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•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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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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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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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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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 

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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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