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J Acupunct Meridian Stud 2016;9(6):297e306

Available online at www.sciencedirect.com

Journal of Acupuncture and Meridian Studies


journal homepage: www.jams-kpi.com

REVIEW ARTICLE

Technical Challenges in Current Primo


Vascular System Research and Potential
Solutions
Kyung A. Kang 1,*, Claudio Maldonado 2, Vitaly Vodyanoy 3,4

1
Department of Chemical Engineering, University of Louisville, Louisville, KY, USA
2
Department of Physiology and Biophysics, University of Louisville, Louisville, KY, USA
3
Department Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, and
School of Kinesiology, Auburn University, Auburn, AL, USA
4
The Research Department, The Edward Via College of Osteopathic Medicine, Auburn, AL, USA
Available online 17 February 2016

Received: Sep 19, 2015 Abstract


Revised: Jan 28, 2016 Since Bonghan Kims discovery of the Bonghan system (BHS) in the 1960s, numerous re-
Accepted: Feb 8, 2016 ports have suggested that the system is fundamental for maintaining mammalian life.
The BHS is a circulatory system independent of the blood or the lymphatic system, forms
KEYWORDS an extensive network throughout the entire mammalian body, has been reported to be
Bonghan system; the acupuncture meridian, stores distinct types of stem cells, and appears to have some
primo vascular system; roles in cancer metastasis. Despite Kims first report having been published as early as
PVS identification; 1962, research progress has been rather slow mainly because the system is very small
PVS harvest; and translucent, making it optically difficult to distinguish it from the hemoglobin-rich
optical characterization surrounding tissues. Unfortunately, Kim did not describe in detail the methods that he
used for identifying and harvesting the system and the components of the system. In
2000, Kwang-Sup Soh reopened the BHS research, and since then, new and important sci-
entific findings on the system have been reported, and many of Kims results have been
verified. In 2010, the BHS was renamed the primo vascular system. Nevertheless, good
tools to properly deal with this system are still lacking. In this article, we address some
of the technical difficulties involved in studying the primo vascular system and attempt to
discuss potential ways to overcome those difficulties.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
* Corresponding author. Department of Chemical Engineering, University of Louisville, Louisville, KY 40292, USA.
E-mail: Kyung.Kang@louisville.edu (K.A. Kang).

pISSN 2005-2901 eISSN 2093-8152


http://dx.doi.org/10.1016/j.jams.2016.02.001
Copyright 2016, Medical Association of Pharmacopuncture Institute.
298 K.A. Kang et al.

As Kims publications were reports (not journal publi-


1. Introduction cations requiring peer review processes), his findings were
to be verified before they were taken as scientific facts.
In 1962, Bonghan Kim [1], a scientist in North Korea, re- Until now, only some of Kims findings have been re-
ported a discovery of a new vascular system in mammals, examined and verified, and many are still to be investi-
which was soon named as the Bonghan system (BHS). The gated. Since 2000, PVS scientists have also reported new,
small size (< 3 mm) and optical transparency of the system important findings about the system, and some of the
were probably the main reasons for its delayed discovery. notable scientific findings on the BHS/PVS are as follows.
Kim and his research team [2e5] published four additional (1) Kim claimed that the superficial BHS was the acupunc-
reports by the end of 1965, and the content of the reports ture meridian [1e5], and, recently, the Ryu team [8] in
strongly implied the systems importance in the mammalian Korea colocalized the acupoints and superficial Bonghan
physiology. Kims claim of the system being the acupuncture corpuscles/PNs in the abdomen of animal models, vali-
meridian particularly surprised the scientific community. dating Kims claim. (2) After 2000, PVS scientists confirmed
Unfortunately, Kims research activities suddenly stopped in it to be independent of the blood or lymphatic system by
1965 [6] and had undergone a dormant period until Kwang- the differences in microanatomical structures and bio-
Sup Soh reopened the research on the system in 2000. By markers [9]. (3) The BHS/PVS is an extensive network
2002, new scientific results on the system started to appear throughout the entire mammalian body [3,7]. (4) The P-
in various journals, and in 2010, the system was renamed as microcells, which Kim named as sanals (live eggs, in
the primo vascular system (PVS) [6,7]. Korean), are now confirmed to be unique types of stem
The BHS/PVS consists of the ducts (vessels), corpuscles cells, and positively confirmed to be performing nonmarrow
(nodes), various fluid-containing cells, and biochemicals hematopoiesis and repairing damaged cells/tissues
that flow throughout the system. The following are the [5,10e12]. (5) Bonghan liquor/P-fluid contains various bio-
terms used during the Kims time and those after the chemicals and cells, particularly those associated with im-
Bonghan system became the PVS, placed side by side: mune responses [2,3,13]. (6) The PVS appears to have a role
in the dynamics of cancer metastasis [9,14], which is a new
Bonghan system Primo vascular system (PVS) finding after 2000.
Bonghan duct Primo vessel (PV) Although only limited facts on the BHS/PVS have been
Bonghan ductule Subprimo vessel (Sub-PV) revealed so far, the nature of these listed above clearly
Bonghan corpuscle Primo node (PN) demonstrates the importance of the system in the
Sanal (or Bonghan sanal) Primo microcell (P-microcell) mammalian physiology. Despite the urgency for a thorough
Bonghan liquor Primo fluid (P-fluid) investigation on this important system, there are still
serious impeding issues in performing PVS research. The
most serious one is lacking scientific tools to properly
Kim also named the subunits of sanal as the sanalosome, handle its extremely small size (PV, 20e50 mm;
sanaloplasm, and sanal membrane, which were not PN < 1500 mm) and optical transparency [1e5], which cause
renamed by PVS scientists. A new term primogenesis was difficulties in identifying and harvesting the system. Kim
created in 2012, by the University of Louisville team presented his study results logically with reasonable de-
(Louisville, KY, USA), to define the phenomenon of gener- tails, but did not describe the methods for identifying and
ating or forming the PVS, while describing the new PVS harvesting the PVS in animal models at a level that future
formation on/in the cancer xenografts. scientists can repeat his studies. New techniques for iden-
Kim [2,3] classified the system into several subtypes by tifying/harvesting the PVS have been reported since 2000,
their locations. As his research progressed and more details but they are still not satisfactory for most of the scientists,
on the system were uncovered, his classification became particularly those who are new to the system, to be able to
also updated. The subtypes are as follows. (1) Internal (or reliably access the PVS sample to perform in-depth
intravascular): floating inside blood and lymphatic vessels investigation.
(LVs). (2) External (or extravascular): outside the blood In this article, in an effort to support the scientific
vessels and nerves, or independently. (3) Intraexternal: advancement in the system, the authors have analyzed
freely on the surface of internal organs in thoracic/ important issues to overcome and attempted to suggest
abdominal cavities. (4) Intraorganic: inside organ walls. (5) potential techniques to resolve these issues. We hope that
Neural: in/on the central and peripheral nervous systems. other PVS scientists also share their ideas with the PVS
(6) Superficial: in the corium or subcutaneous tissue. This community.
subtype was to be surrounded by dense blood vessels and It should be noted that, the term PVS scientists in this
was close to nerves. Kim reported the corpuscles of this article is used to represent the scientists who have been
subtype to be acupoints and the ducts to be the acupunc- involved in PVS research after 2000. For the scientific terms
ture meridian. associated with the system, PVS (not BHS) terms are mainly
Currently, all subtypes except the external subtype are used to avoid unnecessary confusion.
positively identified [7,8]. After observing the path of a
radioactive tracer injected into predetermined Bonghan
corpuscles, Kim [2,3] concluded that the subtypes closely 2. Identifying PVS with nanoparticles
communicated with each other and that the system did not
appear to be controlled by a single central unit, but by For properly performing any scientific research, the target
multicompartmentalized units. sample needs to be identified first. Frequently, utilizing
Technical Challenges in PVS Research and Solutions 299

biomarkers specific for the target sample is helpful, but 1e5 mm, and the size of the PV or the pore does not seem to
identifying/developing PVS biomarkers has not been real- vary significantly among the species studied. According to
ized yet because of the difficulties in obtaining sufficient Kim [3], the interstitial space of sub-PVs (ductules) has a
amount of PVS samples. Identifying the PVS in the entire fibrous structure and is filled with amorphous substances.
body, with the help of appropriate contrast agents, using Therefore, if particles of an appropriate size (i.e., smaller
medical imaging modalities could be a way of obtaining than the pore in the PV wall, but similar to or slightly larger
sufficient samples. However, the usual spatial resolution than the spacing in the fibrous structure) are applied to the
obtainable with these tools would be 100 mm at best [15], PV from outside, they are expected to get into the PV via
which is much bigger than the average PV diameter. the pores and be retained in the interstitial space. If these
Although some advanced modalities such as specially particles possess an appropriate optical property, then they
designed magnetic resonance imaging [16e18] and electron would generate optical contrast for the PV.
or X-ray microscopy [19e21] provide exceptionally good Fig. 2 schematically describes the mechanism of iden-
resolution, they are not readily available to most scientists tifying the PVS inside an LV (intra-LV PVS) using nano-sized
and not practical for real-time sample harvesting from contrast agents. The LV is a good site for identifying the PVS
animal models. Therefore, the main technique for identi- because it has a translucent vessel wall and its fluid is clear,
fying the PVS has been optical (stereo) microscopy, and, therefore, the optically contrasted, intra-LV PVS can
particularly for studies involving surgeries in live animals, be seen through the LV wall. In this procedure, the contrast
which is the current situation for most PVS studies. agent at an appropriate size is first injected into the lymph
Identification of the PVS has been performed by taking node connected to the target LV until the LV is filled with
advantage of its natural anatomical structure, and the PV is the particles (Fig. 2A), and in time, the particles would
known to have at least three unique characteristics (Fig. 1), enter the PV via the pores. The natural flow of lymphatic
according to Kim [2,3] and later PVS scientists [19e22]: (1) fluid clears particles from the LV (outside the PV), while the
multiple sub-PVs in a single PV (Figs. 1A and 1B), and the particles that have already entered the PV remain in the
inter-sub-PV space filled with fibrous, amorphous sub- interstitial space, providing optical contrast to the PV
stances [3]; (2) rod-shaped nuclei of sub-PV endothelial (Fig. 2B).
cells (Fig. 1A) [3]; and (3) pores in the PV external wall One such method exploits the unstable nature of Alcian
(Fig. 1B). The second property is helpful for confirming blue at a pH of > 5.6 [25]. Alcian blue is dark green in color,
whether the harvested sample is a PV or not. The first and, its molecular size is 2e3 nm, and it is frequently used in
particularly, the third properties have been effectively staining acidic mono- or polysaccharides of biological
utilized for placing appropriate optical contrast agents into samples in histological studies [26]. This dye itself, how-
the PV. It is still not confirmed whether these pores exist in ever, does not appear to stain the PVS preferentially to the
the outer membrane of all six PVS subtypes, but this LV. For the purpose of PVS identification, Alcian blue
property has been used for identifying the PVS inside the LV powder is first dissolved in PBS (pH 7.4); the solution is
and the intraexternal PVS [22e24]. Electron microscopy boiled (possibly to maximize its solubility in PBS) and
and X-ray microscopy [19e21] have determined the pore filtered to remove large particles. The Alcian blue solution
size of the outer wall of the rabbit intraexternal PV to be is then injected into the lymph node of the animal until the

Figure 1 Properties unique for the PV. (A) PV illustrated in Kims third report (slightly modified from [3]; 1-sub-PV; 2- sub-PV
endothelial cell; 3-rod shaped nucleus of endothelial cell; 4-PV external layer; 5-nucleus of PV endothelial cell. (B) Schematic
diagram of a PV cross section: pores in the PV outermost wall; multiple sub-PVs in a PV; and fibrous nature of PV interstitial space.
PV Z primo vessel; sub-PV Z subprimo vessel.
300 K.A. Kang et al.

A B

Sub-PV Lymphatic
vessel

Nanocon
trast
agent

Pore on
PV wall
PV
Lyymphatic
flluid flow

Figure 2 Schematic diagrams illustrating the identification of the PVS inside an LV using nano-sized contrast agent. (A) PVS-
containing LV is filled with nanoparticles, by injecting them into the lymph node. The particles get into the PV via the pores of
the PV external vessel wall. (B) The LV is cleared by its natural fluid flow, while the PVS retains the particles trapped in it,
generating optical contrast. LV Z lymphatic vessel; PV Z primo vessel; PVS Z primo vascular system; sub-PV Z subprimo vessel.

LV is filled with it [25]. During the process, the dye forms our experiments, dark green, greenish blue, or dark blue
small particles due to its instability in the PBS and seems to fit well for the purpose.
lymphatic fluid at pH 7.4. The actual size of the formed Up to now, five out of the six PVS subtypes (except the
Alcian blue particles is not known, but they go through the external subtype) have been positively confirmed. The su-
pore in the PV outer membrane and remain in the inter- perficial PVS may also need to be given special attention
stitial space, generating contrast for the PVS. The dye because this subtype is said to be directly related to the
preparation and injection steps should to be carried out acupuncture therapy, and a thorough understanding of this
within a short period of time (not clearly defined yet) subtype may help in optimizing the therapy. Kim [2] illus-
before larger particle formation and/or precipitation trated how the radioactive tracer injected to a Bonghan
starts. corpuscle (PN) in the skin moved to other corpuscles via the
A more controlled and user-friendly method is using meridian. Besides the study by Kim, there have been
nano-sized particles of an appropriate size range and op- several studies on tracing meridian paths using radioactive
tical contrast against hemoglobin. The University of Louis- tracers or dyes, by injecting them into a particular acupoint
ville team has developed a technique utilizing hollow gold (not Bonghan corpuscles or PNs) [29e33]. Since dealing
nanoparticles (HGNs) with a color range from green to blue with radioactive tracers is not ideal, the method developed
[27]. HGNs were selected because gold is biocompatible by Lim et al [8] for the colocalization of PNs and acupoints
and the size and color of HGNs can be controlled well. In a in the abdominal hypodermis region of rats using the three-
preliminary study using the rat model, particles in the size solution Hemacolor system may be a better approach.
range of 50e125 nm worked well, with a success rate of 95% The subtype that is most difficult to identify seems to be
[28]. This method has advantages over the Alcian blue the external PVS subtype, judging from the lack of scientific
method, because the HGNs have a relatively homogeneous articles on this subtype. The external PVS was reported to
size distribution, preparation steps are simpler, and the PVS be covered with connective tissue, according to Kim [3],
in the LV becomes visible within 20 minutes after the and, therefore, accessibility to the pores on its external
particle injection. wall is still not known.
The nanoparticle optical contrast agent used for the
purpose of intra-LV PVS identification requires certain
properties when used in live animals: (1) it must be 3. Harvesting PVS via microelectromechanical
nontoxic, biocompatible, and chemically inert because it is system
to be injected into LVs; (2) the particle surface should be
relatively hydrophilic and should not easily aggregate; and As described in the Introduction, most PVS scientists have
(3) it needs to provide a strong optical contrast against the already recognized the potential health benefits that may
tissue background (i.e., hemoglobin color). According to be achieved by appropriately manipulating the nature of
Technical Challenges in PVS Research and Solutions 301

the PVS, e.g., regenerating tissues using ones own P-


microcells or detecting cancer via the abnormally formed
PVS, etc. However, much of the fundamental information
about the PVS (e.g., micro- and submicroanatomical
structures, biological compositions, biomarkers, physiolog-
ical functions, the entire PVS map in the body, and
communication between the PVS and other organs) still
remains to be studied before any translational work can be
done. Besides, we still need to verify all of Kims findings,
which were published 50 years ago.
To acquire these basic scientific information, PVS sam-
ples need to be available in a reliable manner, but acquiring
a sufficient amount of PVS samples has been difficult
because of its small size as well as its identification-related
issues already described. The sites that may potentially
provide a relatively large amount of PVS samples are the
blood and LVs because the PVS runs inside them and they
are long. Out of the two, LVs are better candidates, as
mentioned earlier.
Identification of the intra-LV PVS can be performed more
easily in live animals because the floating motion of the PVS
in the lymphatic fluid helps identify it better. Once the LV
loses its fluid and becomes flattened, the PVS easily sticks
to the LV inner wall. Live animals, however, breathe and
the target LV also moves with the breathing motion, which Figure 3 A PVS identified inside an LV, using HGNs. (A) The
makes the harvesting tasks more challenging. Therefore, PVS contrasted by the HGNs can be seen inside the LV. (B) After
keeping the target LV still during the breathing motion may the lower end of the PVS bearing LV is cut, the PVS is curled up
be helpful (e.g., fixing the LV on a solid surface). Next, in the LV. HGN Z hollow gold nanosphere; LV Z lymphatic
retrieving the PVS from the LV requires extremely delicate vessel; PN Z primo node; PV Z primo vessel; PVS Z primo
microsurgical techniques with good microsized tools. A few vascular system.
medical tools, such as micro-extra-fine surgical knives and
grippers (e.g., Excelta, Buellton, CA, USA; nanoScience
Instruments, Phoenix, AZ, USA), are currently available for
this type of study, although some of them can be expensive highly desirable to have microliter-sized P-fluid collectors
and may still need to be tailored for PVS research. The tip and/or microinjectors, utilizing MEMS technology, for the
of the atomic force microscope may potentially serve as a P-fluid analysis and applying biochemicals into the PVS.
microknife. A suggested method for the intra-LV PVS, once
it is identified, is as follows: make small incisions at two
positions on the LV, depending upon the desired amount of 4. P-microcell retrieval by cell sorting
PVS samples; then insert the tips of two sets of micro- techniques
grippers (e.g., nanoScience Instruments) into the two
respective incision sites of the LV, to hold the two ends of Kim [4] reported that there are unique, self-regenerating
the PVS; cut the LV along its length between the two inci- cells (P-microcells/sanals) inside the PVS, particularly in
sion sites; and cut the two ends of the PVS (outer sides of the PNs [5], and these cells are now positively confirmed to
the grippers) and retrieve the freed PVS by lifting the be stem cells [11,12]. A thorough understanding of P-
grippers. microcells and their roles would, therefore, be very
One might suggest to first harvest the PVS-containing LV important for the stem cell science and regenerative
and then retrieve the PVS from it, in order to simplify the medicine, and retrieving these cells from PNs in a reliable
process. However, when the LV is cut without holding the manner is the first step toward it. This may be done, first,
two ends of the PVS, the PVS tends to curl up inside the LV by mechanically or enzymatically disintegrating the outer
because of its elastic nature. Fig. 3 shows the PVS optically layer of PNs to obtain all cells inside them, and then
contrasted by HGNs, before (Fig. 3A) and after (Fig. 3B) the separating the P-microcells from other cells. Since the
LV-containing PVS was cut at its lower part, and the PVS is constituents or properties of the PN membrane are still not
shown to curl up after the LV was cut. In addition, if PVS clearly known, development of an optimized process for
retrieval is not performed immediately after the LV is cut, removing the membranes may have to wait until they are
then the PVS may stick to the inner wall of the LV, which well characterized.
makes it difficult to harvest the PVS. After the membranes are removed, P-microcells need to
Since the entire procedure involved in the PVS harvest be separated. Over the past decade, cell-sorting tech-
requires precise operation due to the small target sample niques have been advanced significantly because detecting
size, if the procedure is performed by a micromanipulator circulating cancer cells has been emphasized as an impor-
based on the microelectromechanical system (MEMS) tant research topic for early cancer diagnosis. The P-
technology, human errors may be minimized. It is also microcell is characterized by its small size, 1e3 mm
302 K.A. Kang et al.

[10e12], while most cells are in the range of 10 mm. allow the image edge to become sharper, thus increasing
Therefore, P-microcells may be separated from the cell the resolution [42e46].
population in PNs by flow cytometry, either using the dif- Minimizing the light spot size on the sample and reducing
ference in cell size [34] or by fluorescence [35] after la- the stray light can also improve spatial resolution and
beling the cells with fluorophores via antibodies against contrast: reducing the spot dimension raises the irradiance
stem cell biomarkers that P-microcells express [10,36]. For of the sample and increases the contrast by increasing the
PVS studies, it may also be advantageous to use micro-sized interaction with small particles in the sample. The photon
cell-sorting systems [37,38] instead of using a regular flow density of the system modified by the Auburn University
cytometer, since PNs are very small, and the cell volume team (Auburn, AL, USA) became approximately 150 times
and number in a PN are very small. higher than that of the conventional Nikon darkfield
condenser (Eclipse E400, Oil 1.43-1.20, Japan). This
increased illumination intensity raises the optical differ-
5. Optical microscopy for PVS characterization ence between tiny light-scattering particles and their
background. By doing so, all particles larger than 90 nm,
Optical microscopy was one of the most important methods including small DNA granules and P-microcells, can be
used in the discovery of the BHS in the 1960s, and redis- resolved [42]. A high-output numerical aperture of an illu-
covery of the PVS in the 2000s [39], although it was used mination system allows utilizing high numerical apertures
mainly for PVS identification instead of characterization. If of the microscope objective and produces highly luminous
it is to be used for PVS characterization, the micro- and/or super-resolution images.
submicrostructural components and translucent optical Figs. 4 and 5 are images with significantly enhanced
property of the PVS, however, present serious challenges. resolution and contrast, taken by the Auburn University
In the visible light transmission and reflection microscopy, team with proper adjustments described above. Fig. 4
even for advanced confocal devices, the lateral image shows an image of an intraexternal PN connected to a PV
resolution would not exceed 180 nm [40], requiring sub- [3], which was harvested from the surface of the rat in-
stantially enhanced resolution [41] for detailed structural testine. PNs are usually linked to the PVs at their both ends,
characterization of the PVS. and in some cases, several PVs are connected to a single
Optical resolution up to the submicron level may, how- PN. This particular PN was, however, connected to the PV
ever, be achieved via the annular illumination to increase at its one end only (Figs. 4A and 4B). Figs. 4B and 4D are
the intensity of diffraction fringes, and via the coherent enlarged images of the rectangular sections in Figs. 4A and
illumination of small (compared to wavelength) objects to 4C, respectively. The PVS sample was treated with acridine

Figure 4 Optical images of a nonfixed, intraexternal PVS from a rat, stained with acridine orange. (A) Overall view of the PVS. (B)
A magnified image of the part connecting the PN and PV in Fig. 4A. (C) A magnified image of the branched PV, shown in the dotted
rectangle in Fig. 4A. (D) A further magnified view of the PV shown in the dotted rectangle in Fig. 4C. Its cell nuclei are shown with
arrows. The scale bars in A, B, C, and D are 500 mm, 200 mm, 100 mm, and 50 mm, respectively. PN Z primo node; PV Z primo vessel;
PVS Z primo vascular system.
Technical Challenges in PVS Research and Solutions 303

Figure 5 A transverse sectional view of a small PN. (A) In the brightfield mode, basophilic granules are labeled with black stars.
(B) The same section photographed in the darkfield mode. Vacuoles are indicated by white arrows, intravascular space by a white
arrow head, cluster of granules by a black arrow, a granule in the small lumen by a black arrow head, the largest lumen by a white
star, and the interrupted portion of lumen connected to a vacuole by a dotted white arrow. C Z capsule; L Z lumens; PN Z primo
node.

orange, which is used as a nucleic-acid-selective fluores- As illustrated here, optical microscopy with proper
cent cationic dye. The staining reveals cells fluorescing adjustment can be utilized effectively for PVS character-
with green and red light: the light at a wavelength of ization in a micron/submicron level.
502 nm excites the acridine orange/DNA complex that
emits at 525 nm (green). When it interacts with RNA,
excitation shifts to 460 nm and emission to 650 nm (red) 6. Studying cancer primogenesis via
[47]. In these images, the details of this PVS may be very fluorescent protein
clearly seen: the PN has an oval shape with dimensions of
0.85  1.1 mm2 and the PV length is 1.25 mm. The diameter Most PVS studies performed up to now have focused on the
of the middle part of the vessel is w140 mm. The apical end identification and basic characterization of the system
of the vessel is branched into two smaller vessels. The probably because the system is still new. However, there
branched vessels are connected to other nodes (not shown have been interesting preliminary study results on the be-
here). The green and red colors may be interpreted as DNA haviors of the PVS in/adjacent to cancerous tumors (cancer
and RNA of cell nuclei, but acridine orange also accumu- PVS), at least for six different cancer xenografts. Although
lates and emits red light in mast secretory granules and this topic is not associated with basic characterizations of
other cellular acidic compartments [48,49]. In Fig. 4D, the the normal PVS, we included it in the article because
red color is emitted from the cell cytoplasm rather than cancer affects many people, and a thorough understanding
from the cell nuclei. The nuclei of these cells (shown by of the cancer PVS may lead to better management of the
arrows) are green. These particular cells represent w5% of disease. The cancer xenografts studied up to now are: lung
the entire cell population. Microscopic observation of the (NCI-H460) and ovarian (SK-OV-3) cancer [9,14], cutaneous
mast cells under the acupuncture meridian lines of humans melanoma (B16 F10) [55], breast cancer (MDA-MB231),
and rats revealed that the mast cells were more concen- gastric cancer (MKN12) [56], human leukemia (U937) [57],
trated under the meridian lines in comparison with their and also human breast cancer mimicking cell line 4T1 [58].
control areas. Therefore, the red-stained cells in the figure The PVS in/on normal tissues or organs is usually neither
may be mast cells [50e53]. According to this result, the very densely populated nor easily observed. The cancer
mast cells represent w5% of acridine orange-stained cells PVS, however, has been shown to form at a higher density
in the PVS, which is close to 4%, as quantified by Lim et al in/around the tumor. In addition, cancer PVS has been
[53], but less than 20%, as reported by Kwon et al [13]. The shown to participate in cancer cell transport from the pri-
difference may be due to variations in techniques and mary to the secondary tumors [9,14].
experimental conditions. To learn how the cancer PVS proliferates, Heo et al [55]
Fig. 5 illustrates the transversal section of a PN in the used a combination of green fluorescent protein (GFP)-
bright and dark field modes (Figs. 5A and 5B, respectively). expressing host animals and nonfluorescing human skin
The walls of sub-PVs are smooth, single layered, and un- cancer cell line, and the result showed that GFP PVS was
interrupted, except for the wall of the lumen labeled by the formed inside the tumor. The Soh team [56] used a GFP-
white star. There is a small opening in the wall that leads to a expressing cancer cell line in nonfluorescing animal
vacuole (dotted white arrow). Some sub-PVs run very close to models and observed the GFP-producing cancer cells
each other, with a small intravascular space between them transporting via the PVS. Results of another study, by Islam
(white arrow head). The section also shows randomly et al [57], using a lymphoma xenograft showed that the
scattered single cells and granules, and some granules are in cells in the cancer PN were human-origin, i.e., from the
clusters (black arrow). These features shown in the optical cancer, not from the host. These cells exhibited stem-cell-
image of the PN cross section agree with those described by specific transcription factor KLF4 at a significantly upregu-
electron and light microscopy in general [22,54]. lated level. It is still not clear whether the cancer PVS is
304 K.A. Kang et al.

formed from the host, tumor, or both, and whether these 8. Conclusion
cells are in fact cancer stem cells, all of which are very
important information for the cancer metastasis and the For the past decade, there have been numerous scientific
sources of cancer stem cells. reports and articles on the BHS/PVS. Based on the already
The study using a GFP-expressing host with no- reported findings, the PVS has been proven to be an inde-
fluorescence-expressing cancer cells [55] may not have pendent vascular system and it may have important roles in
complete information on the hostexenograft communica- mammalian physiology, even though its existence is still not
tion because GFP can provide information from the host known to many scientists.
only. By the same reason, the study using a GFP-expressing In this article, we have discussed difficulties to be
cancer cell line in nonfluorescing animal models was not overcome in the current PVS research approach with sug-
able to tell us the roles of the host animal in cancer pri- gested new methods. It is our hope that this article will be
mogenesis. A more complete method suggested here is helpful for PVS scientists in general, and that other scien-
combining the animal model expressing one fluorescing tists would join us to share their ideas to resolve other
protein (e.g., GFP) and the cancer cells expressing another problems so that we can together elucidate the roles and
fluorescing protein (e.g., red fluorescent protein). This functions of the PVS in mammals. Ultimately, we hope that
method can help in finding the origin (i.e., the host or the accumulated knowledge on the PVS, via effective commu-
cancer) of the PVS and its subunits by individual fluores- nication among PVS scientists, is utilized for the preven-
cence. A study of the cancer PVS growth path and pattern, tion, diagnosis, and treatment of diseases.
and cancer P-microcell production by the color of fluores-
cence should tell us the role of the PVS in cancer primo-
genesis and the source of the cancer stem cells. Disclosure statement

The authors declare that they have no conflicts of interest


7. Nontechnical issues and no financial interests related to the material of this
manuscript.
In addition to the numerous technical issues that need to be
overcome, there are also nontechnical issues that affect the References
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