B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells
et al. (2013) B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and
Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells. PLoS ONE 8(10): e76012. doi:10.1371/journal.pone.0076012
B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells
Sheng-Jia Shi 0 1
Li-Juan Wang 0 1
Guo-Dong Wang 0 1
Zhang-Yan Guo 0 1
Ming Wei 0 1
Yan-Ling Meng 0 1
An- 0 1
Gang Yang 0 1
Wei-Hong Wen 0 1
0 Editor: John Souglakos, University General Hospital of Heraklion and Laboratory of Tumor Cell Biology, School of Medicine, University of Crete , Greece
1 1 State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University , Xi'an, China , 2 Department of Oncology, the First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University , Xi'an, China , 3 Department of Urology, Xijing Hospital, Fourth Military Medical University , Xi'an, China , 4 Department of Comprehensive Medicine, 323 Hospital of the Chinese People's Liberation Army , Xi'an , China
Background And Objective: The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells. Methods: Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells. Results: Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition. Conclusions: Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.
Funding: This work was supported by the National Natural Science Foundation of China (No.30873004, 30973000 and 81171924) and Natural Science
Foundation of Shan xi Province (No.2011JM4006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
These authors contributed equally to this work.
Colorectal carcinoma is the third most frequently diagnosed
malignancy in the world and the fifth leading cause of death
among cancer patients in China . Due to a lack of effective
diagnostic markers, most colorectal cancer patients have
distant metastases (stage IV) at diagnosis. The most effective
colorectal cancer treatment is surgery. However, the lack of
accurate prognosis markers makes it difficult to predict patient
survival time after surgery. Thus, new and effective markers for
diagnosis and prognosis are required in the clinic.
The co-stimulatory molecule B7 homolog 1 (B7-H1 or
CD274) is a recently identified ligand for the co-inhibitory
receptor programmed death-1 (PD-1 or CD279) [2,3]. B7-H1 is
expressed on T cells, B cells, macrophages and dendritic cells.
The expression of B7-H1 can be further upregulated upon
lymphocytes, B7-H1 has also been detected at low levels on
pancreatic islets and syncytiotropho-blasts in the placenta [5,6].
Traditionally, the function of B7-H1 on antigen-presenting cells
is achieved through binding with PD-1 on T cell, which is
thought to have an important role in the induction and
maintenance of immune tolerance [7-9].
In addition to the expression on lymphocytes and normal
tissue, aberrant B7-H1 expression has also been found in
various human malignancies. Tumor types including squamous
cell carcinomas of the lung, esophagus, head and neck, and
other types of carcinomas such as ovarian, bladder, breast
cancer, melanoma and glioma also express B7-H1 [10-16]. The
expression of tumor-associated B7-H1 is correlated with poor
prognosis and high malignancy grade. The blockade of
tumorassociated B7-H1 has been shown to promote tumor
regression in vivo in several murine tumor transplants
[10,12,17,18]. PD-1 expression is upregulated on
tumorinfiltrating lymphocytes, and it has been proposed that B7-H1
expressed on cancer cells may inhibit the function of infiltrating
lymphocytes . It has also been demonstrated that
tumorassociated B7-H1 can induce apoptosis of CTL, which
subsequently resulted in an escape from T cell-mediated
immune surveillance [8,10]. Previous study paid excessive
attention to the function of tumor-associated B7-H1 which take
effect as a ligand for PD-1 or CD80, but neglected the effect of
tumor-associated B7-H1 itself on tumor cell. The study
concerning the effect of tumor-associated B7-H1 on tumor cell
is still in its infancy. Thus, tumor-associated B7-H1 may act in
concert with other negative regulators of T cell activation to
dampen the host antitumor immune response , also with
the great possibility, tumor-associated B7-H1 may affect the
process of cancer progression through interfering with the
function of cancer cell.
The expression of B7-H1 in colorectal carcinomas is
inconsistent. Dong et al. failed to detect B7-H1 expression in
normal colon tissues, but the expression of B7-H1 was
detected in a relatively high proportion (10/19) of colorectal
cancer patients . However, another group identified the
expression of B7-H1 on mRNA level but also failed to detect
the surface expression of B7-H1in colonic epithelial cells from
healthy controls . The investigation concerning the B7-H1
expression in colorectal cancer cells is at an early stage. It is
unclear whether B7-H1 expression may have diagnostic or
prognostic value in colorectal carcinoma. Additionally, how
B7H1 is associated with the clinical features of colorectal
carcinoma is not known. In this study, we investigated the
expression profile of B7-H1 in 5 normal colon tissues, 143
colorectal cancer tissues and 44 adjacent tissues. We also
evaluated the predictive value of B7-H1 for prognosis in
colorectal cancer patients and examined whether
tumorassociated B7-H1 itself could directly modulate colorectal
cancer progression rather than through binding to PD-1 on T
Materials and Methods
2.1: Patients and follow-up
As we described previously, this study was approved by the
ethics committee of Fourth Military Medical University and all of
the participating patients have given their written informed
consent for their information and tissue samples to be stored in
the database of Xijing Hospital and used for research . .
The retrospective cohort we investigatedincluded143 patients
with potentially resectable colorectal carcinoma diagnosed from
February 2006 to December 2007. The patients were collected
from the Department of Gastrointestinal Surgery, Xijing
Hospital, Fourth Military Medical University. The exclusion
criteria for recruitment in this study are the following: receiving
adjuvant chemotherapy before surgery, diagnosis of
gastrointestinal stromal tumor or lymphoma, diagnosis with
additional cancers, and any patient who refuses consent. The
clinical specimens were retrieved from the tissue archive in the
Department of Pathology, Xijing Hospital, Fourth Military
Medical University. The follow-up information of all participants
was updated every 3 months by telephone. The overall survival
was defined as the time elapsed from surgery to death.
Information regarding the death of patients was ascertained
from their family.
2.2: Immunohistochemical (IHC) staining and
Paraffin-embedded sections of normal and tumor tissues
were stained for B7-H1 expression. The immunohistochemical
staining for B7-H1 was performed as previously reported with
slight modifications . Briefly, slides were deparaffinized in
xylene and rehydrated in a graded alcohol series before
endogenous peroxidase activity was blocked with 3% H2O2. All
nonspecific protein binding was blocked using pre-immune
rabbit serum. The primary antibody for B7-H1 (Abcam,
ab58810) was diluted according to the recommended
concentration (5g/ml), and the sections were incubated
overnight in a humidified chamber at 4C. The sections were
washed 3 times with PBS, and then a biotinylated secondary
antibody was added and incubated for 30 min at room
temperature. The signal visualization was performed using
DAB chromogen for 2 to 3 min. The negative staining control
was made by replacing the primary antibody with pre-immune
rabbit serum. The B7-H1 staining was scored independently by
two pathologists blinded to the clinical characteristics of the
patients. The scoring system used in grading the B7-H1
expression was described previously . Tumors with strong
and moderate immunostaining intensity were classified as
having positive (+) expression, whereas tumors with absent
and weak immunostaining were classified as having negative
2.3: Cell culture
Human colon cancer HCT116 cells were obtained from the
Cell Bank of the Chinese Academy of Sciences (Shanghai,
China), where they were characterized by mycoplasma
detection, DNA Fingerprinting, isozyme detection and cell
vitality detection. This cell line was immediately expanded and
frozen so that the cell cultures could be restarted every 3 to 4
months from the same batch of frozen vials. The HCT116 cells
were cultured in Dulbeccos modified Eagles medium
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine
serum (FBS) (HyClone, Logan, UT) and cultured in a
humidified incubator at 37C in 5% CO2.
2.4: SiRNA transfection and gene silencing
To silence B7-H1 in cells, a small interfering RNA (siRNA)
was transfected into the cells. The siRNAs duplexes
5CCAGCACACUGAGAAUCAATT-3), targeted the B7-H1 gene.
The negative control duplex
(5UUCUCCGAACGUGUCACGUTT-3) targeted a nonspecific
sequence. The siRNAs were synthesized by Sangon Biotech
(Shanghai, China). The siRNA duplexes (100 nmol/L) were
transfected into HCT116 cells using Lipofectamine 2000
(Invitrogen, Carlsbad, CA) according to the manufacturers
instructions. The HCT116 cells were harvested 48 h after
transfection for further analysis. The inhibition efficiency was
identified by western blot (Figure S1).
2.5: RT-qPCR (reverse transcriptionquantitative PCR)
Total cell RNA was extracted using TRIzol reagent
(Invitrogen, Carlsbad, CA, USA) according to the
manufacturers protocol. The RNA was then reverse
transcribed with the Revert AidTM First Strand cDNA Synthesis
Kit (Fermentas, Sankt Leon-Rot, Germany) according to the
manufacturers instructions. The
reverse-transcriptionquantitative polymerase chain reactions (RT-qPCR) were
performed using a CFX96TM Real-Time PCR system (BioRad,
Valencia, CA) with SYBR Green reagents (#DRR041A; Takara
Bio, Japan) according to the manufacturers instructions. The
RT-qPCR analysis was performed in a total volume of 20 L
with the following amplification steps: an initial denaturation
step at 95C for 10 min, which was followed by 40 cycles of
denaturation at 95C for 15 sec and elongation at 55C for 30
sec. The RT-qPCR gene expression was normalized to human
-actin. The primers used for real-time PCR in this study were
the following: 5- TCAATGCCCCATACAACAAA -3 (sense)
and 5-TGCTTGTCCAGATGACTTCG -3 (antisense) for
B7H1; 5-CGTCTTCCCCTCCATCGT-3 (sense) and
5GAAGGTGTGGTGCCAGATTT-3 (antisense) for -actin.
2.6: Western blot
Cells were harvested in lysis buffer (50 mM NaCl, 50 mM
EDTA, 1% Triton X-100) containing protease inhibitor cocktail
(Roche, Indianapolis, IN, USA). The cell lysates (30 g) were
separated using 10% SDS-PAGE gels and then transferred
onto nitrocellulose membranes (Millipore, Bedford, MA). The
membranes were blocked with 5% nonfat milk diluted in PBS
for 2 h at room temperature before the addition of the
appropriate primary antibody. The antibodies used in this study
included anti-B7-H1 (1:400; Abcam, ab58810) and anti-GAPDH
(1:2,000; Abcam, mAbcam9484). The membranes were then
washed with PBS containing 0.05% Tween and incubated with
the appropriate HRP-conjugated secondary antibody (1:10,000;
Abcam) for 1 h at room temperature. The bands were
visualized using a chemiluminescence reagent (New England
Nuclear, Boston, MA).
2.7: MTT assay
Cell proliferation was analyzed in vitro with the tetrazolium
salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) reagent. HCT116 cells were transfected with
specific siRNA (si-scramble or si-B7-H1) for 24 h, and then
proliferation was examined. Briefly, 2000 cells from each group
(parental, si-scramble or si-B7-H1) were plated in each well of
five 96-well plates in 200 L of medium. To analyze cell
proliferation, 20 L of MTT substrate at a concentration of 2.5
mg/mL in PBS was added to each well. The plates were then
returned to a standard tissue incubator for an additional 4 h.
The medium was then removed, and the cells were solubilized
in 150 L of dimethylsulfoxide for the colorimetric analysis
(wavelength, 490 nm). One plate was analyzed immediately
after the cells adhered (approximately 4 h after plating). Then,
one plate per day was examined for the next 4 consecutive
2.8: Flow cytometric analysis to detect cell apoptosis
HCT116 cells were transfected with specific siRNA
(siscramble or si-B7-H1) for 48 h, and the cells were then
suspended in incubation buffer at a density of 1106 cells/mL.
The cells were incubated with Annexin V-FITC and propidium
iodide (BD Bioscience, San Jose, CA) for 15 min in the dark at
room temperature. Cell apoptosis was then analyzed using a
flow cytometer (BD FACS Aria).
2.9: Migration and invasion assay
Cell migration and invasion capacity were measured in vitro
using transwell migration assays (Millipore, Billerica, MA). The
HCT116 cells were transfected with specific siRNA
(siscramble or si-B7-H1) for 48 h and suspended in DMEM with
10 g/L BSA at a density of 50 cells/mL. Then, cell suspensions
(200L) were seeded in the upper chamber with aporous
membrane coated with (for the transwell invasion assay) or
without (for the migration assay) Matrigel (BD Bioscience, San
Diego, CA). To attract the cells, 500 L of DMEM with 10%
serum was added to the bottom chamber. After allowing the
cells to migrate for 24 h or to invade for 48 h, the penetrated
cells on the filters were fixed in dried methanol and stained in 4
g/L crystal violet. The numbers of migrated or invasive cells
were determined from five random fields using a microscope
(Olympus) at 10 magnification.
2.10: Statistical analysis
Statistical analysis was performed using IBM SPSS
statistical software (version 20.0). Survival curves were
estimated using the Kaplan-Meier method, and distributions
were evaluated by the long-rank test. Cox proportional hazard
models of factors related to survival were used to calculate
HRs and to identify the factors that affect survival. The
differences in characteristics between the 2 groups were
examined by the 2 test and Fishers exact test. All P-values
were determined from 2-sided tests, and statistical significance
was based on a P-value of 0.05.
3.1: Clinical significance of positive B7-H1 expression
in colorectal cancer tissue
To determine the prevalence and clinical significance of
B7H1 expression in colorectal carcinoma, we evaluated the
B7H1 protein level by immunohistochemistry in a retrospective
cohort of 143 colorectal cancer patients after tumor resection.
Among the 143 patients, 64 patients (44.8%) showed positive
B7-H1 expression in the cytoplasm and membrane (Figure 1A).
There were 79 patient samples without detectable B7-H1
expression (Figure 1C). Additionally, only 5 (11.4%) of 44
adjacent tissues showed positive B7-H1 expression (Figure
1B& D). Our study failed to detect B7-H1 expression in normal
colon tissues. We also evaluated the relationship between
B7H1 expression and clinical features using a Pearson chi-square
test or Fishers exact test. We found a trend of increased
B7H1 expression between well and poorly differentiated
carcinoma and from TNM stage i to iv. These results suggest
that B7-H1 expression was significantly associated with cell
differentiation status and TNM stage in colorectal carcinoma
(P=0.030 and 0.034, respectively). However, B7-H1 expression
is not significantly correlated with gender, age, tumor location,
or lymph node status (Table 1).
3.2: Positive expression of B7-H1 is associated with
poor overall survival
To determine the prognostic value of B7-H1 expression in
colorectal carcinoma, we analyzed the relationship between the
B7-H1 expression and clinical outcome. The overall median
patient survival time in our retrospective cohort was 43 months
(range: 1-56 months). Of the 143 patients, 73 patients were
alive and 70 patients were deceased at the time of analysis (60
months). The relationship between B7-H1expression and
overall survival was investigated using Kaplan-Meier analysis
and a log-rank test. The patients were divided into 2 groups
based on whether B7-H1 was present or absent, which was
defined as B7-H1 positive or B7-H1 negative. A statistically
significant difference in overall survival was found between the
B7-H1 positive and negative groups (Figure 1E, log-rank test:
P=0.0169). The patients with positive B7-H1expression tended
to have an increased risk of death compared to patients with
negative B7-H1expression. The unadjusted HR was
2.611(95%CI: 1.008-3.576; p=0.006). As shown in Table 2, cell
differentiation and TNM stage were also associated with the
prognosis of colorectal carcinoma. In multivariate analysis, we
found that positive B7-H1 expression was associated with a
decreased overall survival. The adjusted HR was 2.771
(95%CI: 1.048-2.994; p=0.003), indicating that B7-H1
expression could be a prognostic factor independent of these
adjusted clinicopathologic characteristics (Table 2).
3.3: Effective knockdown of B7-H1 by siRNA in HCT116
It has been reported that tumor-associated B7-H1 could
promote T cell apoptosis , but there are no studies
indicating a role for B7-H1 expression on tumor cells. As a
commonly used human colorectal cancer cell line, HCT116
cells have been shown to be invasive and highly motile in vitro
[24-26]. Thus, to examine the function of B7-H1 in colorectal
cancer cell biology, we used siRNAs targeting B7-H1 to inhibit
the B7-H1 expression. We then examined the tumor cell
characteristics including cell proliferation, apoptosis, migration
and invasion. The effective knockdown of B7-H1 was
confirmed by qRT-PCR, western blot and flow cytometry
analysis. Compared to cells transfected with scrambled siRNA,
cells transfected with siRNAs to B7-H1 showed significantly
reduced B7-H1 expression (each experiment was performed
three times, and the typical result is present as Figure 2A, 2B
3.4: Effect of B7-H1 knockdown on cell proliferation
After confirming the knockdown efficiency of the siRNAs
targeting B7-H1, we determined the effect of a reduced B7-H1
level on cell proliferation using an MTT assay. HCT116 cells
that were transfected with siRNA targeting B7-H1 showed
significantly less proliferation than the parental or scrambled
siRNA-transfected cells (Figure 3A). This result demonstrated
the B7-H1 had a direct effect on cell proliferation in HCT116
cells and that a high B7-H1 protein level is correlated with
increased cell proliferation.
3.5: Effect of B7-H1 knockdown on cell apoptosis
We have demonstrated that the B7-H1 expression level is
correlated with cell proliferation. Therefore, we tested whether
the inhibited cell proliferation may be caused by increased cell
apoptosis in B7-H1 knockdown cells. HCT116 cells transfected
with scramble siRNA or siRNA targeting B7-H1 for 48 h were
analyzed for apoptosis. The results indicate that compared with
the parental or scrambled siRNA-transfected cells, cells
transfected with siRNA targeting B7-H1 had increased
apoptosis index which calculated by add the cells in the 1 and
the cells in the 2(12.35.9% and 17.2%6.2 vs. 1.10.4%, P<
0.05; 12.35.9% and 17.2% vs. 0.90.5%, P< 0.05; each
experiment was performed three times, and the typical result is
present as Figure 3B). Collectively, these results suggest that
the expression of B7-H1 in HCT116 cells is important for both
cell proliferation and apoptosis.
3.6: Effect of B7-H1 knockdown on cell migration and
B7-H1 has been previously shown to regulate cell migration
and invasion in pancreatic carcinoma cells . Our
observations that B7-H1 expression played an important role in
HCT116 cell proliferation and apoptosis led us to assess the
function of B7-H1 on cell migration and invasion in colon
cancer cells. To test migration, we used standard
Matrigelcoated or uncoated transwell chamber assays. We found that
compared with the scrambled siRNA-transfected cells, HCT116
cells transfected with siRNAs targeting B7-H1 had reduced
migration and invasion ability (Figure 4A-4D), and a reduced
invasive index (invasion cell number/migration cell number,
Figure S2). In conclusion, our results indicate that B7-H1
expression in HCT116 cells is correlated with cell proliferation,
apoptosis, migration and invasion.
Figure 1. Immunohistochemical staining of B7-H1 and its correlation with survival in colorectal cancer patients. (A-D)
Representative immunohistochemiscal staining of positive and negative expression of in colorectal cancer or adjacent tissue
(original magnification 100). (A) B7-H1 positive tumour tissue, (B) B7-H1 positive adjacent tissue, (C) B7-H1 negative tumour
tissue, and (D) B7-H1 negative adjacent tissue. Representative pictures were shown. (E) Association between the B7-H1
expression and cancer specific death in 143 colorectal cancer specimens.
This increased risk was independent of gender, age, tumor
size, tumor location, differentiation status and TNM stage.
These results provided the first evidence supporting B7-H1 as
a predictor of poor prognosis in colorectal carcinoma.
The most exciting and unexpected finding was our results
demonstrated that B7-H1 itself correlated with cell proliferation,
apoptosis, migration and invasion. Though some research
groups have confirmed that tumor cells expressing B7-H1 had
a high proliferative index [14,27], most groups tended to
believe B7-H1 prevent tumor destruction only by forming a
molecular shield [18,29] but not forming a more powerful
spear [18,30]. Our finding provides powerful evidence that
B7H1 may have oncogenic function during colonic
carcinogenesis, which shed a new light on the function of
B7H1 in colorectal cancer development.
The recurrence rate of colorectal carcinoma is relatively high.
One possible reason for recurrence is that the residual tumor
may evade host immunesurveillance. Previous studies have
confirmed that high T cell infiltration into colorectal cancer
tissue is correlated with an improved 5-year overall survival. A
high level of T cell infiltration may serve as a better predictor of
prognosis than conventional histopathological staging .
However, it has also been shown that colorectal cancer
patients have an expanded Treg population. The increased
number of Treg cells can suppress CD4+T cell function in
response to tumor-associated antigens . It has also been
reported that the frequency of Tregs in TDLNs was correlated
with disease stage . Colorectal cancer patients with high
expression of Th1 or cytotoxic cluster genes have a prolonged
disease-free survival. Conversely, high expression of the Th17
cluster genes results in a poor prognosis [34,35]. The
relationship between tumor-associated B7-H1 and the function
of infiltrated T cells in the tumor microenvironment has been
well established. It is also well accepted that tumor-associated
B7-H1 can help the tumor cells evade immune surveillance by
inhibiting the function of effector T cells and enhancing the
function of Tregs in colorectal cancer . And our results
support this notion for high expression of B7-H1 which may
paralyze the host immunesurveillance is associated with poor
prognosis in colorectal cancer.
B7-H1 is highly expressed in different types of tumors.
However, the correlation between B7-H1 expression and
colorectal cancer progression has not been well studied
[20,28]. In this study, we confirmed that the expression of
B7H1 could be detected in both colorectal cancer and adjacent
tissues but at a different frequency. We also provided evidence
that positive B7-H1 expression was correlated with adverse
clinical and pathologic features in colorectal carcinoma.
Moreover, we demonstrated that the B7-H1 expression level
was also predictive of disease progression and cancer-specific
death. Our results showed the patients with positiveB7-H1
expression are usually at a significantly higher risk of cancer
progression, cancer-specific death and shorter overall survival.
Figure 3. Effect of B7-H1 knockdown on cell proliferation and apoptosis in HCT 116 cells. (A) MTT analysis to detect cell
proliferation. Parental or HCT116 cells were transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in
96-well plates and cell proliferation was detected by MTT. Data were presented as means SD, *P<0.05 versus the si-scramble
group. (B) Flow cytometric analysis to detect cell apoptosis. Parental or HCT116 cells transfected with scrambled siRNA or siRNA
targeting B7-H1 for 48 h were collected and stained with Annexin-V-FITC and PI before flow cytometric analysis. Data were
presented as means SD, *P<0.05 versus the si-scramble group.
However, the function of B7-H1in the suppression of tumor
immunity is still not fully understood. For example, how B7-H1
regulates T cell function and the putative non-PD-1 receptor
must be identified. The identification of B7-1 as a new B7-H1
receptor made the issue more complex, and the complexity of
these molecular interactions suggests that B7-H1 or PD-1
blockade alone may not be sufficient to block the inhibitory
pathways. And our results make this question even become
more complicated. Our results showed that the cells with
reduced B7-H1 expression had impaired cell proliferation,
migration and invasion. Additionally, reducing B7-H1expression
led to increased cell apoptosis (Figures 3 & 4). Although a
more detailed mechanism must be discovered to explain our
results, these results provide evidence that B7-H1 may be not
only a ligand for PD-1 or a putative non-PD-1 receptor but also
function as an oncogenic molecule. Taken together our results
and previous literatures, we can get the conclusion that B7-H1
can accelerate the progression of colorectal cancer though
inhibiting the function of T cell and enhancing the degree of
malignancy of colorectal cancer cell, thus making it associated
with the poor prognosis in colorectal cancer patients.
We also noticed a recently literature get a contradictory
conclusion with our study. This study found B7-H1 expression
is associated with early tumor stage, absence of lymph node
metastases, lower tumor grade, and a significantly improved
5year survival . The discrepancies between this study and
our work may reflect the different pathological features selected
by each study. The cohort of their study was consist of
unselected, non-consecutive CRC patients, but the patients in
our cohort were recruited with a more rigorous standards,
which excluded the patients receiving adjuvant chemotherapy
before surgery, diagnosis of gastrointestinal stromal tumor or
lymphoma, diagnosis with additional cancers. As known to us
all, accurate results are based on the rigorous exclusion criteria
in retrospective study. We also notice B7-H1 expression was
found in normal colonic tissues in their study, but both our
group and Dong et al fail to detect immunoreactivity in normal
colonic mucosa . The different antibody and score system
may account for this discrepancy. They also found PD-L1
expression correlates with high CD8+ T-cell infiltration in
MMRproficient CRC, and previous study had proven that a high level
of T cell infiltration may serve as a better predictor of prognosis
than conventional histopathological staging . From this
point, it is possible that the benefit of high level of T cell
infiltration compensate the adverse effect of tumor-associated
B7-H1 in their study. Thus we tend to believe the better
outcome of patients with MMR-proficient CRC maybe due to
the higher infiltration of T cell, but not due to B7-H1 high
expression. But considering the contradict conclusion, a
retrospective or prospective studies with a sufficient number of
samples should be conducted to make clear the role of B7-H1
played in colorectal cancer. And more research at the cell level
should be conducted to confirm the effect of B7-H1 on the
biology of the others cancer cell lines.
In conclusion, our retrospective study provides evidence that
B7-H1 functions as an independent predictor of prognosis of
colorectal cancer. In addition to the traditional immune
inhibitory function, we also provided evidence that B7-H1 itself
may have oncogenic function during colonic carcinogenesis by
directly regulating cell proliferation, apoptosis, migration and
invasion. These new findings will improve our understanding of
the relationship between B7-H1 and the progression of
colorectal cancer. We agreed with Dr. Kwon that we are nave
if we think that one bullet will bring down cancer . However,
we also believe that B7-H1 alone or in combination with other
cancer biomarkers may be extremely useful in predicting the
outcome for high-risk patients.
Figure S1. Kinetic analysis of B7-H1 expression. A, Kinetic
analysis of B7-H1 expression by western blotting in HCT116
cells treated with si-B7-H1-1 and si-B7-H1-2. B, Optical density
value of si-B7-H1-1 and -actin. C, Optical density value of
siB7-H1-2 and -actin. The maximum transient inhibition with
both si-B7-H1-1 -1 and si-B7-H1-2 occurs at 48h.
SS, LW, GW, ZG, YM, and MW had full access to all the data
in the study and taken responsibility for the integrity of the data
and the accuracy of the data analysis. The authors thank the
local doctors and the patients who participated in our study.
Conceived and designed the experiments: WW AY. Performed
the experiments: SS LW GW. Analyzed the data: SS ZG.
Contributed reagents/materials/analysis tools: MW YM. Wrote
the manuscript: SS LW.
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