Urologic Oncology: Seminars and Original Investigations ] (2014) ∎∎∎–∎∎∎

Original article
Effect of platelet-derived growth factor-B on renal cell carcinoma
growth and progression
Wenling Wang, M.D.a,b,1, Lifeng Qi, M.D.b,c,1, Minhan Tan, M.D., Ph.D.d,
Zhenting Zhang, M.D.a, Ju Du, M.D., Ph.D.a, Xiaona Wei, M.D., Ph.D.d, Xin Yao, M.D., Ph.D.a,b,*
a
Department of Genitourinary Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer,
Tianjin, China
b
Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
c
Department of Gynecology, Cangzhou Central Hospital, Hebei Medical University, Cangzhou, China
d
Institute of Bioengineering and Nanotechnology, Singapore
Received 16 October 2014; received in revised form 22 December 2014; accepted 22 December 2014

Abstract
Background: Platelet-derived growth factor-B (PDGF-B) expression promotes the proliferation of mural cells surrounding the blood
vessels during angiogenesis. The effect of PDGF-B involved in angiogenesis on tumor growth and progression in clear cell renal cell
carcinoma (ccRCC) is unknown.
Methods: We examined the expression of PDGF-B and its receptor PDGFR-β in 174 patients with ccRCC by microarray analysis.
Cancer-specific survival was estimated using the Kaplan-Meier method. PDGF-B–transfected and mock-transfected ACHN cells were
implanted into mice to induce tumor formation and tumor growth, respectively, and progression in mice models was assessed using
immunohistochemistry and histomorphology. The role of PDGF-B during angiogenesis in vitro was evaluated by flow cytometry analysis,
cell migration, and tube formation assay.
Results: High expression of PDGF-B was associated with significantly decreased risk of cancer-specific mortality (P r 0.001). The data
indicated significant inhibition of tumor growth (P r 0.05) and a reduction in proliferating tumor cells (P ¼ 0.019) in vivo. PDGF-B also
inhibits tumor metastasis and invasion events in tumor-bearing mice models. In vitro studies revealed that the tube formation capability of
vascular smooth muscle cells (VSMCs), which are believed to be the precursors to pericytes in vivo, significantly induced by PDGF-B. The
PDGF-B overexpression also results in a tendency to reside in S and G2/M phases of the cell cycle (P ¼ 0.001) and increasing migration
capability of VSMCs (P r 0.001).
Conclusion: Our results demonstrated that PDGF-B, which increased VSMCs proliferation and migration capability during angiogenesis,
limited tumor growth and progression in ccRCC. Therefore, PDGF-B may be a novel and promising prognostic marker. r 2014 Elsevier
Inc. All rights reserved.

Keywords: Platelet-derived growth factor-B; Angiogenesis; Clear cell renal cell carcinoma; Metastasis; Progression

This project was supported by the National Natural Science Foundation
of China, China (Grant no.: 81072090).
Dr. Minhan Tan has filed for molecular diagnostics patents in kidney
cancer and has received research funding from Pfizer for research on
kidney cancer.
1
These authors contributed equally to this work and should be
considered co-first authors.
* Corresponding author. Tel.: þ86-222-334-0123; fax: þ86-222-353-
7796.
E-mail address: yaoxin1969@hotmail.com (X. Yao).
1. Introduction
In the past decades, the incidence of renal cell carcino-
mas (RCC) has steadily increased. It was predicted that an
estimated 63,920 new cases of kidney and renal pelvis
cancer would be diagnosed and 13,860 would die of this
disease in the United States in 2014 [1]. Although a higher
incidence of small renal masses are being detected, this
disease continues to have a significant public health effect
[2]. Despite this, targeted therapy has significantly changed

http://dx.doi.org/10.1016/j.urolonc.2014.12.015
1078-1439/r 2014 Elsevier Inc. All rights reserved.
2 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11
the life expectancy of patients with metastatic RCC, the
median disease-specific survival of patients with metastatic
RCC diagnosed from 2005 to 2009 is only 16 months [3].
RCC are characterized by their hypervascularity and their
particular angiogenic microenvironment [4]. Angiogenesis is
required for tumor growth, and the differentiation and
recruitment of mural cells are essential steps during tumor
angiogenesis. Mural cells of the vascular system include
vascular smooth muscle cells (VSMCs) and pericytes.
VSMCs are believed to be the precursors to pericytes
in vivo and pericytes are important structural and functional
components of blood vessels [5,6]. Therefore, in the era of
targeted therapies, combination therapy of targeting vascular
endothelial growth factor signaling in the endothelium and
PDGFR-β signaling in the pericytes to inhibit tumor angio-
genesis and tumor growth as antiangiogenesis therapy yields
encouraging results in cancer. However, our previous study
revealed that 2 distinct types of blood vessels, including
differentiated and undifferentiated microvascular density
(MVD) in clear cell RCC (ccRCC), predicted contrasting
prognostic implications, and relatively higher differentiated
MVD was a favorable prognostic factor, which indicated the
undifferentiated blood vessels rather than differentiated blood
vessels are potentially therapeutic targets [7]. Thus, a well-
differentiated and stable vasculature may play a beneficial
role for tumor treatment. Several signaling systems, through
the interaction between endothelial cells and mural cells,
have been implicated in regulating vessel maturation and
stability. Platelet-derived growth factor-B (PDGF-B), which
is expressed by a number of cells including endothelial cells
and mural cells, is responsible for their proliferation and
migration during vascular maturation [8]. Through the
interaction with PDGF receptor (PDGFR)-β expressed on
mural cells, PDGF-B appears to be the crucial player in
recruitment of pericytes to newly formed vessels [9]. How-
ever, the role of the PDGF-B and PDGFR-β signaling
pathway in ccRCC has not yet been explained. A previous
study found that higher PDGF-B expression in ccRCC was
an independent prognostic factor for longer survival, suggest-
ing that PDGF-B is a novel and promising prognostic marker
[10]. Our present study focused on the effects of PDGF-B on
pericyte functions in vitro and the role of pericyte recruitment
induced by PDGF-B in tumor growth and progression
(metastasis and invasion) in ccRCC.
2. Materials and methods
2.1. Expression of PDGF-B and its receptor in 174 cases of
ccRCC microarray samples and survival analysis
The messenger RNA expression of PDGF-B and PDGFR-β
was measured in 174 cases of ccRCC accrued at the Van
Andel Research Institute using Affymetrix HGU133 Plus 2.0
microarray analysis [11]. More than 90% of the patients (n ¼
157) were diagnosed before 2005. To evaluate the effect of
PDGF-B and PDGFR-β expression levels on clinical outcomes,
patients were divided into low and high expressers based on
the median expression level of PDGF-B and PDGFR-β,
respectively (low and high PDGFB groups; low and high
PDGFR-β groups). Cancer-specific survival was estimated
using the Kaplan-Meier method; cancer-specific survival differ-
ences between low and high expression of PDGF-B were
analyzed using the log-rank test. The same survival analysis
was used in the low and high PDGFR-β groups.
3. Research in vivo
3.1. Cell culture and transfection
RCC cell lines (786-O, CRC-1932, CRC-1933, HTB-47,
ACHN, and HTB-46) were obtained from Bin Tean Teh in Van
Andel Research Institute, United States. Experiments were
conducted on cell lines passed for a maximum of 6 months
postresuscitation. All cell lines were cultured and maintained in
Dulbecco's modified Eagle's medium (DMEM) and 10% fetal
bovine serum (FBS). The ACHN cell line was chosen for
further studies, as it was cultured well and the expression of
PDGF-B was lower on Western blotting. ACHN cells were
propagated in DMEM with 10% FBS and standard supplements.
The cells were transfected with a vector containing the PDGF-B
gene using Lipofectamine reagent according to the manufac-
turer's instructions. Scrambled RNA (scr) was used as a negative
control. For selection, ampicillin resistance was used. The
PDGF-B–transfected ACHN cells were also selected in a second
round using Western blotting to confirm PDGF-B expression.
VSMCs are believed to be the precursors to pericytes in vivo
and are used to detect the effects of PDGF-B on pericytes.
VSMCs and human vascular endothelial cells (HUVECs) were
obtained from American Type Culture Collection (ATCC: CRL-
1999, ATCC: CRL-1730) and the cells were dyed fluorescent
green (VSMCs) and fluorescent red (HUVECs), respectively
(sigma, MIDI67IKT; sigma, MINI26IKT). Then VSMCs and
HUVECs were added to the upper and lower chamber of culture
dish (BD, 353180), respectively. They were cultured in DMEM
and 10% FBS supplemented with 3 different culture-conditioned
media (control group: conditioned medium from scr-transfected
ACHN cells; PDGFB group: conditioned medium from PDGF-
B–transfected ACHN cells; Imatinib group: conditioned medium
from PDGF-B–transfected ACHN cells and inhibitor of PDGF-
B). The Imatinib group, conditioned with inhibitor of PDGF-B,
is used to detect the change of VSMCs and HUVECs
capabilities in vitro when PDGF-B expression is down-
regulated. Hence, we can observe the effects of PDGF-B on
VSMCs and HUVECs functions in vitro in 3 different
expression levels.
3.2. Animal studies
A total of 20 nude mice that were 4 to 6 weeks old with
immune deficiency were divided into 2 groups randomly
 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11
(PDGFB group and control group). All animal studies
conformed to the guidelines of our laboratory animal ethics
committee of Tianjin Medical University. PDGF-B–trans-
fected and scr-transfected ACHN cells were rinsed with
phosphate buffered saline (PBS) solution, resuspended, and
injected onto the left groin of mice and each injection of
ACHN cells contained 0.2  106 cells. Tumor size was
measured every 3 days. The tumor size was calculated using
the formula V ¼ a2  b/2, where a and b represent the
shortest and longest diameter of tumors, respectively. The
mice were then killed on the 60th day. Tumor tissue was
fixed in 4% buffered formalin for paraffin fixation accord-
ing to standard procedure and 4-mm paraffin sections of
tumors were used for subsequent immunohistochemistry
and histomorphometry analyses.
3.3. Immunohistochemistry and histomorphometry
Immunohistochemistry to assess tumor cells proliferation
and hematoxylin/eosin (H&E) staining to observe the histo-
morphology of tumor progression (metastasis and invasion)
were performed using paraffin sections of 4-mm in thickness
from tumors. Immunohistochemistry: After deparaffinization,
sections were steam pretreated in a citrate buffer at pH 6.0 for
2.5 minutes. The endogenous peroxidase activity was blocked
with 3% hydrogen peroxide. The conventional tissue sections
were then incubated overnight at 41C with one of the primary
antibodies—rabbit anti-mouse Ki-67 (CST, #9027S, 1:50).
The sections were incubated for 1 hour at room temperature
and then washed using PBS with 0.1% for 15 minutes; they
were then incubated at room temperature with rabbit secon-
dary antibody for 1 hour followed by PBS washes. They were
stained with 0.02% diaminobenzidine solution followed by
counterstaining with hematoxylin, and then washed in tap
water for 10 minutes. The slides were dehydrated with ethanol
and xylene. Glass coverslips were mounted with neutral gums
routinely. Histomorphology analysis of tumor metastasis and
invasion: Briefly, after deparaffinization, sections were stained
with hematoxylin, and then washed in tap water for 10
minutes. Eosin staining was carried out for 5 minutes. The
slides were dehydrated with ethanol and xylene. Glass cover-
slips were mounted with neutral gums routinely.
4. Research in vitro
4.1. Cell proliferation analysis of transfected cells
Proliferation analysis of transfected cells was performed
using the MTT assay. A stock solution was prepared by
dissolving 5 mg of MTT (3-[4,5-dimethylthiazol-2-yl]-2,
5-diphenyltetrazolium bromide; sigma, M2128) in 1 ml of
PBS and filtering the solution to remove particulates. The
solution was protected from light, stored at 41C, and used
within 1 month. Cells were seeded into 96-well plates and
after 24, 48, 72, 96, and 120 hours, absorbance was
3
determined by the MTT assay. After 4 hours of incubation
in a medium containing MTT, the cells were lysed using
dimethyl sulfoxide. The conversion of MTT to formazan by
metabolically viable cells was monitored at 490 nm by a
microplate reader (Molecular Devices, SpectraMaxM2).
4.2. Immunoblotting analysis
The phosphorylation PDGFR-β (p-PDGFR-β) expression
levels of VSMCs and HUVECs in 3 different culture-
conditioned media (control group, PDGFB group, and
Imatinib group) were detected using Western blotting. The
lysates were prepared, and equal amounts of protein were
used for blotting with anti–p-PDGFR-β antibody (CST,
#4549S, 1:200). The level of p-PDGFR-β were standardized
against β-actin level. p-PDGFR-β was imaged using the
Electrochemiluminescence system (Bio-Rad, VersSa Doc).
4.3. Effects of PDGF-B overexpression on VSMC and
HUVEC functions in vitro
To detect the effects of PDGF-B overexpression on
mural cells and endothelial cells in vitro, a variety of cell
proliferation, migration, and tube formation assays of
VSMCs and HUVECs were performed as follows.
Flow cytometry (FCM) analysis was performed to
measure the cell cycle distribution of VSMCs and HUVECs
in 3 different culture-conditioned media (control group,
PDGFB group, and Imatinib group). Cells were grown in
plates for 48 to 72 hours, washed in PBS, and fixed in 95%
ethanol at 41C more than 12 hours. Cells were washed in
PBS again. Propidium iodide was added and 30 minutes
later, FCM analysis was performed using Multicycle soft-
ware to analyze the cell cycle.
The effects of tumor cell–secreted PDGF-B on VSMCs
and HUVECs migration in vitro were investigated using
Transwell Cell Culture Chambers (BD, 353097) with
uncoated inserts (8.0-μm pores). Equal numbers (1  105
per ml) in 200 ml of cells (VSMCs or HUVECs) were
placed in the upper chamber. Furthermore, 300-ml DMEM
and 20% FBS supplemented with 3 different culture-
conditioned media (control group, PDGFB group, and
Imatinib group) were plated into the lower chamber. After
20 hours of incubation in 5% CO2 at 371C; nonmigrating
cells were scraped from the upper surface of the filter. Cells
on the lower surface were fixed and stained (Richard-Allan,
139786). The number of cells on the lower surface of the
filter was determined microscropically by counting 5
random fields per insert at 400 magnification.
Matrigel 3-dimensional systems were used to detect tube
formation capability of VSMCs and HUVECs in 3 different
media (control group, PDGFB group, and Imatinib group).
A quantity of 150 μl of Matrigel was added to 24-well cell
culture plate, and the culture plate was placed at 371C
incubator for 30 minutes to let the Matrigel solidify.
Cells were trypsinized and adjusted to a concentration of
4 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11

1  105 per ml and 1-ml cell suspension was added in
Matrigel. Then it was cultured in 5% CO2 incubator at 371C.
4.4. Statistical analysis
Cancer-specific survival was calculated using the Kaplan-
Meier method, and the log-rank test evaluated differences
between survival distributions. A multivariate analysis of
variance was conducted for comparisons between 3 different
groups. Statistical differences between PDGF-B–transfected
and scr-transfected tumor cells were examined using the
2-tailed Student t test or the chi square test. A P o 0.05 was
considered statistically significant. These analyses were
conducted in SPSS Statistics 17.0 software.
5. Results
5.1. Microarray analysis showed high expression of
PDGF-B and PDGFR-β associated with decreased risks of
cancer-specific mortality
We found that high expression of PDGF-B remained
associated with significantly decreased risk of death due to
cancer. Cancer-specific survival was significantly different
between high and low PDGFB groups (P r 0.001; Fig. 1A
and B). It also showed the superiority of high expression of
PDGFR-β over low expression of PDGFR-β, although a
statistically significant difference was not observed in these
2 groups (P ¼ 0.056; Fig. 1C and D).
6. Research in vivo
6.1. Expression of PDGF-B in transfected ACHN cells
The morphology of 6 renal cell lines is showed in
Supplementary Fig. S1 (available online). The ACHN cell
line was chosen for further studies as it was cultured well
and the expression of PDGF-B was low. PDGF-B expres-
sion of PDGF-B–transfected and scr-transfected ACHN
cells was verified by Western blotting (Fig. 2A and B).
VSMCs and HUVECs cultured in 3 different media and
dyed fluorescent green (VSMCs) and fluorescent red
(HUVECs), respectively (Fig. 2C and D).
6.2. Effects of PDGF-B overexpression on ACHN tumor
growth and tumor cells proliferation in vivo
All mice were killed on the 60th day, and the tumors in 2
groups were showed in Fig. 3A. Tumor volumes of PDGFB

Fig. 1. (A) The mRNA expression of PDGF-B in low and high expression groups. (B) Cancer-specific survival of patients with ccRCC according to PDGF-B
expression levels. (C) The mRNA expression of PDGFR-β in low and high expression group. (D) Cancer-specific survival of patients with ccRCC according
to PDGFR-β expression levels. mRNA ¼ messenger RNA. (Color version of figure is available online.)
 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11 5

Fig. 2. (A) The ACHN and HTB-46 cell lines among 6 human renal cancer cell lines had lower PDGF-B expression, as evaluated by Western blotting
method. (B) The PDGF-B expression of PDGF-B–transfected and scr-transfected ACHN cells was verified using Western blotting. VSMCs and HUVECs
were dyed fluorescent green (C) and fluorescent red (D), respectively. (Color version of figure is available online.)

group were smaller than control ones (P r 0.05; Fig. 3B).
Furthermore, data indicated a significant reduction of
proliferating tumor cells in PDGF-B–transfected tumors
compared with control tumor cells, which was confirmed
by immunohistochemistry staining with Ki-67 (P ¼ 0.019;
Fig. 3C and D).
proliferation in vitro revealed that there was no significant
difference in OD value between PDGF-B–transfected and
scr-transfected ACHN cells (P 4 0.05; Fig. 4).
7.2. The p-PDGFR-β expression level of VSMCs and
HUVECs in 3 different culture-conditioned media

6.3. Effects of PDGF-B overexpression on tumor
metastasis and invasion
To detect the metastasis and invasion in tumor-
bearing mice models, tumors sections stained with H&E
(Fig. 3E) showed that in PDGFB group, 1 mouse was
found to have invasion in fibrofatty tissue around the tumor.
In the control tumors, 1 mouse had invasion in fibrofatty
tissue; 1 mouse had skeletal muscle metastasis, tumor
embolus in lymph vessel, and lymph node metastasis; and
1 mouse had pulmonary metastasis and fibrofatty tissue
invasion.
7. Research in vitro
7.1. Effects of PDGF-B overexpression on transfected
tumor cells proliferation in vitro
MTT assay was performed to assess whether over-
expression of PDGF-B would promote ACHN cells
Up-regulation of p-PDGFR-β was observed in VSMCs
cultured in PDGFB group and VSMCs cultured in Imatinib
group expressed lower PDGFR-β than control group, as
detected by immunoblotting. For HUVECs, the expression
of p-PDGFR-β in PDGFB group increased slightly and the
expression almost disappears when cultured with Imatinib
(Fig. 5A).
7.3. Effects of PDGF-B overexpression on VSMCs and
HUVECs functions in vitro
FCM analysis was performed to determine whether
overexpression of PDGF-B affects cell cycle distribution
of mural cells and endothelial cells. Proliferation capability
of VSMCs in PDGFB group was significantly increased, as
VSMCs have a tendency to reside in S and G2/M phases of
cell cycle than the control group (P ¼ 0.001). But for
HUVECs, there was no significant difference (P 4 0.5) in
proliferation capability among these 3 different media
(Table and Supplementary Fig. S2).
6 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11

Fig. 3. (A) A total of 20 mice was divided into 2 groups (PDGFB group and control group). Tumors were successfully induced in 9 mice in the PDGFB group
and 10 mice in the control group. (B) Tumor size in the PDGFB group was significantly smaller than in the control tumors. *P r 0.05. (C and D) There was a
significant reduction in proliferating tumor cells in PDGF-B–transfected ACHN tumors compared with the control ones, which was confirmed by staining with
Ki-67, *P r 0.05 vs control group. (E) Tumors sections were stained for H&E to detect metastasis and invasion in ACHN tumor-bearing mice models. The
results revealed that in the PDGFB group tumors, 1 mouse showed with invasion in the fibrofatty tissue around the tumor. In the control tumors,
1 mouse had invasion in the fibrofatty tissue; 1 mouse had skeletal muscle metastasis, tumor embolus in lymph vessel, and lymph node metastasis; and
1 mouse had pulmonary metastasis and fibrofatty tissue invasion. (Color version of figure is available online.)

Cell migration assay showed that the migration of
VSMCs from the upper chamber was significantly increased
in the PDGFB group than in control group (P r 0.001); the
migration of VSMCs in Imatinib group was significantly
inhibited than in control group (P ¼ 0.007; Fig. 5B and C).
Nevertheless, the migration capability of HUVECs has no
significant difference among the 3 groups (P 4 0.5;
Fig. 5B and C).
Tube formation assay revealed that VSMCs cultured in
PDGF-B–overexpressed conditioned medium stimulated the
tube formation, whereas VSMCs in the other 2culture-
conditioned media did not form tube (Fig. 5D). The tube
formation capability in HUVECs showed no significant
difference among the 3 different media (Fig. 5D).
8. Discussion
Our microarray analysis performed to investigate the
PDGF-B and PDGFR expression of ccRCC and survival
analysis showed that high expression of PDGF-B and
PDGFR-β was associated with decreased risk of death due
to cancer. The studies in vivo further showed that PDGF-B
overexpression significantly inhibited tumor growth and
 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11
Fig. 4. MTT assay to assess ACHN tumor cells proliferation revealed no
significant difference in proliferation capability between PDGF-B–trans-
fected and scr-transfected ACHN cells, P 4 0.05 (MTT proceded for 5
days and the OD value at 120th hour exceeded the effective measurement
range of MTT for both).
progression in ccRCC. Our studies in vitro revealed that the
capabilities of proliferation, migration, and tube formation
of mural cells in PDGF-B–overexpressed conditioned
medium were increased, which may be associated with
blood vessel maturation and stability by pericyte recruit-
ment and lead to decreased tumor metastasis and invasion
events directly. PDGF-B is further integral to pericyte and
endothelial cell interactions and inhibits tumor growth and
proliferation of tumor cells. All these account for the results
in microarray experiments that patients with up-regulation
of PDGF-B or PDGFR have beneficial outcomes.
Several ligand-receptor systems have been implicated in
the interaction between endothelial cells and pericytes to
regulate vessel maturation and stability. The prior study
investigated the effect of PDGF-induced cellular migration
during angiogenesis in vitro and showed that PDGF-BB
possibly contributes to endothelial repair and angiogenesis
by direct effects on proliferation and composite movements
of endothelial cells and cords [12]. Pericytes are ubiquitous
in the tumor microenvironment [13]. The PDGF-B and
PDGFR-β signaling has been shown to be directly involved
in pericyte recruitment into tumor blood vessels. The
functional significance of PDGFR-β signaling in tumor
pericytes was confirmed by studies in genetic ablations of
PDGF-B or PDGFR-β. Mice lacking PDGF-B or PDGFR-β
showed a severe deficit in pericytes and develop an
abnormal vasculature with many features in common with
tumor vessels, such as irregular vessel diameter and
increased leakiness. And the cause of microvascular dys-
function in the mutant mice is severe pericyte deficiency
[8,14]. Recent experiments suggest that PDGF-B produced
by tumor cells stimulates pericyte recruitment in gastric
cancer as well [15]. And other studies also revealed the
increased pericyte recruitment induced by PDGF-B in
different tumors [16,17].
Though our current study did not detect the pericyte
recruitment in xenografted tumors as no single marker is
definitive in identifying pericytes, the in vitro studies
determined the increased capabilities of proliferation,
7
migration, and tube formation of VSMCs induced by
PDGF-B. Mural cells (VSMCs and pericytes) migrate
almost simultaneously along endothelial cells during blood
vessel formation and PDGF-B appears to play a major role.
It is important to note that pericytes are unable to expand
and spread along the newly formed vessels because of their
reduced proliferation capability and reduced migration
capability, suggesting that PDGFB/PDGFR-β signaling is
required for the proper attachment and for pericyte migra-
tion and proliferation [18]. Our research in vitro also
confirmed the results of previous work that suggested that
PDGF-B might be involved in promoting VSMC prolifer-
ation and this reduced proliferation rate of VSMCs was
found in PDGF-B mutants [14]. All these effects of PDGF-
B on VSMCs possibly facilitate pericyte recruitment during
vessel maturation and stability, which is involved in the
inhibition of metastatic evens in cancer. Tumor cells in a
fibrosarcoma model (T241) limit tumor cell metastasis by
stabilizing the microvessel wall with improved pericyte
recruitment and increased perivascular deposition of extrac-
ellular matrix molecules [19]. This is in agreement with
another study suggesting a significant negative correlation
between pericyte coverage and metastasis [20]. Conversely,
Nakamura et al. [21] stated that PDGF-B may be associated
with vascular invasion in clinical cases of colorectal cancer
and the extent of invasion and migration of PDGF-B–
transfected tumors was obviously greater than that of scr-
transfected ones, suggesting that cells expressing PDGF-B
were more malignant than those not expressing PDGF-B.
Moreover, it has been reported that PDGF-B induces
intratumoral lymphangiogenesis and promotes lymphatic
metastasis in murine experiments [22]. These discoveries
further make the scenario more complicated and need
further investigation as PDGF-B and PDGFR-β signal
pathway may have a different role in tumor microenviron-
ment.
In our studies, we also demonstrated that overexpression
of PDGF-B by human kidney cancer cells inhibited tumor
growth in vivo, which also may be attributed to up-
regulation of PDGF-B expression. Tumor volumes of
PDGF-B–overexpressed tumors were smaller than in the
control ones, and there was a significant reduction in
proliferating tumor cells in PDGF-B–transfected tumors
compared with control tumors, which was confirmed by
immunohistochemistry by staining with Ki-67. The inhib-
ition of tumor growth induced by PDGF-B attracted our
attention. The PDGF and PDGFR signaling has previously
been shown to be directly involved in oncogenic trans-
formation [23,24]. There is extensive evidence for the
PDGF-B and PDGFR-β signaling leading to enhanced
tumor growth or progression in many tumor types, such
as lung cancer and breast cancer [25,26]. In a study by Guo
et al. [16], transfection of PDGF-B into glioblastoma cells
led to increased pericyte coverage, but with enhanced
angiogenesis and tumor growth in the brain microenviron-
ment. The discrepancies between our results and those of
8 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11

Fig. 5. (A) Up-regulation of p-PDGFR-β was observed in VSMCs cultured in the PDGFB group, and VSMCs cultured in the Imatinib group expressed lower
PDGFR-β. The expression of p-PDGFR-β of HUVECs in PDGFB group increased slightly, and it almost disappeared when cultured with Imatinib. (B)
Migration of VSMCs from the upper chamber was significantly increased in PDGF-B over-expressed conditioned medium counted at 400 magnification,
while the migration of HUVECs had no significant difference among the 3 groups. (C) Quantitative data of average number of cell migrating. **P r 0.01 vs.
control group; ***P r 0.001 vs. control group. (D) Tube formation assay showed stimulation of VSMCs capability to form tubes induced by PDGF-B and no
tube formation in the other 2 groups; the HUVECs tube formation showed no significant difference among the 3 different groups— original
magnification, 100. (Color version of figure is available online.)

Guo et al. are probably because of the activation of an
autocrine loop, which resulted in increased tumor cell
proliferation, as the U87MG glioblastoma cells used in
the study by Guo et al. express high levels of PDGFR-β.
Our current study demonstrated that positive staining with
PDGFR-β was detected in the stromal cells (including
pericytes) rather than transfected tumor cells, and there
was no significant difference in proliferation between
PDGF-B–transfected and scr-transfected ACHN cells, sug-
gesting the potential role of the PDGF-B and PDGFR-β
loop in the RCC microenvironment is paracrine and there
was no autocrine effect of PDGF-B on ACHN cells
 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11
Table
The role of PDGF-BB on the cell cycle of VSMCs and HUVECs
Groups G0/G1(%) S(%) G2/M(%)
VSMC
Control 66.99 ⫾ 1.53 17.82 ⫾ 0.89 15.20 ⫾ 1.02
PDGFB 51.54 ⫾ 2.48 30.48 ⫾ 1.49a 17.97 ⫾ 0.99a
Imatinib 78.19 ⫾ 2.03 10.82 ⫾ 1.17b 10.94 ⫾ 1.39b
HUVEC
Control 56.84 ⫾ 2.03 27.45 ⫾ 0.71 15.72 ⫾ 1.33
PDGFB 58.54 ⫾ 0.44 22.51 ⫾ 2.49 19.16 ⫾ 1.96
Imatinib 58.00 ⫾ 1.15 26.81 ⫾ 0.54 15.18 ⫾ 0.68
a not influence the outcome of therapeutic approaches based
VSMCs in PDGFB group have a tendency to reside in the S and G2/M
phases of cell cycle than control group (P ¼ 0.001).
b
Less VSMCs in the Imatinib group were distributed into the S or G2/M
phases of the cell cycle than in the control group (*P r 0.05).
proliferation in vitro. Depletion of pericytes led to an
unexpected increase in tumor growth in some models
[27,28]. An increase in pericytes associated with PDGF-B
overexpression led to a decrease in tumor growth was
determined in other studies as well [17,29]. Their studies
in vitro suggested that the observed reduction of tumor
growth might be because of pericyte-mediated inhibition of
endothelial cell proliferation [17]. This role of pericytes in
inhibiting endothelial cell proliferation was also revealed by
other studies [30,31]. The proliferating endothelial cells is
reduced by half when tumor vessels are covered by pericytes,
and this function may be mediated by the cell-cell contact
and through the production of soluble mediators [30].
The idea of disrupting tumor angiogenesis to treat cancer
has been explored for many years. Accordingly, the
correlation of PDGF-B and tumor vasculature also initiates
lots of research. MVD is a quantified variable of tumor
vasculature, and its variety may be influenced by PDGF-B
expression. In the study, the relatively high expression of
PDGF-B associated with inhibition of microvessel counts
marked by CD31 antibody, suggesting that PDGF-B might
inhibit the angiogenic process [17]. On an average, micro-
vessel counts in PDGF-B–expressing tumors were only
41% of those in control tumors (P o 0.02). The study
revealed that a higher MVD score (stained with CD34)
correlated with high expression of PDGF-B in intestinal-
type gastric cancers, but this correlation was not found in
diffuse-type cancers [15]. No significant association was
observed between survival and PDGF-B. Thus, studies
assessing the correlation of PDGF-B and tumor vasculature
may differ, depending on different tumor types and the
prognostic value of MVD as a predictor of prognosis may
be controversial, which may be caused partly by the
different methodologies used both to stain and to count
the microvessels in the tumors. This may question the value
of MVD as a predictor of prognosis.
Inhibition of tumor angiogenesis is an encouraging
treatment for many types of cancer, and the vascular
endothelial growth factor signaling pathway is a logical
9
starting point as a target for exploring antiangiogenic
therapy [32]. Published data suggest that pericytes might
confer resistance to VEGF withdrawal in tumors, and lots of
studies have explored strategies that target both VEGF
signaling and PDGFR-β signaling to inhibit tumor angio-
genesis and tumor growth accordingly [33–35]. But there
were observations that suggested that additional targeting of
pericytes does not increase the antitumor effect already
generated by anti-VEGF drugs [27]. And our current study
further verified the role of PDGF-B in the inhibition of
tumor metastasis and growth. Thus, tumor pericytes might
on VEGF-A inhibition and might contribute to vessel
maturation and stability. However, most patients in our
microarray analysis were diagnosed before the era of
targeted therapy and a large proportion of these individuals
may have limited exposure to targeted therapy. The lack of
evaluation of the prognostic value of PDGF-B in patients
with targeted therapy may be an important limitation and
our present data warrant further investigation into the role
of PDGF-B in the era of targeted therapy.
9. Conclusion
Our results demonstrated that PDGF-B, which increased
VSMC proliferation and migration capability during angio-
genesis, limited tumor growth and progression in ccRCC.
PDGF-B appears to be the crucial player in recruitment of
pericytes to newly formed vessels, which may lead to
decreased tumor metastasis and invasion events directly.
Therefore, PDGF-B may be a novel and promising prog-
nostic marker. But further investigations will be needed to
clarify the potential roles of PDGF-B and pericyte in tumors.
9.1. Ethical standards
All persons involved in our study have given their
informed consent before their inclusion in the study. All
animal studies conformed to the guidelines of our labora-
tory animal ethics committee of Tianjin Medical University.
Acknowledgment
We thank all individuals who participated in this study
and all the people who helped us to successfully complete
this research. We also thank Bin Tean Teh for introducing
RCC cell lines into our laboratory and for making the VARI
dataset available to researchers.
Appendix A. Supplementary Materials
Supplementary material cited in this article is available
online at http://dx.doi.org/10.1016/j.urolonc.2014.12.015.
10 W. Wang et al. / Urologic Oncology: Seminars and Original Investigations ] (2014) 1–11
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