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ONCOLOGY REPORTS
Abstract. Malignant mesothelioma is an aggressive neoplasm
for which effective treatments are lacking. We often encounter
mesothelioma cases with a profound desmoplastic reaction,
suggesting the involvement of cancer‑associated broblasts
(CAFs) in mesothelioma progression. While the roles of CAFs
have been extensively studied in other tumors and have led to
the view that the cancer stroma contains heterogeneous popu‑
lations of CAFs, their roles in mesothelioma remain unknown.
We previously showed that connective tissue growth factor
(CTGF), a secreted protein, is produced by both mesothelioma
cells and broblasts and promotes the invasion of mesothe‑
lioma cells in vitro. In this study, we examined the clinical
relevance of CAFs in mesothelioma. Using surgical specimens
of epithelioid malignant pleural mesothelioma, we evalu‑
ated the clinicopathological signicance of the expression of
α‑smooth muscle actin (αSMA), the most widely used marker
of CAFs, the expression of CTGF, and the extent of brosis by
immunohistochemistry and Elastica‑Masson staining. We also
analyzed the expression of mesenchymal stromal cell‑ and
broblast‑expressing Linx paralogue (Mein; ISLR), a recently
reported CAF marker that labels cancer‑restraining CAFs and
differ from αSMA‑positive CAFs, by in situ hybridization.
The extent of brosis and CTGF expression in mesothelioma
cells did not correlate with patient prognosis. However, the
expression of αSMA and CTGF, but not Meflin, in CAFs
correlated with poor prognosis. The data suggest that CTGF+
CAFs are involved in mesothelioma progression and represent
a potential molecular target for mesothelioma therapy.
Introduction
Malignant mesothelioma is a tumor that is primarily caused by
exposu re to asbestos, including croc idol ite, amosite, and ch r ys‑
otile (1). Mesothelioma is one of the most lethal tumors, with
an expected median survival time of 4‑18 months for pleural
forms (2). There are three main histological types of meso‑
thelioma: Epithelioid, biphasic and sarcomatoid (2). Clinically,
patients with the sarcomatoid subtype have the poorest prog‑
nosis (3). The molecular mechanisms of asbestos‑induced
mesothelial carcinogenesis have been recently revealed to
include oxidative stress, chronic inflammation, molecular
adsorption, and chromosome tangling (4‑8). It is necessary to
understand the molecular mechanisms that regulate mesothe‑
lial carcinogenesis (9‑14) and to develop molecular‑targeted
drugs (15) to improve the prognosis of patients.
Mesothelioma often features a profound desmoplastic
reaction since asbestos can develop brotic diseases before
mesothelial carcinogenesis (1,2,11), suggesting the involve‑
ment of cancer‑associated broblasts (CAFs) in its progression.
CAFs occupy the majority of the area in the tumor stroma
and produce extracellular matrix (16‑20). One of the most
well‑known CAF markers is α‑smooth muscle actin (αSMA). In
Connective tissue growth factor produced by
cancer‑associated broblasts correlates with poor prognosis
in epithelioid malignant pleural mesothelioma
YUUKI OHARA1,2, ATSUSHI ENOMOTO3, YUTA TSUYUKI3, KOTARO SATO1, TADASHI IIDA3,
HIROKI KOBAYASHI3, YASUYUKI MIZUTANI3, YUKI MIYAI3, AKITOSHI HARA3, SHINJI MII3,
JUN SUZUKI2, KYOKO YAMASHITA1, FUMIYA ITO1, YASHIRO MOTOOKA1, NOBUAKI MISAWA1,
TAKAYUKI FUKUI4, KOJI KAWAGUCHI4, KOHEI YOKOI4 and SHINYA TOYOKUNI1,5
1Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine,
Nagoya 466‑8550; 2Division of Pathology and Molecular Diagnosis, National Cancer Center Hospital East,
Kashiwa 277‑8577; Departments of 3Pathology and 4Thoracic Surgery, Nagoya University Graduate School of Medicine,
Nagoya 466‑8550, Japan; 5Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia
Received October 24, 2019; Accepted April 29, 2020
DOI: 10.3892/or.2020.7669
Correspondence to: Dr Yuuki Ohara or Professor Shinya
Toyokuni, Department of Pathology and Biological Responses,
Nagoya University Graduate School of Medicine, 65 Tsurumai‑cho,
Showa‑ku, Nagoya 466‑8550, Japan
E‑mail: yuuki.oohara.1196@gmail.com
E‑mail: toyokuni@med.nagoya‑u.ac.jp
Abbreviations: αSMA, α‑smooth muscle actin; CAFs,
cancer‑associated fibroblasts; CTGF, connective tissue growth
factor; DAB, 3,3'‑diaminobenzidine; H&E, hematoxylin and eosin;
IHC, immunohistochemistry; ISH, in situ hybridization; Meflin,
mesenchymal stromal cell‑ and fibroblast‑expressing Linx paralogue;
ROS, reactive oxygen species
Key word s: connective tissue growth factor, CTGF, cancer‑
associated fibroblasts, tumor microenvironment, malignant
mesothelioma, mesenchymal stromal cell‑ and fibroblast‑expressing
of a Linx paralogue, Meflin, ISLR, molecular target therapy
OHARA et al: CTGF+ CAFs AS A MOLECULA R TARGET FOR MESOTHELIOMA
2
general, CAFs have been shown to exert protumorigenic effects
by promoting cancer cell proliferation and invasion. However,
recent studies have shown that CAFs are heterogeneous, and
the existence of a type of CAF with antitumor functions has
been proposed (19‑23). Studies of CAF heterogeneity have led
to the proposal of multiple CAF markers (16‑23). Connective
tissue growth factor (CTGF) is known as a protumorigenic
CAF marker (18‑20). CTGF is a 36‑38 kDa multifunctional
secretory protein involved in various functions, including cell
proliferation, cell invasion and myobroblast differentiation.
We previously demonstrated that CTGF expression is corre‑
lated with the malignant behavior of mesothelioma cells (13)
and that a CTGF‑specific monoclonal antibody (FG‑3019,
pamrevlumab), which is currently under clinical trials for
idiopathic pulmonary brosis (24,25) and pancreatic ductal
adenocarcinoma (26), was found to inhibit mesothelioma
growth (15). Interestingly, we found that CTGF is expressed
in both mesothelioma cells and CAFs (15). In contrast,
mesenchymal stromal cell‑ and fibroblast‑expressing Linx
paralogue (Mein; ISLR) is a glycosylphosphatidylinositol
(GPI)‑anchored membrane protein, which has been identied
as a marker of mesenchymal stem cells and tissue‑resident
broblasts (27,28). The results of our recent study suggest that
Mein is a potential new marker of antitumorigen ic CAFs (29).
Although the functions and heterogeneity of CAFs have
been recognized in other tumors, those in the mesothelioma
microenvironment have not yet been addressed. We aimed to
understand the signica nce of stromal remodeling during meso‑
thelioma progression. In the present study, we examined the
correlations between patient prognosis and the extent of brosis,
the expression of CAF markers (αSMA, CTGF and Mein), and
the expression of a cell proliferation marker (Ki‑67).
Materials and methods
Patients. A total of 37 patients underwent surgery for malignant
pleural mesothelioma at the Nagoya University Hospital between
January 2007 and December 2016. All patients were reviewed
for age, sex, histological subtype, pathological invasion (pT),
lymph node metastasis (pN), and neoadjuvant therapy. Tumor
classication was performed based on the TNM Classication
of Malignant Tumors (UICC) 7th edition (30). Patients who had
another cancer, who had undergone several surgeries, or who had
undergone only cytoreductive surgery were excluded. Based on
histological and immunohistochemical analyses, patients with
the biphasic or sarcomatoid subtype were also excluded because
of the difculty in distinguishing the mesothelioma cells from
CAFs. In total, 22 samples were ultimately analyzed. Human
mesothelioma tissues were obtained with informed patient
consent at the time of surgery at Nagoya University Hospital
(Nagoya, Japan). This study was carried out in accordance with
the principles of the Helsinki Declaration for human research
and approved by the Ethics Committee of Nagoya University
Graduate School of Medicine (protocol no. 2017‑0127).
Histologic and immunohistochemical analysis. Fou r‑micron‑
thick serial sections were cut from formalin‑fixed and
parafn‑embedded tissue and were stained with hematoxylin
and eosin (H&E) or Elastica‑Masson or for immunohisto‑
chemistry (IHC). The following antibodies were used for
IHC: Anti‑CTGF (goat polyclonal, dilution 1:50; Santa Cruz
Biotechnology, Inc.; cat. no. 14939), anti‑AE1/AE3 (mouse
monoclonal, dilution 1:100; Biocare Medical; cat. no. ACR011A,
B, C), anti‑Ki‑67, clone SP6 (rabbit monoclonal, dilution 1:100;
Abcam; cat. no. 16667), and anti‑αSMA (mouse monoclonal,
dilution 1:50; Dako; cat. no. M0851). High‑temperature
antigen retrieval for CTGF and Ki‑67 was performed using
Immunosaver (Nisshin EM, Tokyo, Japan) and that for αSMA
and A E1/AE3 was performed using 10 mM Tr i s (hydroxy‑
methyl) aminomethane/1 mM ethylenediaminetetraacetic acid
(TE) buffer, pH 9.0. Following antigen retrieval, the sections
were dipped for 30 min in methanol containing H2O2 (0.3%
vol/vol) to quench endogenous peroxidase activity and subse‑
quently blocked with Protein Block Serum‑Free Ready‑to‑use
(Dako). For CTGF staining, the avidin‑biotin complex method
using peroxidase was employed, as previously described (31).
For AE1/AE3, Ki‑67, and αSMA staining, Histone Simple
Stain MAX‑PO (Multi; Nichirei, Tokyo, Japan) was used as
the secondary antibody. Color development was performed
with 3,3'‑diaminobenzidine (DAB, Dako) or HistoGreen
(EUROBIO/ABCYS, Courtaboeuf, France). All images were
obtained using an Olympus BX53 microscope (Olympus,
Japan; objective lens PlanApo N 2X and U PlanApo 4X and
10X) and a DP22/U‑TV0.5XC camera.
Double staining IHC with primary antibodies raised in the
same species. We performed double staining for αSMA and
AE1/AE3. DAB was used to stain αSMA in the rst step, and
HistoGreen was used to stain AE1/AE3 in the second step.
Because both antibodies are mouse monoclonal, high‑temper‑
ature TE buffer was used to inactivate the anti‑αSMA antibody
after DAB staining.
Semiquantitative imaging analysis of the brotic and αSMA
area indices in tumors. The entire tumor mass of each
specimen was digitized using the microscope and camera
described above. Based on previous studies (15,32), we
analyzed the images using ImageJ 1.50i (http://rsb.info.nih.
gov/ij/) and the color deconvolution plugin (http://imagej.
net/Colour_Deconvolution) for ImageJ and Fiji to imple‑
ment staining separation via the method of Ruifrok and
Johnston (33). Fibrosis was detected as a light green color
in Elastica‑Masson staining. The light‑green positive area
was extracted with the color deconvolution plugin (vector:
Feulgen Light Green), and the area was converted to black
(threshold: Upper cutoff, 214; lower cutoff, 0). After this
processing, most of the remaining pixels in the image were
those originally stained in light green, and we calculated the
total area occupied by these pixels. This area was divided
by the entire tumor area, and the extent of brosis (brotic
area index) was determined as previously described (15).
To obtain the αSMA‑positive area (αSMA area index) and
AE1/AE3‑positive area in the entire tumor, we used the same
method used to determine the brotic area index (vector: H
DAB; threshold: Upper cutoff, 200; lower cutoff, 0). Most of
the cases had the reactive pleural brosis with mesothelioma
cell invasion. For the brotic and αSMA area indices, we
evaluated the entire tumor mass including pleural brosis
since invasion to parietal and visceral pleura is a common
pattern of mesothelioma invasion (2) and since the brosis
ONCOLOGY REPORTS 3
can contribute to mesothelioma progression. To minimize the
inuence of tumor heterogeneity, all the elds in the entire
tumor were evaluated. The average of each entire tumor mass
area was 141.7 mm2. We used a 2x objective lens and the
average elds of view for each tumor mass was 7.6.
Semiquantitative analysis of CTGF expression in tumors.
We used the entire tumor mass of each specimen. The CTGF
immunostaining intensity in mesothelioma cells and CAFs
was assessed as follows: 0, negative; 1, weak; 2, moderate;
and 3, strong. In addition, the H‑score for CTGF (CTGF
score) was calculated using the following formula: [1x (% of
cells with an intensity of 1)+2x (% of cells with an intensity
of 2)+3x (% of cells with an intensity of 3)] (34,35). All the
elds were evaluated by a registered pathologist (YO). The
average of each entire tumor mass area was 141.7 mm2. We
used a 4x objective lens and the average elds of view for
each tumor mass was 24.4. For the Kaplan‑Meier survival
curve, the CTGF score for mesothelioma cells and CAFs
was modied by the tumor area (AE1/AE3‑positive area)
and stromal area (αSMA‑positive area) as follows: (CTGF
score x AE1/AE3 or αSMA area index). Using these modied
CTGF scores, the patients were divided into two groups (low
or high).
Figure 1. Fibrotic and αSMA area indices in mesothelioma. (A) Calculation of the brotic area index using ImageJ. The image was obtained from mesothe‑
lioma in pleural brosis. (B) Analysis of Elastica‑Masson staining. Fibrotic area indices for all cases are plotted as a histogram. (C) Calculation of the αSMA
area index using ImageJ. DAB solution was used to stain αSMA, and HistoGreen was used to stain AE1/AE3. Mesothelioma cells were positive for AE1/AE3.
The image was obtained from mesothelioma in pleural brosis. (D) Analysis of αSMA staining. The αSMA a rea indices for all cases are plotted as a h istogram.
(E) Fibrotic area index and patient prognosis based on Kaplan‑Meier survival curves. There were no signicant differences in prognosis based on the brotic
area index (low, <40%; high, ≥40%). (F) αSMA expression and patient prognosis based on Kaplan‑Meier survival curves. There was a signicant difference
(P=0.0262) in prognosis based on the αSMA area index (low, <20%; high, ≥20%). αSMA, α‑smooth muscle actin; DAB, 3,3'‑diaminobenzidine.
OHARA et al: CTGF+ CAFs AS A MOLECULA R TARGET FOR MESOTHELIOMA
4
In situ hybridization of Meflin. In situ hybridization
(ISH) analysis was performed using four‑micron‑thick
formalin‑xed and parafn‑embedded human tissue sections
with the RNAscope technology (RNAscope 2.5 HD Detection
Kit; Advanced Cell Diagnostics) and a custom‑designed probe
of human Meflin according to the manufacturer's instruc‑
tions, as previously described (28,29). Briey, tissue sections
were baked in a dry oven (HybEZ II Hybridization System;
Advanced Cell Diagnostics) at 60˚C for 1 h, deparafnized,
and incubated with H2O2 solution (Pretreat 1 buffer) for
10 min at room temperature. The slides were boiled in target
retrieval solution (Pretreat 2 buffer) for 30 min, incubated
with protease solution (Pretreat 3 buffer) for 30 min at 40˚C,
incubated with the probe for 2 h at 40˚C, and then successively
incubated with Amp1 to 6 reagents. Staining was visualized
with DAB, followed by counterstaining with hematoxylin.
The RNAscope probe was as follows: Human Mein (ISLR)
(NM_005545.3, region 275‑1322; cat. no. 455481). The slides
were evaluated, as previously described (29).
ImageJ software was used for obtaining the merged image
of αSMA + AE1/AE3 and Mein. To detect the Mein‑positive
area, we used the same method used to determine the αSMA
area index (vector: H DAB; threshold: Upper cutoff, 190; lower
cutoff, 0). The Mein‑positive area was indicated by red color
and merged with αSMA + AE1/AE3 using Image Calculator.
Statistical analysis. The data were analyzed using GraphPad
Prism 5 (GraphPad Software). The correlation between the
expression of CAF markers and various clinicopathological
features was analyzed by the Fisher's exact test. Correlation
analysis was performed using non‑parametric method
(Spearman's rank correlation coefcient). The overall survival
rate was calculated according to the Kaplan‑Meier method and
compared using the Log‑rank test if not indicated otherwise.
Gehan‑Breslow‑Wilcoxon test was used if crossover between
the groups was observed at late timepoints. A P‑value of <0.05
was considered as indicative of statistical signicance.
Results
αSMA‑positive (αSMA+) CAFs correlate with patient prog‑
nosis. Through H&E, IHC staining of αSMA + AE1/A E3,
and Elastica‑Masson staining, a high extent of fibrosis was
observed in the reactive pleura present adjacent to mesothelioma
Table I. Fibrotic area index and clinicopathological features of
the mesothelioma cases.
Characteristics Low High P‑value
Age (years) 0.670
<65 6 5
≥65 4 7
Sex 0.571
Male 8 11
Female 2 1
Pathological invasion 0.074
pT1 or pT2 6 2
pT3 or pT4 4 10
Lymph node metastasis 0.391
pN0 4 8
pN1 or pN2 6 4
Stage 0.624
I or II 3 2
III or IV 7 10
Neoadjuvant chemotherapy 0.348
Absent 4 2
Present 6 10
Chemosensitivity >0.999
Grade 0 or 1a 4 7
Grade 1b or 2 2 3
The brotic area index was calculated by Elastica‑Masson staining.
The index did not correlate with clinicopathological features.
Cisplatin and pemetrexed were administered to 16 out of 22 patients
as neoadjuvant chemotherapy. One patient was readministered carbo‑
platin and pemetrexed due to cisplatin‑induced vomiting. Fibrotic
area index: Low, <40%; high, ≥40%.
Table II. αSMA area index and clinicopathological features of
the mesothelioma cases.
Characteristics Low High P‑value
Age (years) >0.999
<65 6 5
≥65 5 6
Sex >0.999
Male 10 9
Female 1 2
Pathological invasion 0.183
pT1 or pT2 6 2
pT3 or pT4 5 9
Lymph node metastasis 0.670
pN0 7 5
pN1 or pN2 4 6
Stage 0.311
I or II 4 1
III or IV 7 10
Neoadjuvant chemotherapy 0.635
Absent 4 2
Present 7 9
Chemosensitivity 0.596
Grade 0 or 1a 4 7
Grade 1b or 2 3 2
The αSMA area index was calculated by the immunohistochemical
staining of αSMA. The index did not correlate with clinicopathological
features. Cisplatin and pemetrexed were administered to 16 out of
22 patients as neoadjuvant chemotherapy. One patient was readministered
carboplatin and pemetrexed due to cisplatin‑induced vomiting. αSMA
area index: Low, <20%; high, ≥20%. αSMA, α‑smooth muscle actin.
ONCOLOGY REPORTS 5
(Fig. S1). To conrm the correlation between brosis or CAFs
and mesothelioma patient features, we rst evaluated the density
of brosis and the presence of CAFs expressing αSMA using
parafn‑embedded sections of mesothelioma samples. Although
epithelioid mesothelioma is positive for AE1/AE3 (2,36),
approximately 20% of sarcomatoid mesothelioma is negative for
AE1/AE3 (37,38). In addition, it has been reported that reactive
spindle cells can be positive for AE1/AE3 in sarcomatoid meso‑
thelioma (39). Although the H&E and IHC staining distinguished
mesothelioma cells from CAFs in epithelioid mesothelioma
(Fig. S2), our cases of biphasic or sarcomatoid mesothelioma
contained cells for which it was difcult to clarify whether they
were mesothelioma cells or CAFs. Thus, we excluded cases of
biphasic and sarcomatoid mesothelioma. The area indices of
brosis and αSMA were quantied based on color deconvolution
(Fig. 1A‑D), and the clinicopathological ndings are summa‑
rized (Tables I and II). The indices of brosis and αSMA did
not correlate with the clinicopathological features. In addition,
no signicant differences in overall survival were found based on
the brotic area index (Fig. 1E). However, a signicant difference
(P=0.0262) was found based on the αSMA area index (Fig. 1F).
CTGF‑positive (CTGF+) CAFs correlate with mesothelioma
cell proliferation and patient prognosis. To conrm CTGF
as a prognostic factor and potential targets, we next evaluated
the expression of CTGF and Ki‑67 using parafn‑embedded
sections. Mesothelial cells, which are nontumorous, did not
express CTGF (Fig. S3), whereas obvious CTGF expres‑
sion was observed in both mesothelioma cells and CAFs
(Figs. 2A‑F and S2). Biphasic and sarcomatoid mesotheliomas
were excluded after the IHC staining results were examined,
as described above. Heterogeneity in CTGF expression was
observed in both mesothelioma cells and CAFs. We therefore
adopted a semiquantitative scoring system (CTGF score; see
Materials and methods) to quantify the expression of CTGF
in each tumor sample (Fig. 3A and B) and compared these
scores with the numbers of Ki‑67‑positive cells (Ki‑67 index).
The CTGF score for CAFs but not that of mesothelioma cells
was correlated with the Ki‑67 index for mesothelioma cells
(Fig. 3C and D). The CTGF score for CAFs was also corre‑
lated with the αSMA area index, while that for mesothelioma
cells was not (Fig. 3E and F). No signicant correlations were
found between CTGF expression in mesothelioma cells and
patient prognosis (Fig. 3G). However, CTGF expression in
CAFs correlated with poor prognosis (Fig. 3H). Notably, the
clinicopathological features (pathological invasion, lymph
node metastasis, and stage) and sensitivities to neoadjuvant
chemotherapy of the examined mesothelioma cases did not
Figure 2. Immunohistochemical staining of CTGF. Both mesothelioma cells and CAFs were stained for CTGF. (A and B) Weak staining; score=1. The arrows
in B indicate CAFs. (C and D) Moderate staining; score=2. (E and F) Strong staining; score=3. The arrows in E indicate mesothelioma cells. The arrows in F
indicate CAFs. All images are shown at the same magnication. CAFs, cancer‑associated broblasts; CTGF, connective tissue growth factor.
OHARA et al: CTGF+ CAFs AS A MOLECULA R TARGET FOR MESOTHELIOMA
6
correlate with CTGF expression in either mesothelioma cells
or CAFs (Tables III and IV), suggesting that CTGF in CAFs
could be used as a marker that specically predicts patient
prognosis.
Figure 3. CTGF expression in CAFs correlates with mesothelioma patient prognosis. (A and B) Analysis of immunohistochemical staining of CTGF. The
H‑score of CTGF (CTGF score) for all cases is plotted as a histogram. (C) CTGF expression in mesothelioma cells and the Ki‑67 index, indicating no
signicant differences. (D) CTGF expression in CAFs and the Ki‑67 index, indicating a positive correlation (r=0.533, P=0.0107; Spearman's correlation test).
(E) CTGF expression in mesothelioma cells and the αSMA area index, indicating no signicant differences. (F) CTGF expression in CAFs and the αSMA area
index, indicating a positive correlation (r=0.502, P=0.0172; Spearman's correlation test). (G) CTGF expression in mesothelioma cells and patient prognosis
based on Kaplan‑Meier survival curves. CTGF scores were modied by the AE1/AE3 area index values. There were no signicant differences in prognosis
based on modied CTGF scores (low, <10; high, ≥10). (H) CTGF expression in CAFs and patient prognosis based on Kaplan‑Meier survival curves. CTGF
scores were modied by the αSMA area index values. There was a signicant difference (P=0.0186) in prognosis based on modied CTGF score (low, <30;
high, ≥30). αSMA, α‑smooth muscle actin; CAFs, cancer‑associated broblasts; CTGF, connective tissue growth factor.
ONCOLOGY REPORTS 7
Meflin as a potential marker for mesothelioma patient
prognosis. We next investigated Mein expression by RNA
ISH using paraffin‑embedded sections. Mesothelial cells,
which are nontumorous, did not express Meflin (Fig. S2),
while CAFs expressed Mein (Fig. 4A and B). More Mein+
CAFs were observed in the αSMA‑negative area than in the
αSMA‑positive area. Meflin expression did not correlate
with the clinicopathological features (Table V). Additionally,
no significant differences were found in patient prognosis
according to Mein expression (Fig. 4C).
Discussion
In the present study, we demonstrated that not only mesothe‑
lioma cells but also cancer‑associated broblasts (CAFs) in
mesothelioma express connective tissue growth factor (CTGF).
CTGF expression in CAFs was found to be correlated with
patient prognosis although CTGF expression in mesothelioma
cells did not. The CTGF score for mesothelioma cells did
not correlate with the Ki‑67 index, but that for CAFs did. In
addition, CTGF expression did not correlate with tumor stage.
If a marker correlates with poor prognosis and tumor stage,
it is possible that the correlation is driven by tumor stage. In
other words, the marker is highly expressed in advanced stage
tumors, which results in a correlation of marker expression
with poor prognosis. In the present study, CTGF expression
was found to be correlated with poor prognosis after surgery
irrespective of the tumor stage diagnosed at surgery. Therefore,
CTGF‑positive (CTGF+) CAFs are directly correlated with
tumor malignancy/progression and CTGF may be a molecular
target for this disease.
Using tissue or serum samples, previous studies have
revealed that sarcomatoid mesothelioma expresses higher
levels of CTGF than the epithelioid subtype (13,40). In
another study, however, all human mesothelioma cell lines
expressed CTGF irrespective of histological subtype (15).
This appa rent inconsist ency can be explained by the result s of
the present st udy, that is, based on CTGF expression by CAFs
in vivo. Cells of sarcomatoid mesothelioma are commonly
spindle‑shaped and accompanied by proliferating nonneo‑
plastic CAFs, making it difficult to distinguish between
these two cell types. Moreover, CTGF‑specic monoclonal
antibody (FG‑3019, pamrevlumab) was reported to exhibit
little effect on cancer cell proliferation in conventional
Table III. CTGF in mesothelioma cells and clinicopathological
features of the mesothelioma cases.
Characteristics Low High P‑value
Age (years) 0.670
<65 7 4
≥65 5 6
Sex 0.571
Male 11 8
Female 1 2
Pathological invasion 0.675
pT1 or pT2 5 3
pT3 or pT4 7 7
Lymph node metastasis 0.691
pN0 6 6
pN1 or pN2 6 4
Stage >0.999
I or II 3 2
III or IV 9 8
Neoadjuvant chemotherapy >0.999
Absent 3 3
Present 9 7
Chemosensitivity 0.308
Grade 0 or 1a 5 6
Grade 1b or 2 4 1
The modied CTGF score of mesothelioma cells did not correlate
with clinicopathological features. Cisplatin and pemetrexed were
administered to 16 out of 22 patients as neoadjuvant chemotherapy.
One patient was readministered carboplatin and pemetrexed due to
cisplatin‑induced vomiting. Modied CTGF score: Low, <10; high,
≥10. CTGF, connective tissue growth factor.
Table IV. CTGF in CAFs and clinicopathological features of
the mesothelioma cases.
Characteristics Low High P‑value
Age (years) 0.198
<65 8 3
≥65 4 7
Sex >0.999
Male 10 9
Female 2 1
Pathological invasion 0.675
pT1 or pT2 5 3
pT3 or pT4 7 7
Lymph node metastasis 0.691
pN0 6 6
pN1 or pN2 6 4
Stage >0.999
I or II 3 2
III or IV 9 8
Neoadjuvant chemotherapy 0.646
Absent 4 2
Present 8 8
Chemosensitivity >0.999
Grade 0 or 1a 6 5
Grade 1b or 2 2 3
The modied CTGF score of CAFs did not correlate with clinico‑
pathological features. Cisplatin and Pemetrexed were administered to
16 out of 22 patients as neoadjuvant chemotherapy. One patient was
readministered carboplatin and pemetrexed due to cisplatin‑induced
vomiting. Modied CTGF score: Low, <30; high, ≥30. CAFs,
cancer‑associated broblasts; CTGF, connective tissue growth factor.
OHARA et al: CTGF+ CAFs AS A MOLECULA R TARGET FOR MESOTHELIOMA
8
Figure 5. Fibroblasts in mesothelial carcinogenesis. Previous studies suggest the existence of three phenotypes of broblasts: Quiescent, wound healing‑asso‑
ciated, and mesothelioma cell‑educated. Asbestos bers, which contain iron as a component, can directly induce reactive oxygen species (ROS) generation via
catalysis of the Fenton reaction by iron on the surface. Macrophages phagocytose asbestos bers and form granulomas. These macrophages can also produce
ROS. ROS can induce quiescent broblasts to differentiate into myobroblasts. These αSMA+ broblasts can contribute to carcinogenesis by secreting CTGF
and cytokines. Fibroblasts educated by mesothelioma cells express CTGF and have protumorigenic roles. Mein+ broblasts may have antitumorigenic roles.
αSMA, α‑smooth muscle actin; CTGF, connective tissue growth factor; Mein, mesenchymal stromal cell‑ and broblast‑expressing Linx paralogue.
Figure 4. Mein expression in mesothelioma. (A) RNA ISH of Mein. DAB solution was used to stain αSMA, and HistoGreen was used to stain AE1/AE3.
Mesothelioma cells were positive for AE1/AE3. CAFs and vessels were positive for αSMA. More Mein‑positive (Mein+) CAFs were observed in the
αSMA‑negative area (top), which is the invasive front of mesothelioma. Less Mein+ CAFs were observed in the αSMA‑high area (bottom), which is the
proximal side of mesothelioma. The merged images of αSMA + AE1/AE3 and Mein were obtained using ImageJ software. Mein‑positive area is indicated
by red color in the merged images. All images are shown at the same magnication. (B) Analysis of RNA ISH data for Mein. The proportions of the Mein+
CAFs for all cases are plotted as a histogram. (C) Mein expression in CAFs and patient prognosis based on Kaplan‑Meier survival curves. There was no
signicant difference in prognosis based on the proportions of the Mein+ CAFs (low, <10%; high, ≥10%). Gehan‑Breslow‑Wilcoxon test was used for analysis.
αSMA, α‑smooth muscle actin; CAFs, cancer‑associated broblasts; H&E, hematoxylin and eosin; ISH, in situ hybridization; Mein, mesenchymal stromal
cell‑ and broblast‑expressing Linx paralogue.
ONCOLOGY REPORTS 9
2‑dimensional cell culture in vit ro, whereas it strongly inhib‑
ited cancer growth in vivo (15,41‑43). These results can also
be due to the existence of CTGF+ CAFs.
In the present study, the αSMA area index was found to be
correlated with prognosis, as shown previously (16‑20,44‑47).
Although brosis in mesothelioma is distinctive from that in
other tumors, it is suggested that αSMA+ broblasts correlate
with mesothelioma growth. Inhaled asbestos can rst result
in benign pleural brosis and then in mesothelioma (1,2,11).
Thus, mesothelioma tissues may exhibit substantial brosis
from the precancerous lesion/early mesothelioma stage in situ,
although tumor cells in other tumors involve broblasts and
form stroma only when they invade. In addition, reactive
oxygen species (ROS), playing a key molecular mechanism
in mesothelial carcinogenesis (4‑8), can activate quiescent
fibroblasts to form myofibroblasts (18). Therefore, not all
αSMA+ broblasts in mesotheliomas may be CAFs that are
under the command of mesothelioma cells, as some of these
cells may be wound healing‑associated (related to granulation
for asbestos) broblasts (Fig. 5). These myobroblasts may
also express CTGF, because we previously conrmed that
normal broblasts can also express CTGF in vitro (15). T h ese
cells can contribute to carcinogenesis by secreting CTGF and
cytokines.
For this study, we used immunohistochemistry (IHC) to
demonstrate the roles of CAFs in mesothelioma progression
as IHC rarely decreases the signal compared to immuno‑
uorescence (IF). IHC made it possible to evaluate all of the
specimens. In contrast, IF of αSMA/CTGF may be useful for
studying the differentiation of CAFs from mesenchymal stem
cells and for classi f yi ng CAFs (αSMA/CTGF‑, αSMA+/CTGF‑,
αSMA‑/CTGF+, and αSMA+/CTGF+). We will perform such
studies and develop the IF of αSMA/CTGF in the future.
A limitation in this study is that the eventual number of
cases was not large. In our hospital, surgical cases of meso‑
thelioma are rare because of the rareness of the disease and
because the majority of cases were at the advanced stage at
diagnosis. In the stroma, Mein expression appeared posi‑
tive where αSMA expression was negative in some lesions of
the mesothelioma tissue samples. The present study did not
elucidate whether Mein correlates with patient survival. We
were able to collect samples from only 15 patients as RNA
ISH needs to be performed on tissue samples within ve years
of sample collection. CTGF expression did not correlated
with sensitivities to neoadjuvant chemotherapy. This may be
also because the number of cases is small for analysis. We
will gather more samples to examine the expression of CAF
markers (αSMA, CTGF, and Mein) and the correlations of
CAFs and chemotherapy in the future.
In conclusion, CTGF+ CAFs are important for mesothe‑
lioma growth and correlate with patient prognosis. Thus, these
cells may be a potential target for drugs. Our previous study
demonstrated that FG‑3019 was effective for mesothelioma
in a murine orthotopic implantation model, and the results of
the present study suggest that FG‑3019 targets CTGF+ CAFs.
Thus, whether FG‑3019 has therapeutic effects in human
mesothelioma patients warrants further investigation.
Acknowledgements
We thank Ms Tomomi Aoyama, Ms Naomi Tagami, and
Dr Hideki Tsubouchi (Nagoya University) for technical assis‑
tance. YO was a recipient of the Takeda Science Foundation
Fellowship (April 2014‑March 2018).
Funding
The present study was supported in part by in part, from JST
CREST (grant no. JPMJCR19H4) and JSPS Kakenhi (grant
nos. JP17H04064, JP19H05462 and JP20H05502) to ST and
by a Meidai Ishikai Funding 2016 to YO.
Availability of data and materials
Data and materials are available upon request to the
corresponding author.
Authors' contributions
YO and ST designed the experiments. YO performed the
experiments and data analysis, wrote the main manuscript text,
and prepared the gures. AE, YT, KS, TI, HK, YMiz, YMiy,
AH, SM, JS, KY, FI, YMo, NM, TF, KK and KY provided
administrative, technical, and material support. All authors
Table V. Mein and clinicopathological features of the
mesothelioma cases.
Characteristics Low High P‑value
Age (years) >0.999
<65 4 2
≥65 6 3
Sex 0.333
Male 10 4
Female 0 1
Pathological invasion >0.999
pT1 or pT2 4 2
pT3 or pT4 6 3
Lymph node metastasis 0.580
pN0 5 4
pN1 or pN2 5 1
Stage >0.999
I or II 3 1
III or IV 7 4
Neoadjuvant chemotherapy 0.600
Absent 4 1
Present 6 4
Chemosensitivity >0.999
Grade 0 or 1a 4 3
Grade 1b or 2 2 1
Mein did not correlate with clinicopathological features. Cisplatin
and pemetrexed were administered to 10 out of 15 patients as
neoadjuvant chemotherapy. Mein: Low, <10%; high, ≥10%. Mein,
mesenchymal stromal cell‑ and broblast‑expressing Linx paralogue.
OHARA et al: CTGF+ CAFs AS A MOLECULA R TARGET FOR MESOTHELIOMA
10
read and approved the manuscript and agree to be accountable
for all aspects of the research in ensuring that the accuracy or
integrity of any part of the work are appropriately investigated
and resolved.
Ethics approval and consent to participate
Human mesothelioma tissues were obtained with informed
patient consent at the time of surgery at Nagoya University
Hospital (Nagoya, Japan). This study was carried out in accor‑
dance with the Helsinki Declaration for Human Research
and approved by the Ethics Committee of Nagoya University
Graduate School of Medicine (protocol no. 2017‑0127).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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