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Connective tissue growth factor produced by cancer‑associated fibroblasts correlates with poor prognosis in epithelioid malignant pleural mesothelioma

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  • Nagoya University Graduate School of Medicine, Japan

Abstract and Figures

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 fibroblasts (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 populations 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 fibroblasts and promotes the invasion of mesothelioma cells in vitro. In this study, we examined the clinical relevance of CAFs in mesothelioma. Using surgical specimens of epithelioid malignant pleural mesothelioma, we evaluated the clinicopathological significance of the expression of α‑smooth muscle actin (αSMA), the most widely used marker of CAFs, the expression of CTGF, and the extent of fibrosis by immunohistochemistry and Elastica‑Masson staining. We also analyzed the expression of mesenchymal stromal cell‑ and fibroblast‑expressing Linx paralogue (Meflin; ISLR), a recently reported CAF marker that labels cancer‑restraining CAFs and differ from αSMA‑positive CAFs, by in situ hybridization. The extent of fibrosis 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.
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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 signicance 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 (Mein; 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 myobroblast 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 (Mein; ISLR) is a glycosylphosphatidylinositol
(GPI)‑anchored membrane protein, which has been identied
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 signica 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 Mein), 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
classication was performed based on the TNM Classication
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 difculty 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. 20170127).
Histologic and immunohistochemical analysis. Fou r‑micron‑
thick serial sections were cut from formalin‑fixed and
parafn‑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, Histone 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
inuence 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 modied 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 modied
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 signicant 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 signicant 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 parafn‑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). Briey, tissue sections
were baked in a dry oven (HybEZ II Hybridization System;
Advanced Cell Diagnostics) at 60˚C for 1 h, deparafnized,
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 Mein (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 Mein. To detect the Mein‑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 Mein‑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 coefcient). 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 signicance.
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 conrm 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
parafn‑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 difcult 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 quantied 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 signicant differences in overall survival were found based on
the brotic area index (Fig. 1E). However, a signicant 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 conrm CTGF
as a prognostic factor and potential targets, we next evaluated
the expression of CTGF and Ki‑67 using parafn‑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 signicant 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 magnication. 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 specically 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
signicant 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 signicant 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 modied by the AE1/AE3 area index values. There were no signicant differences in prognosis
based on modied CTGF scores (low, <10; high, ≥10). (H) CTGF expression in CAFs and patient prognosis based on Kaplan‑Meier survival curves. CTGF
scores were modied by the αSMA area index values. There was a signicant difference (P=0.0186) in prognosis based on modied 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 Mein expression by RNA
ISH using paraffin‑embedded sections. Mesothelial cells,
which are nontumorous, did not express Meflin (Fig. S2),
while CAFs expressed Mein (Fig. 4A and B). More Mein+
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 Mein 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‑specic 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 modied 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. Modied 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 modied 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. Modied 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 myobroblasts. These αSMA+ broblasts can contribute to carcinogenesis by secreting CTGF
and cytokines. Fibroblasts educated by mesothelioma cells express CTGF and have protumorigenic roles. Mein+ broblasts may have antitumorigenic roles.
αSMA, α‑smooth muscle actin; CTGF, connective tissue growth factor; Mein, mesenchymal stromal cell‑ and broblast‑expressing Linx paralogue.
Figure 4. Mein expression in mesothelioma. (A) RNA ISH of Mein. 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 Mein‑positive (Mein+) CAFs were observed in the
αSMA‑negative area (top), which is the invasive front of mesothelioma. Less Mein+ CAFs were observed in the αSMA‑high area (bottom), which is the
proximal side of mesothelioma. The merged images of αSMA + AE1/AE3 and Mein were obtained using ImageJ software. Mein‑positive area is indicated
by red color in the merged images. All images are shown at the same magnication. (B) Analysis of RNA ISH data for Mein. The proportions of the Mein+
CAFs for all cases are plotted as a histogram. (C) Mein expression in CAFs and patient prognosis based on Kaplan‑Meier survival curves. There was no
signicant difference in prognosis based on the proportions of the Mein+ 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; Mein, 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 (16‑20,4447).
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 myobroblasts may
also express CTGF, because we previously conrmed 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, Mein expression appeared posi
tive where αSMA expression was negative in some lesions of
the mesothelioma tissue samples. The present study did not
elucidate whether Mein 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 Mein) 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. Mein 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
Mein did not correlate with clinicopathological features. Cisplatin
and pemetrexed were administered to 10 out of 15 patients as
neoadjuvant chemotherapy. Mein: Low, <10%; high, ≥10%. Mein,
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. 20170127).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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... Nonetheless, it does help explain why chemotherapy is not as effective in elderly hosts, as even though there is likely elevated tumour antigen presentation, on account of increased tumour cell death, CTL functionality remained compromised in elderly mice [59] . In contrast, chemotherapy enhanced CTL function in young mice; CD8 + T cell depletion studies confirmed that these CTLs were responsible for chemotherapy-induced mesothelioma regression [59,64] . ...
... Furthermore, a higher microvessel density (MVD) has been reported in mesothelioma biopsies compared to other malignancies [194] which was independently related to poor survival, even when adjusted for other known prognostic factors, including age. These data prompted testing of several antiangiogenic drugs with or without chemotherapy in mesothelioma patients, but the effects were unremarkable and sometimes led to significant toxicity [157,64,[195][196][197] . Perhaps age played a role in this poor response. ...
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... Our cohort of 54 patients well reflects this heterogeneity with an OS ranging from 1.3 to 106.4 months. No definite molecular and/or biological prognostic biomarkers have been reported, even if some have been suggested (i.e., CTGF-Connective Tissue Growth Factor-protein [37] and the VISTA immune-related protein [38]). Recently, a higher number of B lymphocytes and a prevalence of tertiary lymphoid structures were present in long survivors (>36 months) versus short survivors (>12 months) [36], which has been reported that in a retrospective series of MPM. ...
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Aim: DNA repair has an important role in malignant pleural mesothelioma (MPM) tumorigenesis and progression. Prognostic/predictive biomarkers for better management of MPM patients are needed. In the present manuscript, we analyzed the expression of more than 700 genes in a cohort of MPM patients to possibly find biomarkers correlated with survival. Methods: A total of 54 MPM patients, all with epithelioid histology, whose survival follow-up and formalin-fixed paraffin-embedded tumors were available, were included in the study. Gene expression profiles were evaluated using a Nanostring platform analyzing 760 genes involved in different cellular pathways. The percentages of proliferating tumor cells positive for RAD51 and BRCA1 foci were evaluated using an immunofluorescence assay, as a readout of homologous recombination repair status. Results: Patient median survival time was 16.9 months, and based on this value, they were classified as long and short survivors (LS/SS) with, respectively, an overall survival ≥ and <16.9 months as well as very long and very short survivors (VLS/VSS) with an overall survival ≥ than 33.8 and < than 8.45 months. A down-regulation in the DNA damage/repair expression score was observed in LS and VLS as compared to SS and VSS. These findings were validated by the lower number of both RAD51 and BRCA1-positive tumor cells in VLS as compared to VSS. Conclusions: The down-regulation of DNA repair signature in VLS was functionally validated by a lower % of RAD51 and BRCA1-positive tumor cells. If these data can be corroborated in a prospective trial, an easy, cost-effective test could be routinely used to better manage treatment in MPM patients.
... Therefore, high FAP expression in CAFs can be considered a marker of aggressive tumor behavior and poor prognosis [20]. There is often a desmoplastic reaction suggesting that CAFs play a role in mesothelioma cases [21]. In recent studies, it has been reported that 68 Ga-FAPI-04 PET/CT is more effective than FDG in many epithelial tumor types [12,13,22,23]. ...
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Objective: In this study, we aimed to compare the role of 68Ga-labeled FAP inhibitor (68Ga-FAPI)-04 PET/computed tomography (CT) and 18F-fluorodeoxyglucose (18F-FDG) PET/CT in the evaluation of primary tumor and metastases in patients diagnosed with malignant mesothelioma. Materials and methods: Our prospective study included 21 patients with histopathological diagnosis of malignant mesothelioma who underwent both 68Ga-FAPI-04 PET/CT and 18F-FDG PET/CT imaging between April 2022 and September 2022. Maximum standardized uptake value (SUVmax), metabolic tumor volume, total lesion glycolysis, tumor-to-background ratio (TBR) and highest SUVpeak (HPeak) values and lesion numbers were calculated from primary and metastatic lesions on FDG and FAPI PET/CT images. Findings obtained from FAPI and FDG PET/CT were compared. Results: More lesions were detected in 68Ga-FAPI-04 PET/CT compared to 18F-FDG PET/CT in primary tumor and lymph node metastases. Statistically significantly higher SUVmax and TBR values were found with FAPI PET/CT (primary lesion SUVmax and TBR, P = 0.001 and P < 0.001, respectively; lymph node SUVmax and TBR, P = 0.016 and P = 0.005, respectively). With FAPI PET/CT, upstage was observed according to tumor-node-metastasis staging in a total of seven patients including three patients with pleural origin, three patients with peritoneal origin and one patient with pericardial origin. Conclusion: In addition to the stage change with 68Ga-FAPI-04 PET/CT in malignant mesothelioma patients, a statistically significant superiority was observed in SUVmax, TBR and volumetric parameters in primary tumors and metastases.
... Ohara et al. reported that proliferation, migration, and invasion of PM cells was enhanced by the presence of normal human lung fibroblasts in a partly CTGF-dependent manner [42]. The same group also found in an immunohistochemistry study that CTGF expression in fibroblasts of PM specimens correlated with poor prognosis [43]. Recently, medium conditioned by cultures of the human lung fibroblasts MRC-5 and IMR-90 was shown to regulate the activity of multiple kinases in PM cells, especially those connected to the mitogen-activated protein kinase (MAPK) pathway [44]. ...
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Background Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis . Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells. Methods Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. Results Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib. Conclusions Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.
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The incidence of malignant mesothelioma (MM), a disease linked to refractory asbestos exposure, continues to increase globally and remains largely resistant to various treatments. Our previous studies have identified a strong correlation between connective tissue growth factor (CTGF) protein expression and MM malignancy, underscoring the importance of understanding CTGF regulation in MM cells. In this study, we demonstrate for the first time that stimulation with platelet-derived growth factor receptor (PDGFR) ligand, PDGF-BB, increases CTGF protein expression levels without affecting CTGF mRNA levels. Inhibition of PDGFR resulted in a reduction of CTGF protein expression, indicating that PDGFR activation is essential in regulating CTGF protein expression in MM cells. PDGF-BB also activated the protein kinase B (AKT) pathway, and inhibition of AKT phosphorylation abolished the PDGFR-induced CTGF protein expression, suggesting that PDGFR acts upstream of CTGF via the AKT pathway. This reinforces the role of CTGF protein as a key regulator of MM malignancy. Additionally, PDGFR activation led to the phosphorylation of mTOR and 4E-BP1, critical regulators of protein synthesis downstream of AKT, suggesting that PDGFR controls CTGF protein expression through the regulation of CTGF mRNA translation.
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Mesothelioma is an aggressive cancer of the mesothelial layer associated with an extensive fibrotic response. The latter is in large part mediated by cancer-associated fibroblasts which mediate tumour progression and poor prognosis. However, understanding of the crosstalk between cancer cells and fibroblasts in this disease is mostly lacking. Here, using co-cultures of patient-derived mesothelioma cell lines and lung fibroblasts, we demonstrate that fibroblast activation is a self-propagated process producing a fibrotic extracellular matrix (ECM) and triggering drug resistance in mesothelioma cells. Following characterisation of mesothelioma cells/fibroblasts signalling crosstalk, we identify several FDA-approved targeted therapies as far more potent than standard-of-care Cisplatin/Pemetrexed in ECM-embedded co-culture spheroid models. In particular, the SRC family kinase inhibitor, Saracatinib, extends overall survival well beyond standard-of-care in a mesothelioma genetically-engineered mouse model. In short, we lay the foundation for the rational design of novel therapeutic strategies targeting mesothelioma/fibroblast communication for the treatment of mesothelioma patients.
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Despite centuries since the discovery and study of cancer, cancer is still a lethal and intractable health issue worldwide. Cancer-associated fibroblasts (CAFs) have gained much attention as a pivotal component of the tumor microenvironment. The versatility and sophisticated mechanisms of CAFs in facilitating cancer progression have been elucidated extensively, including promoting cancer angiogenesis and metastasis, inducing drug resistance, reshaping the extracellular matrix, and developing an immunosuppressive microenvironment. Owing to their robust tumor-promoting function, CAFs are considered a promising target for oncotherapy. However, CAFs are a highly heterogeneous group of cells. Some subpopulations exert an inhibitory role in tumor growth, which implies that CAF-targeting approaches must be more precise and individualized. This review comprehensively summarize the origin, phenotypical, and functional heterogeneity of CAFs. More importantly, we underscore advances in strategies and clinical trials to target CAF in various cancers, and we also summarize progressions of CAF in cancer immunotherapy.
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The diverse members of the CCN family now designated as CCN1(CYR61), CCN2 (CTGF), CCN3(NOV), CCN4(WISP1), CCN5(WISP2), CCN6(WISP3) are a conserved matricellular family of proteins exhibiting a spectrum of functional properties throughout all organs in the body. Interaction with cell membrane receptors such as integrins trigger intracellular signaling pathways. Proteolytically cleaved fragments (constituting the active domains) can be transported to the nucleus and perform transcriptional relevant functional activities. Notably, as also found in other protein families some members act opposite to others creating a system of functionally relevant checks and balances. It has become apparent that these proteins are secreted into the circulation, are quantifiable, and can serve as disease biomarkers. How they might also serve as homeostatic regulators is just becoming appreciated. In this review I have attempted to highlight the most recent evidence under the subcategories of cancer and non-cancer relevant that could lead to potential therapeutic approaches or ideas that can be factored into clinical advances. I have added my own personal perspective on feasibility.
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In malignant tumors, cancer cells adapt to grow within their host tissue. As a cancer progresses, an accompanying host stromal response evolves within and around the nascent tumor. Among the host stromal constituents associated with the tumor are cancer-associated fibroblasts, a highly abundant and heterogeneous population of cells of mesenchymal lineage. Although it is known that fibroblasts are present from the tumor's inception to the end-stage metastatic spread, their precise functional role in cancer is not fully understood. It has been suggested that cancer-associated fibroblasts play a key role in modulating the behavior of cancer cells, in part by promoting tumor growth, but evolving data also argue for their antitumor actions. Taken together, this suggests a putative bimodal function for cancer-associated fibroblasts in oncogenesis. As illustrated in this Review and its accompanying poster, cancer-associated fibroblasts are a dynamic component of the tumor microenvironment that orchestrates the interplay between the cancer cells and the host stromal response. Understanding the complexity of the relationship between cancer cells and cancer-associated fibroblasts could offer insights into the regulation of tumor progression and control of cancer.
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Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.
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Malignant mesothelioma (MM) is a rare but socially important neoplasm due to its association with asbestos exposure. While MM is difficult to diagnose at an early stage, there are no particularly effective treatments available at the advanced stage, thus necessitating efficient strategies to prevent MM in individuals already exposed to asbestos. We previously showed that persistent oxidative damage caused by foreign body reaction and affinity of asbestos both to hemoglobin and histones is one of the major pathogeneses. Accordingly, as an effective strategy to prevent asbestos-induced MM, we undertook the use of an iron chelator, deferasirox, which decreased the epithelial-mesenchymal transition in a crocidolite-induced rat MM model. However, this agent may exhibit adverse effects. Here, we studied the effects of iron removal by phlebotomy as a realistic measure on the same rat model. We injected a total of 5 mg of crocidolite intraperitoneally to F1 hybrid rats between the Fischer-344 and Brown-Norway strains at the age of 6 weeks. We repeated weekly or biweekly phlebotomy of 6 to 8 ml/kg/time from 10 to 60 weeks of age. The animals were observed until 120 weeks. In male rats, phlebotomy significantly decreased the weight and nuclear grade of MM, and modestly reduced the associated ascites and the fraction of more malignant sarcomatoid subtype. Weekly phlebotomy prolonged the long-term survival. Our results indicate that appropriate phlebotomy may be a practical preventive measure to attenuate the initiation and promotion capacity of asbestos towards MM by reducing iron in individuals exposed to asbestos.
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Objective: To develop a flexible method of separation and quantification of immunohistochemical staining by means of color image analysis. Study design: An algorithm was developed to deconvolve the color information acquired with red-green-blue (RGB) cameras and to calculate the contribution of each of the applied stains based on stain-specific RGB absorption. The algorithm was tested using different combinations of diaminobenzidine, hematoxylin and eosin at different staining levels. Results: Quantification of the different stains was not significantly influenced by the combination of multiple stains in a single sample. The color deconvolution algorithm resulted in comparable quantification independent of the stain combinations as long as the histochemical procedures did not influence the amount of stain in the sample due to bleaching because of stain solubility and saturation of staining was prevented. Conclusion: This image analysis algorithm provides a robust and flexible method for objective immunohistochemical analysis of samples stained with up to three different stains using a laboratory microscope, standard RGB camera setup and the public domain program NIH Image.
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Background: Connective tissue growth factor (CTGF) is a secreted glycoprotein that has a central role in the process of fibrosis. This study was designed to assess the safety, tolerability, and efficacy of pamrevlumab (FG-3019), a fully recombinant human monoclonal antibody against CTGF, in idiopathic pulmonary fibrosis. The aim was to establish whether pamrevlumab could slow, stop, or reverse progression of idiopathic pulmonary fibrosis. Methods: The phase 2, randomised, double-blind, placebo-controlled PRAISE trial was done at 39 medical centres in seven countries (Australia, Bulgaria, Canada, India, New Zealand, South Africa, and the USA). Patients with idiopathic pulmonary fibrosis and percentage of predicted forced vital capacity (FVC) of 55% or greater were enrolled and randomly assigned (1:1) by use of interactive responsive technology to intravenous infusion of pamrevlumab 30 mg/kg or placebo every 3 weeks over 48 weeks (16 infusions). The primary efficacy outcome was change from baseline in percentage of predicted FVC at week 48. Disease progression (defined as a decline from baseline in percentage of predicted FVC of ≥10%, or death) at week 48 was a key secondary efficacy outcome. All patients in the pamrevlumab group received at least one dose of the study drug and were analysed for safety. Two patients in the placebo group were excluded from the intention-to-treat population for the efficacy analyses because of enrolment error. This trial is registered with ClinicalTrials.gov, NCT01890265. Findings: Between Aug 17, 2013, and July 21, 2017, 103 patients were randomly assigned (50 to pamrevlumab and 53 to placebo). Pamrevlumab reduced the decline in percentage of predicted FVC by 60·3% at week 48 (mean change from baseline -2·9% with pamrevlumab vs -7·2% with placebo; between-group difference 4·3% [95% CI 0·4-8·3]; p=0·033). The proportion of patients with disease progression was lower in the pamrevlumab group than in the placebo group at week 48 (10·0% vs 31·4%; p=0·013). Pamrevlumab was well tolerated, with a safety profile similar to that of placebo. Treatment-emergent serious adverse events were observed in 12 (24%) patients in the pamrevlumab group and eight (15%) in the placebo group, with three patients on pamrevlumab and seven on placebo discontinuing treatment. Of the three (6%) deaths in the pamrevlumab group and six (11%) in the placebo group, none was considered treatment related. Interpretation: Pamrevlumab attenuated progression of idiopathic pulmonary fibrosis and was well tolerated. Now in phase 3 development, pamrevlumab shows promise as a novel, safe, and effective treatment for idiopathic pulmonary fibrosis. Funding: FibroGen.
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Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. Significance Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.
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Rationale: Myofibroblasts (MFs) have roles in tissue repair following damage associated with ischemia, aging, and inflammation, and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of MFs is mesenchymal stromal/stem cells (MSCs) that exist as resident fibroblasts in multiple tissues. We previously identified meflin, a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of MSCs and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. Objective: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure (HF). Methods and results: We found that meflin has an inhibitory role in MF differentiation of cultured MSCs. Meflin expression was downregulated by stimulation with transforming growth factor (TGF)-β, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction (MI) and pressure-overload HF mouse models. Analysis of meflin knockout (KO) mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after MI induction. When subjected to pressure overload by transverse aortic constriction, meflin KO mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin KO mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an anti-fibrotic cytokine that counteracts the action of TGF-β and augments its intracellular signaling. Conclusions: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in MF differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.
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Cancer is the primary cause of human mortality in most countries. This tendency has increased as various medical therapeutics have advanced, which suggests that we cannot escape carcinogenesis, although the final outcome may be modified by exposomes and statistics. Cancer is classified by its cellular differentiation. Mesothelial cells are distinct in that they line somatic cavities, facilitating the smooth movement of organs, but are not exposed to the external environment. Malignant mesothelioma, or simply mesothelioma, develops either in the pleural, peritoneal or pericardial cavities, or in the tunica vaginalis testes. Mesothelioma has been a relatively rare cancer but is socially important due to its association with asbestos exposure, caused by modern industrial development. The major pathogenic mechanisms include oxidative stress either via catalytic reactions against the asbestos surface or frustrated phagocytosis of macrophages, and specific adsorption of hemoglobin and histones by asbestos fibers in the presence of phagocytic activity of mesothelial cells. Multiwall carbon nanotubes of ~50 nm-diameter, additionally adsorbing transferrin, are similarly carcinogenic to mesothelial cells in rodents and were thus classified as Group 2B carcinogens. Genetic alterations found in human and rat mesothelioma notably contain changes found in other excess iron-induced carcinogenesis models. Phlebotomy and iron chelation therapies have been successful in the prevention of mesothelioma in rats. Alternatively, loading of oxidative stress by non-thermal plasma to mesothelioma cells causes ferroptosis. Therefore, carcinogenesis by foreign fibrous inorganic materials may overlap the uncovered molecular mechanisms of birth of life and its evolution.
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Purpose: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a central role in tumor progression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. Experimental design: 140 cases of esophageal cancer were analyzed for CAFs and CD8+or forkhead box protein 3 (FoxP3+) TILs by immunohistochemistry. We analyzed cytokines using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c-nu/numice, and the tumors treated with recombinant interleukin 6 (IL-6) or anti-IL-6 antibody. Results: CD8+TILs and CAFs were negatively correlated in intra-tumoral tissues (P< 0.001), while FoxP3+TILs were positively correlated (P< 0.001) in esophageal cancers. Co-cultured Colon26 cancer cells and fibroblasts resulted in accelerated tumor growth in BALB/c mice, along with decreased CD8+and increased FoxP3+TILs, compared with cancer cells alone. In vitro, IL-6 was highly secreted in both murine and human cancer cell/fibroblast co-cultures. IL-6 significantly increased Colon26 tumor growth in immune-competent BALB/c (P< 0.001) with fewer CD8+TILs than untreated tumors (P< 0.001), whereas no difference in BALB/c-nu/numice. In contrast, FoxP3+TILs increased in IL-6-treated tumors (P< 0.001). IL-6 antibody blockade of tumors co-cultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8+TILs in intra-tumoral tissues. Conclusions: CAFs regulate immunosuppressive TIL populations in the TME via IL-6. IL-6 blockade, or targeting CAFs, may improve pre-existing tumor immunity and enhance the efficacy of conventional immunotherapies.