm1.5 mm500 μm1.0 mmBHDs as possible pathways for cancer metastasis 121tissue to the skin. Thus this could be an effective method for detecting a BHD externally, i.e., by following a blood vessel or a nerve.A trypan blue positive BHD (Figure 1B) was reex-amined histologically with H&E staining (Figure 2), revealing several ductules that appeared in both cross- and oblique-sections. The lumina of the duc-tules (arrows in Figure 2) contained endothelial cells in the wall of ductules (W in Figure 2) and flowing cells in the space of lumina (arrowheads in Figure 2). The histological nature of the trypan blue positive BHD was distinctively different from the fascia, blood vessels, or lymph vessels, but was quite consistent with the BHDs on internal organ surfaces [15].A cross-section image of a trypan blue stained BHC identified in a section (Figure 1B) showed the pres ence of many ductules (Figure 3) and its his tolo-gical nature was again consistent with other obser-vations involving BHCs on the surfaces of various organs [15].4.  DiscussionA critical goal in verifying the novel metastatic path hypothesis proposed in this article would be to di-rectly show the movement of cancer cells from tu-mor tissue to other sites via BHDs. Another goal would be to examine whether this route is more efficient than the blood or lymph routes. These goals would require techniques for identifying cancer cells in a BHD and, thus require appropriate markers. The po-ten tial use of quantum dot materials engulfed by cancer cells is currently under consideration.Besides being a potential metastatic path for the spread of cancer cells, BHDs may play other roles in connection with tumor tissues. The liquid flowing in BHDs may play important roles in tumor growth as it contains hyaluronic acid, adrenalin, noradrena-lin, albumin, and microcells (or sanals). In addition, a pro teomic analysis of tissues and liquid from BHDs on rabbit intestinal surfaces [22] revealed a chemical A BC DWW100 μm 20 μm10 μm 10 μmFigure 2   Photomicrographs of cross-sections of Bonghan duct found on the surface of solid mouse tumor with H&E staining. Sample of Figure 1B. (A) Duct (double arrow) located on fascia (dashed arrows) covering solid tumor. (B) Pho tomicrograph showing several ductules (arrow) in duct, in both cross- and oblique-sections; flowing cells (arrowhead) with round or oval nuclei observed in some duct lumina; nuclei in lumen endothelial cells. (C, D) Duct histological characteristics; high magnification; W, wall of the ductule; arrow and arrowhead, ductule and flowing cells in lumen, respectively. Magnifications: A, 100˜; B, 400˜; C and D, 1,000˜.122 J.S. Yoo et alBC DA***50 μm 20 μm20 μm 20 μmFigure 3   Photomicrographs of Bonghan corpuscle cross-sections. Sample from Figure 1B. (A) Light microscope image of general corpuscle histology. (B−D) Magnified images of Figure 3A; presence of many ductules (arrow) in corpuscles, appear in cross- and oblique-sections; ductule diameter from 5−50 μm; parts of corpuscle boundary surrounded by external connective tissue membrane (arrow heads); parts not (asterisks). Magnifications: A, 200˜ and B−D, 400˜.composition similar to that usually associated with stem cells [23,24], cancer cells [25], and differen-tiated myeloid cells [26]. These cells, with vigorous proliferative abilities, show a similar abundance of carbohydrate- or energy-related processes [23−26]. Tumor tissues are likely to take advantage of the nutrients supplied through BHDs.Another hypothetical significance of the Bonghan circulatory system in relation to tumor tissues is its potential use as a drug delivery path for anticancer medicine or even as a route for acupuncture treat-ment because the BHD is an anatomical structure of acupuncture collaterals. A host of items, such as migration, invasion, relation to angiogenesis, prolif-eration, and growth of cancer tissue, have imme-diate importance in connection with the Bonghan system.AcknowledgmentsThis research was supported by a “Systems Bio-logy In frastructure Establishment Grant” from the Gwangju Institute of Science and Technology. We also appreciate the generous support of President B. J. Son of Mobase Co. Ltd.References1. King RJB. Cancer Biology, 2nd ed. Harlow: Prentice Hall, 2000:213−4.2. Kim BH. The Kyungrak system. J Acad Med Sci DPR Korea 1963;90:1−41.3. Kim BH. The Kyungrak system. J Acad Med Sci DPR Korea 1965;168:1−38.4. Fujiwara S, Yu SB. Bonghan theory: morphological studies. Igakuro Ayum 1967;60:567−77. [In Japanese]5. Kellner G. Bau und funktion der haut. Deutsh Z Akupunkt 1966;15:1−31. [In German]6. Kroger WS. Acupuncture analgesia: its explanation by con-ditioning theory, autogenic training and hypnosis. Am J Psychiatry 1973;130:855−60.7. Jiang X, Kim HK, Shin HS, Lee BC, Choi C, Soh KS, et al. Method for observing intravascular Bonghan duct. J Kor Orient Prevent Med Soc 2002;6:162−6.8. Lee BC, Baik KY, Cho S, Min C, Johng HM, Hahm J, et al. Comparison of Intravascular Bonghan Ducts from Rats and Mice. J Kor Orient Prevent Med Soc 2003;7:47−53.BHDs as possible pathways for cancer metastasis 1239. Lee BC, Yoo JS, Baik KY, Kim KW, Soh KS. Novel threadlike structures (Bonghan ducts) inside lymphatic vessels of rab-bits visualized with a Janus Green B staining method. Anat Rec B New Anat 2005;286:1−7.10. Yoo JS, Johng HM, Yoon TJ, Shin HS, Lee BC, Lee C, et al. In vivo fluoresence imaging of threadlike tissues (Bonghan ducts) inside lymphatic vessels with nanoparticles. Curr Appl Phys 2007;4:342−8.11. Lee C, Seol SK, Lee BC, Hong YK, Je JH, Soh KS, et al. Alcian blue staining method to visualize Bonghan threads inside large caliber lymphatic vessels and X-ray microtom-ography to reveal their microchannels. Lymphat Res Biol 2006;4:181−90.12. Lee BC, Soh KS. Contrast-enhancing optical method to observe a Bonghan duct floating inside a lymph vessel of a rabbit. Lymphology 2009; accepted.13. Lee BC, Kim S, Soh KS. Novel anatomic structure in the brain and spinal cord of rabbit that may belong to the Bonghan system of potential acupuncture meridians. J Acupunct Meridian Stud 2008;1:29−35.14. Shin HS, Johng H, Lee BC, Cho S, Baik KY, Yoo JS, et al. Feulgen reaction study of novel threadlike structures on the surface of rabbit livers. Anat Rec B New Anat 2005;284:35−40.15. Ogay V, Bae KH, Kim KW, Soh KS. Comparison of the charac-teristic features of Bonghan ducts, blood and lymphatic capillaries. J Acupunct Meridian Stud 2009;2:107−17.16. Lee BC, Yoo JS, Ogay V, Kim KW, Dobberstein H, Soh KS, et al. Electron microscopic study of novel threadlike struc-tures on the surfaces of mammalian organs. Microsc Res Tech 2007;70:34−43.17. Sung B, Kim MS, Lee BC, Yoo JS, Lee SH, Kim YJ, et al. Mea-surement of flow speed in the channels of novel threadlike  structures on the surfaces of mammalian organs. Naturwissenschaften 2008;95:117−24.18. Han HJ, Ogay V, Park SJ, Lee BC, Kim KW, Soh KS. Potential new flow path from testis to the abdominal organ via Bonghan ducts. J Anat 2009; submitted.19. Park SH, Lee BC, Choi CJ. Bioelectrical study of Bonghan corpuscles on organ surfaces in rat. J Kor Phy Soc 2009; submitted.20. Park SH, Lee BC, Choi CJ, Choi JH, Lee SY, Soh KS, Ryu PD. Electrophysiological study of Bonghan corpuscles with drug stimulation. J Acupunct Meridian Stud 2009; submitted.21. Lee BC, Kim KW, Soh KS. Visualizing the network of Bonghan ducts in the omentum and peritoneum by using Trypan blue. J Acupunct Meridian Stud 2009;1:66−70.22. Lee SJ, Lee BC, Nam CH, Lee WC, Jhang SU, Park HS, Soh KS, et al. Proteomic analysis for tissues and liquid from Bonghan ducts on rabbit intestinal surfaces. J Acupunct Meridian Stud 2008;1:1−13.23. Chen EI, Hewel J, Krueger JS, Weber MR, Tiraby C, Kralli A, et al. Adaptation of energy metabolism in breast cancer brain metastasis. Cancer Res 2007;67:1472−86.24. Lian Z, Wang L, Yamaga S, Bonds W, Beazer-Barclay Y, Kluger Y, et al. Genomic and proteomic analysis of the myeloid differentiation program. Blood 2002;98:513−24.25. Forte G, Carotenuto F, Pagliari F, Pagliari S, Cossa P, Fiaccavento R, et al. Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and dif-ferentiation. Stem Cells 2006;24:23−33.26. Wuchter P, Boda-Heggemann J, Straub BK, Grund C, Kuhn C, Krause U, et al. Processus and recessus adhaerentes: giant adherens cell junction systems connect and attract human mesenchymal stem cells. Cell Tissue Res 2007;328:499−514.

Bonghan Ducts as Possible Pathways for Cancer Metastasis

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Bonghan Ducts as Possible Pathways for Cancer Metastasis

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