OPEN 3 ACCESS Freely available online
•0-PLOS I ONE
Identification of Biological Properties of Intralymphatic
Tumor Related to the Development of Lymph Node
Metastasis in Lung Adenocarcinoma
Keisuke Kirita 1 ' 2 ' 4 , Genichiro Ishii 1 *, Rie Matsuwaki 1 ' 3 , Yuki Matsumura 3 , Shigeki Umemura 2 ,
Shingo Matsumoto 2 , Kiyotaka Yoh 2 , Seiji Niho 2 , Koichi Goto 2 , Hironobu Ohmatsu 2 , Yuichiro Ohe 2 ' 4 ,
Kanji Nagai 3 , Atsushi Ochiai 1
1 Pathology Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba, Japan, 2 Division of Thoracic Oncology, National Cancer
Center Hospital East, Chiba, Japan, 3 Division of Thoracic Surgery, National Cancer Center Hospital East, Chiba, Japan, 4 Juntendo University Graduate School of Medicine,
Tokyo, Japan
Abstract
Background: Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph
node metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the
development of lymph node metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC)
related markers in cancer cells and the number of infiltrating stromal cells.
Material and Methods: Primary lung adenocarcinomas with lymphatic permeation in the extratumoral area were
retrospectively examined (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4,
NANOG, SOX2 and Caveolin-1 in the intralymphatic cancer cells to evaluate their relationship to lymph node metastasis.
Moreover, the number of infiltrating stromal cells expressing CD34, oc-smooth muscle actin, and CD204 were also evaluated.
Results: Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and
a high number of CD204(+) macrophages were independent predictive factors for lymph node metastasis (P = 0.004,
P = 0.008, and P = 0.028, respectively). Among these factors, only low ALDH1 expression in cancer cells was significantly
correlated with the farther spreading of lymph node metastasis (mediastinal lymph node, pathological N2) (P = 0.046) and
the metastatic lymph node ratio (metastatic/resected) (P = 0.028). On the other hand, in the primary tumors, ALDH1
expression in the cancer cells was not associated with lymph node metastasis. Intralymphatic cancer cells expressing low
ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015).
Conclusions: Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204
have a critical impact on lymph node metastasis. Our study also highlighted the significance of evaluating the biological
properties of intralymphatic tumors for tumor metastasis.
Citation: Kirita K, Ishii G, Matsuwaki R, Matsumura Y, Umemura S, et al. (2013) Identification of Biological Properties of Intralymphatic Tumor Related to the
Development of Lymph Node Metastasis in Lung Adenocarcinoma. PLoS ONE 8(12): e83537. doi:10.1371/journal.pone.0083537
Editor: Kaustubh Datta, University of Nebraska Medical Center, United States of America
Received June 23, 2013; Accepted November 5, 2013; Published December 23, 2013
Copyright: © 2013 Kirita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Cancer Center Research and Development Fund (23-A-1 2 and 23-K-1 8), the Foundation for the Promotion of
Cancer Research, 3rd-Term Comprehensive 1 0-Year Strategy for Cancer Control, the Advanced research for medical products Mining Programme of the National
Institute of Biomedical Innovation (NIBIO), and JSPS KAKENHI (24659185). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gishii@east.ncc.go.jp
Introduction
Lung adenocarcinoma is the most common histological type
among primary lung cancers and is one of the most frequent
causes of death among cases with advanced disease [1,2]. Lymph
node (LN) metastasis is conceivably the preliminary step of distant
metastasis and is directly linked with a poor prognosis [3,4] . The
process of LN metastasis starts with the intravasation of cancer
cells followed by escape from anoikis in the lymphatics, and
transmigration to the LNs [5,6].
Recently, investigators studying the cancer initiating/ stem cells
(CIC/CSCs) hypothesis have suggested the existence of a subset of
cancer cells with the ability to undergo metastasis initiation,
including those characterized by a high migration potential and an
ability to adapt to the metastatic site [7] . In particular, cancer cells
involved in metastasis formation, which are also called metastasis-
initiating cells, were identified by their close relationship with
CIC/CSCs, and the elucidation of these properties was regarded
as being critically important. In non-small cell lung cancer
(NSCLC), the expression of CIC/CSCs-related markers, for
example aldehyde dehydrogenase 1 (ALDH1), octamer-binding
transcription factor 4 (OCT4), NANOG, SRY-box 2 (SOX2), and
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Caveolin-1, were reportedly correlated with treatment resistance,
and disease recurrence, and survival [8-11].
Recent reports have revealed that cancer-associated stromal
cells present with the primary or circulating tumor cells play an
important role in cancer cell survival and transmigration [12,13].
Another study revealed that circulating stromal cells with tumor
fragments promote the rapid growth of accompanying metastatic
cancer cells [6].
The lymphatics within the extratumoral area have been
postulated to act as conduits connecting the primary site with
the metastatic LN (Figure 1). We hypothesized that the biological
characteristics of intralymphatic tumor cells in the extratumoral
area may be more informative than those of the primary tumors
with regard to clarifying the process of cancer metastasis. The aim
of the present study was to identify how the immunophenotypic
features of cancer cells and infiltrating stromal cells in the
extratumoral lymphatics are correlated with LN metastases.
Materials and Methods
Ethic Statement
Collection and the use of biopsies from lung adenocarcinoma
patients were approved by the National Cancer Center Institu-
tional Review Board (approval number: 2013-026). The written
informed consent was obtained from all patients involved in the
study.
Patients
A total of 2087 consecutive adenocarcinoma patients underwent
complete resections involving a lobectomy or more extensive
procedures and systematic LN dissections between May 1 998 and
December 2012 at the National Cancer Center Hospital East.
Patients who received preoperative therapy (chemotherapy or
thoracic radiotherapy), who underwent a limited surgery (seg-
mentectomy or wedge resection), or in whom a mediastinal
lymphadenectomy was not performed were excluded. Among the
remaining cases, we selected those that had been diagnosed as
having lymphatic permeation in the extratumoral area according
to previously reported criteria [14]. A total of 127 cases were
selected, but 20 cases with poor-quality specimens were subse-
quently excluded. The remaining 107 cases were included in the
present study. The clinicopathological features of the cases were
collected from the clinical records.
We defined a pNl state as the involvement of an ipsilateral
intrapulmonary, peribronchial, or hilar LN metastasis and a pN2
state as the involvement of a mediastinal or subcranial LN
metastasis according to the 7 th edition of the TNM classification.
The percentage of metastatic LNs was calculated by dividing the
number of metastatic LNs by the number of dissected LNs and
Figure 1. Scheme showing the process of lymphogenic metastasis.
doi:1 0.1 371 /journal.pone.0083537.g001
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Intralymphatic Tumor and Lymphogenic Metastasis
multiplying by 100. The median number of dissected LNs was 14
nodules.
Histopathological Studies
Surgical specimens were fixed in 10% formalin or methanol and
were embedded in paraffin. The specimens were then sectioned
(4 urn) and stained using hematoxylin and eosin. Vascular invasion
and pleural invasion were also evaluated using Verhoeff-Van
Gieson staining.
The histologic diagnoses were based on the fourth-revised
World Health Organization's histologic classification. The disease
stages were based on the 7 th edition of the TNM classification. All
extratumoral lymphatic permeations were confirmed by immuno-
staining with anti D2-40 antibody. The median number of
evaluated lymphatic vessels that contained tumor tissue was 7.
Immunohistochemistry
The markers used in this study were ALDH 1 (clone 44ALDH;
BD Bioscience, San Jose, GA, USA), OCT4 (clone 4H2; Applied
Biological Materials, Richmond, BC, Canada), NANOG (clone
2C4; Applied Biological Materials), SOX2 (clone 3A2; Applied
Biological Materials), Caveolin-1 (clone D46G3; Cell Signaling
Technology, Danvers, MA, USA), a-Smooth muscle actin (oc-
SMA; clone 1A4; DAKO, Glostrup, Denmark), CD34 (clone
QBENT/10; Acris antibodies, San Diego, CA, USA), CD204
(Scavenger Receptor class A-E5; Trans Genie Hyogo, Japan), and
E-cadherin (clone NCH-38; DAKO). The slides were deparaffi-
nized with xylene and rehydrated in a graded ethanol series. For
antigen retrieval, the sections were placed in citrate buffer and
were heated at 95°C for 20 minutes. After the inhibition of
endogenous peroxidase activity, individual slides were incubated
overnight at 4°C with primary antibodies. The slides were washed
with phosphate-buffered saline and then incubated with EnVision
(DAKO) for 1 hour at room temperature, and the color reaction
was developed in 2% 3, 3'-diaminobenzidine in 50 mM Tris
buffer (pH 7.6) containing 0.3% hydrogen peroxidase. Finally, the
sections were counterstained with Meyer hematoxylin, dehydrat-
ed, and mounted. Omission of primary antibody served as a
negative control in each marker and we confirmed there was no
positive product. The normal bronchial epithelial cells (in ALDH1
and SOX2), alveolar macrophages (in CD204 and ALDH1) were
used as internal positive controls.
Evaluation of immunohistochemistry
Two pathologists (K.K. and G.I.) who had no knowledge of the
patient's clinicopathological data evaluated the individual sections
under a light microscope. The labeling scores for the cancer cells
were calculated by multiplying the percentage of positive cancer
cells per each lesion (0%-100%) by the staining intensity level (0 =
negative; 1 = weak; 2 = strong). As for ALDH1, the nuclear and
cytoplasmic expressions were judged as positive. OCT4, NANOG,
and SOX2 expression were judged as positive when nuclear
expression was observed. On the other hand, Caveolin-1 and E-
cadherin expressions were positive when the cellular membrane
and cytoplasmic expressions were observed (Figure 2A-E, I). The
cut-off values were defined as the median staining score. For a-
SMA, CD34 and CD204, the number of positive infiltrating cells
were counted under a microscope at x400 (area = 0.0625 mm ;
Figure 2G-I). The cut-off values were defined as the median
number of positive infiltrating cells. The inter-observer variability
of staining evaluation was very little differentiation and the
concordance was high (Cohen's kappa coefficients stayed with in
0.88 to 1.00 in each evaluated proteins).
Statistical Analysis
The significance of differences between two groups was
evaluated using the Fisher exact test, a multinomial logistic
regression analysis, or the Student t-test. All the reported P-values
were two sided, and the significance level was set at <0.05. The
statistical analyses were performed using JMP® for Macintosh,
version 9.0 (SAS Institute Inc., Cary, NC, USA).
Results
Relationship between clinicopathological characteristics
and lymph node metastasis
Ipsilateral intrathoracic LN metastases were detected in
specimens from 86 patients (80%). Intrapulmonary metastases
were occasionally detected in resected specimens from 35 patients
(33%). The disease-free survival and overall survival periods were
significantly shorter in the group with lymph node metastasis
(P= 0.010 and P= 0.018, respectively) (Figure SI).
A univariate analysis revealed that only intrapulmonary
metastasis was significantly associated with LN metastasis. Sex,
age, smoking history, tumor size, vascular invasion, and pleural
invasion were not significantly associated with LN metastasis in
this cohort (Table 1).
Univariate and multivariate analyses of cancer cells and
stromal cells for the presence of LN metastasis
The median staining scores (cut-off value) were 0 for ALDH 1 ,
90 for OCT4, 20 for NANOG, 30 for SOX2, and 0 for Caveolin-
1, respectively. In univariate analyses, a low ALDH1 expression
(P= 0.004) and a high SOX2 expression (P= 0.008) in cancer cells
were significandy correlated with LN metastasis. However, no
significant correlations were observed between LN metastasis and
the expression levels of OCT4, NANOG, or Caveolin-1 (Table 2).
Table 3 shows the relationship between infiltrating stromal cells
and LN metastasis. The median number of CD204(+) macro-
phages was 4.5 (cells/0.625 mm 2 ). However, the number of cases
with CD34(+) endothelial cells and a-SMA(+) myofibroblasts were
9 and 7 cases, respectively. In the univariate analyses, only a high
number of intralymphatic CD204(+) macrophages was signifi-
candy correlated with LN metastasis (P— 0.023).
Multivariate logistic regression models were used to determine
the independent factors affecting LN metastasis. A low ALDH1
expression, high SOX2 expression, and a higher number of
CD204(+) macrophages were independent predictive factors for
LN metastasis (odds ratio [95%CTJ = 3.25 [1.11-9.82], P= 0.031
for ALDH1; 4.09 [1.38 - 13.4], P= 0.011 for SOX2; and 3.45
[1.16 - 1 1.4], P= 0.026 for CD204(+) macrophages).
We also evaluated the relationship between these factors and
LN metastasis in the primary tumor (Table 4). However, only a
high SOX2 expression level in the cancer cells within the primary
tumor was significandy correlated with LN metastasis (p = 0.008);
ALDH1 expression in the cancer cells and the number of
CD204(+) macrophages were not correlated with LN metastasis
(P= 0.230 and P= 0.088, respectively). Relationship between
other clinicopathological characteristics and immunohistochemi-
cal staining in primary tumors were shown in Table S1-S3.
Correlation of ALDH1, SOX2 expression in cancer cells
and the number of CD204(4-) macrophages with the
aggressiveness of LN metastasis
We investigated the expansion of thoracic LN metastasis based
on the pNl and pN2 classification in patients with LN metastasis.
pN2 disease, which is characterized by the more distant spreading
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Intralymphatic Tumor and Lymphogenic Metastasis
Figure 2. Immunohistochemical staining of intralymphatic cancer cells (A, B, C, D) and stromal cells (E, F). (A) high ALDH1 expression
level, (B) low ALDH1 expression level, (C) high SOX2 expression level, (D) low SOX2 expression level, (E) higher number of CD204-positive
macrophages, (F) lower number of CD204-positive macrophages.
doi:10.1371/journal.pone.0083537.g002
of LN metastasis, is known to represent a higher degree of
malignancy in lung adenocarcinomas [4] .
A total of 20 patients were diagnosed as pNl, and 66 patients
were diagnosed as pN2. A low ALDH1 expression level was
significandy more frequent among the pN2 patients (P= 0.046);
however, the high expression of SOX2 and a higher number of
CD204(+) macrophages were not associated with a pN2 diagnosis
(P=0.440 and 0.121, respectively)(Table 5).
The metastatic LN ratio can also reflect the aggressiveness of
metastasis [15—18]. A low ALDH1 expression level was signifi-
cantly correlated with a high percentage of metastasis (P— 0.015),
but the expression of SOX2 and a higher number of CD204(+)
macrophages were not (P= 0.372 and 0.054, respectively)
(Table 6). Based on the above findings, a low ALDH1 level in
cancer cells was suggested to have the strongest impact on
lymphogenic metastasis.
Correlation between ALDH1 expression and E-cadherin
expression in intralymphatic cancer cells
Recent studies have linked the epithelial-mesenchymal transi-
tion (EMT) process to the induction of metastasis-initiating
features [19,20]. We analyzed the expression of E-cadherin in
the cancer cells and its association with the ALDH1 expression
status.
As shown in Figure 3, the cancer cells in the low ALDH1
expression group had a significantly lower E-cadherin score (mean
± SE, 27.8+3.1 for low ALDH1 group and 40.8±4.3 for high
ALDH1 group, P= 0.014). The expressions of other CIC/CSCs
related markers, including OCT4, NANOG, SOX2, and
Caveolin-1, were not correlated with the E-cadherin score (Figure
S2).
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Intralymphatic Tumor and Lymphogenic Metastasis
Table 1. Relationship between clinicopathological characteristics and lymph node metastasis.
Category
Subcategory
LN meta. (+) N = 86
LN meta. (-) N = 21
p-value
Sex
Male
61
13
0.438
Female
25
8
Age, year
Median (range)
67 (41-86)
>70
32
8
1
70>
54
13
Smoking
Ex or current
59
13
0.608
Never
27
8
Tumor size, cm
&3.0
38
12
0.286
3.0>
48
9
Histology
Mixed subtype
77
20
0.899
Others **
9
1
Vascular invasion
Positive
65
15
0.78
Negative
21
6
Pleural invasion
Positive
48
13
0.806
Negative
38
8
Pulmonary metastasis
Positive
34
1
0.002*
Negative
52
20
Considered to be statistically significant (p<0.05).
••Solid adenocarcinoma with mucin.
doi:l 0.1 371 /journal.pone.0083537.t001
Discussion
Since tumors in the extratumoral lymphatics are considered to
represent the preceding phase of LN metastasis, examining the
biological characteristics of these cells is likely to be important for
elucidating the mechanism of lymphogenic metastases. The
current study demonstrated that the low expression of ALDH1
in cancer cells within the lymphatics was an independent
predictive factor of LN metastasis. Furthermore, low ALDH1
expression impacts metastatic aggressiveness, including pN2
disease with farther spreading and the metastatic LN ratio. We
also found that a higher number of CD204(+) macrophages in the
Table 2. Univariate analysis about relationship of immunohistochemical staining of intralymphatic tumor and LN metastasis
(Cancer cells).
Antibodies
Score
LN meta. (+) N = 21
LN meta. (-) N = 86
p-value, univariate
Odds ratio (95%CI)
p-value, multivariate
ALDH1
high
13
23
•0.004
low/high, 3.25 (1.11-
9.28)
*0.031
low
8
63
OCT4
high
8
46
0.232
low
13
40
NANOG
high
10
53
0.323
low
11
33
SOX2
high
6
53
*0.008
high/low, 4.09 (1.38-
13.4)
•0.01 1
low
15
33
Caveolin-1
high
1
12
0.456
low
20
74
•Considered to be statistically significant (p<0.05).
doi:1 0.1 371 /journal.pone.0083537.t002
extratumoral lymphatics was associated with LN metastasis. The
results of this study suggest the possibility that the microenviron-
ment of the tumor tissue created by intralymphatic cancer cells
and stromal cells in the extratumoral area has a considerable
impact on LN metastasis.
We performed survival analysis, however, only intralymphatic
SOX2 expression significandy associated with overall survival, but
intralymphatic ALDH1 and CD204 positive macrophages did not.
Because this study population included the cases performed
operation until quite recently, we considered the reason of those
results was affected by insufficient follow up time and many
censored cases.
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Intralymphatic Tumor and Lymphogenic Metastasis
Table 3. Univariate analysis about relationship of immunohistochemical staining of intralymphatic tumor and LN metastasis
(Infiltrating stromal cells).
Antibodies Score
LN meta. (+) N = 21 LN meta. (— ) N = 86 p-value, univariate
Odds ratio (95%CI) p-value, multivariate
SMA high
1 6 1
low
20 80
CD34 high
1 8 0.685
low
20 78
CD204 high
6 49 *0.028
low/high, 3.45 (1.16-11.4) 0.026
low
15 37
Considered to be statistically significant (p<0.05).
doi:1 0.1 371 /journal.pone.0083537.t003
Table 4. Univariate analysis about relationship of immunohistochemical staining of primary tumor and LN metastasis.
Antibodies Score
LN meta. (+) N = 21 LN meta. (-) N = 86
p-value, univariate
ALDH1 high
14 44
0.23
low
7 42
SOX2 high
6 51
•0.008
low
15 35
CD204 high
7 48
0.088
low
14 38
•Considered to be statistically significant (p<0.05).
doi:1 0.1 371/journal.pone.0083537.t004
Table 5. The aggressiveness of lymph node metastasis (Pathological N stage).
Antibodies Score
pN1:N = 20 pN2:N = 66
p-value, univariate
ALDH1 high
9 14
•0.046
low
11 52
SOX2 high
14 39
0.44
low
6 27
CD204 high
8 41
0.121
low
12 25
•Considered to be statistically significant (p<0.05).
doi:1 0.1 371 /journal.pone.0083537.t005
Table 6. The aggressiveness of lymph node metastasis (Metastatic lymph node ratio).
Antibodies Score
Percentage of metastatic LN (%) Mean, (±SE)
p-value, univariate
ALDH1 high
23.0 (±4.7)
•0.015
low
37.2 (±3.3)
SOX2 high
34.6 (±3,7)
0.372
low
29.6 (±4.2)
CD204 high
37.6 (±3.8)
0.054
low
26.9 (±3.9)
•Considered to be statistically significant (p<0.05).
doi:1 0.1 371 /journal.pone.0083537.t006
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Intralymphatic Tumor and Lymphogenic Metastasis
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Figure 3. Relationship between ALDH1 and E-cadherin expression. (A) intralymphatic cancer cells showing a high level of ALDH1 expression,
and (B) intralymphatic cancer cells showing a high level of E-cadherin expression. The images shown in A and B were obtained from the same case.
(C) Intralymphatic cancer cells with a low level of ALDH1 expression. (D) Intralymphatic cancer cells with a low level of E-cadherin expression. The
images shown in C and D were also obtained from the same case. (E) Comparison of E-cadherin expression between cancer cells with low and high
levels of ALDH1 expression.
doi:1 0.1 371 /journal.pone.0083537.g003
The prognostic value of ALDH 1 expression in cancer cells in
the primary lesion is controversial. A high ALDH1 expression
level has been used as a biomarker predicting a poor prognosis in
breast cancer, serous ovarian cancer, colorectal cancer, and
several other tumors [21-23]. In contrast, Dimou et al. noted that
the expression of ALDH 1 was independently associated with a
better prognosis in patients with NSCLC, especially those with
adenocarcinoma. In melanoma cells, both ALDH-positive and
ALDH-negative cells have exhibited similarly high clonal forma-
tion abilities in vitro and cancer initiation abilities in vivo when
isolated from melanoma xenografts [24]. Okudela et al. demon-
strated that the forced expression of ALDH1A1 in a NSCLC cell
line remarkably reduced clonogenicity and prolonged the doubling
time in vivo [25], and these results are compatible to those obtained
in the present study. To clarify the role of ALDH1 in
intralymphatic cancer cells more clearly, the in vivo and/ or in vitro
relevance of ALDH1 to tumorigenicity should be examined in
ALDH 1 -sorted cancer cells.
In recent studies examining the EMT, the loss of E-cadherin
expression has been shown to play a key role in the metastatic
process [19,20]. This reflection in part supports our results that a
low ALDH1 expression in intralymphatic cancer cells was
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Intralymphatic Tumor and Lymphogenic Metastasis
correlated with a low E-cadherin expression. Another noteworthy
fact is that the expression of ALDH 1 in cancer cells in the primary
tumor was not correlated with the expression of E-cadherin
(Figure S3) and was not a significant predictive factor for LN
metastasis. Thus, our new findings suggest the possibility that a low
ALDH1 expression level in cancer cells present only in the
extratumoral lymphatics might induce the EMT, leading to the
acquisition of tumorigenic capacity.
About ALDH1, downregulated cases (staining score in intra-
lymphatics decreased less than one-half in primary tumors)
significandy associated with lymph node metastasis than other
cases (p = 0.012) (Table S4). Otherwise, about the group with
SOX2 upregulation or increased CD204+ macrophages (staining
score in intralymphatics increased more than double in primary
tumors), the frequency of lymph node metastasis was almost
equivalent than other cases (p = 0.440 and 0.584, respectively). It is
plausible to think that deregulation of ALDH1 expression in
intralymphatics tumor cells may impact on lymphogenic metas-
tasis by occurring EMT.
Macrophage M1/M2 polarization with either pro-inflammato-
ry or anti-inflammatory properties impact on malignant neoplasm
progression and could be discriminated by immunohistochemical
staining. CD204 was macrophage scavenger receptor, and thought
to be expressed not only M2 macrophages but dendritic cells. We
examined the concordance between number of CD204+ cells and
another M2 macrophage marker using CD 163. As shown in figure
S4, significantly high correlation between CD204 number and
CD 163 number in both primary site (N = 40) and intralymphatics
(N = 40) (R-value were 0.86 and 0.89, respectively), suggesting that
CD204 cells that we counted were M2 macrophages but not
dendritic cells.
In the current study, a higher number of intralymphatic
CD204(+) macrophages was significantly correlated with LN
metastasis. We previously reported that the number of CD204(+)
macrophages was correlated with an intralymphatic tumor
morphology consisting of multiple small nests, which tends to
occur in cases with pulmonary metastasis [6]. CD204(+) macro-
phages reportedly exhibit a tumor-promoting function in several
tumors by secreting matrix metalloproteinase (MMP)-1, MMP-3,
MMP-7, MMP-9, MMP-12, vascular endothelial growth factor
(VEGF), and transforming growth factor (TGF)-|3 [26,27]. Taken
together, intralymphatic CD204(+) macrophages may also play an
important role in the early lymphogenic metastatic process
through the promotion of extracellular matrix remodeling,
angiogenesis, and the EMT at a secondary site [28,29].
SOX2 has been considered as a CIC/CSCs related molecule.
Xiang et al noticed that the knockdown of SOX2 in cancer cells
suppressed experimental pulmonary metastases in an animal
model, suggesting that cancer cells in which SOX2 expression is
upregulated might be a candidate for metastasis-initiating cells.
[30] Singh et al. demonstrated that SOX2 expression had a more
critical impact on cancer cell-proliferation than OCT4 and
NANOG [31,32], and a high level of SOX2 expression is also
known to be a prognostic factor in NSCLC [33]. Our result
confirms that SOX2 also plays an important role in the
progression of cancers caused by lymphogenic metastasis. Addi-
tionally, a high SOX2 expression level in primary cancer cells was
also associated with LN metastasis, which differs from ALDH1
and CD204(+) macrophages in this respect. Cancer cells with high
References
1. Jcmal A, Sicgcl R, Ward E, Hao Y, Xu J, ct al. (2009) Cancer statistics, 2009.
CA Cancer J Clin 59: 225-249.
levels of SOX2 expression might also have an impact on the
lymph vessel invasion process at the primary site.
In conclusion, this study showed the significance of evaluating
the biological properties of intralymphatic tumors. Moreover, the
microenvironment of intralymphatic tumors created by cancer
cells with a low level of ALDH1 expression and a number of
CD204(+) macrophages has a significant impact on LN metastasis.
The current results also suggest the possibility that intralymphatic
cancer cells with a low level of ALDH1 expression and CD204(+)
macrophages could be useful as a new molecular target, especially
as an adjuvant therapy, in patients exhibiting lymphatic perme-
ation. Further in vivo investigation of the metastatic mechanism will
be important.
Supporting Information
Figure SI Kaplan-Meier analysis for disease-free sur-
vival and overall survival stratified according to the
existence of lymph node metastasis. The median follow-up
period was 22.4 months. (A) Disease-free survival for all patients.
(B) Overall survival for all patients.
(TIF)
Figure S2 Comparison of E-cadherin expression be-
tween cancer cells with high and low levels of CIC/
CSCs-related markers. (A) OCT4, (B) NANOG, (C) SOX2,
and (D) Caveolin-1.
(TIF)
Figure S3 Comparison of E-cadherin expression be-
tween ALDH1 in primary tumor cells.
(TIF)
Figure S4 Relationship between CD204+ cells and
CD 163+ cells. (A) Primary tumor tissue. (B) Intralymphatic
tumor tissue.
(TIF)
Table SI Relationship between clinicopathological
characteristics and ALDH1 expression (intralymphatic
tumor cells).
(DOCX)
Table S2 Relationship between clinicopathological
characteristics and SOX2 expression (intralymphatic
tumor cells).
(DOCX)
Table S3 Relationship between clinicopathological characteris-
tics and CD204-positive macrophages expression (intralymphatic
tumor cells).
(DOCX)
Table S4 Molecular expression changes and lymph
node metastasis.
(DOCX)
Author Contributions
Conceived and designed the experiments: KK GI SU SM KY SN KG HO
YO KN AO. Performed the experiments: KK GI RM YM. Analyzed the
data: KK GI. Contributed reagents/materials/analysis tools: KK GI RM
YM YO KN AO. Wrote the paper: KK GI.
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