- Open Access
SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway
- Yi-Jun Shu†1, 2,
- Hao Weng†1, 2,
- Yuan-Yuan Ye†1, 2,
- Yun-Ping Hu1, 2,
- Run-Fa Bao1, 2,
- Yang Cao1, 2,
- Xu-An Wang1, 2,
- Fei Zhang1, 2,
- Shan-Shan Xiang1, 2,
- Huai-Feng Li1, 2,
- Xiang-Song Wu1, 2,
- Mao-Lan Li1, 2,
- Lin Jiang1, 2,
- Wei Lu1, 2,
- Bao-San Han1,
- Zhi-Gang Jie3Email author and
- Ying-Bin Liu1, 2Email author
© Shu et al.; licensee BioMed Central. 2015
Received: 6 August 2014
Accepted: 22 December 2014
Published: 27 January 2015
Gallbladder cancer (GBC) is a leading cause of cancer-related death worldwide, and its prognosis remains poor, with 5-year survival of approximately 5%. In this study, we analyzed the involvement of a novel proteoglycan, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), in the tumor progression and prognosis of human GBC.
SPOCK1 expression levels were measured in fresh samples and stored specimens of GBC and adjacent nontumor tissues. The effect of SPOCK1 on cell growth, DNA replication, migration and invasion were explored by Cell Counting Kit-8, colony formation, EdU retention assay, wound healing, and transwell migration assays, flow cytometric analysis, western blotting, and in vivo tumorigenesis and metastasis in nude mice.
SPOCK1 mRNA and protein levels were increased in human GBC tissues compared with those in nontumor tissues. Immunohistochemical analysis indicated that SPOCK1 levels were increased in tumors that became metastatic, compared with those that did not, which was significantly associated with histological differentiation and patients with shorter overall survival periods. Knockdown of SPOCK1 expression by lentivirus-mediated shRNA transduction resulted in significant inhibition of GBC cell growth, colony formation, DNA replication, and invasion in vitro. The knockdown cells also formed smaller xenografted tumors than control GBC cells in nude mice. Overexpression of SPOCK1 had the opposite effects. In addition, SPOCK1 promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of relevant genes. We found that activation of the PI3K/Akt pathway was involved in the oncogenic functions of SPOCK1 in GBC.
SPOCK1 activates PI3K/Akt signaling to block apoptosis and promote proliferation and metastasis by GBC cells in vitro and in vivo. Levels of SPOCK1 increase with the progression of human GBC. SPOCK1 acts as an oncogene and may be a prognostic factor or therapeutic target for patients with GBC.
Gallbladder cancer (GBC) is the most common biliary tract malignancy and the seventh most common gastrointestinal cancer . The Surveillance, Epidemiology, and End Results (SEER) program estimates the incidence of GBC at 2.5 cases per 1 × 105 people. Despite the relatively low incidence rate, GBC-associated mortality is higher than that of other cancers . The prognosis of advanced gallbladder carcinoma is very poor, and the 5-year survival rate is only approximately 5% . This poor survival rate is because of the early spread of tumors via lymphatic, perineural, and hematogenous routes as well as direct invasion into the liver . Therefore, patient prognoses may be improved by identifying novel and effective therapeutic targets for the treatment of this disease and increasing our understanding of biomarkers that can predict therapeutic responses.
The human genome sequencing project has found that 70% of the genome is transcribed, but only up to 2% of the human genome serves as blueprints for proteins [5,6]. One oncogene, sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), has been found to play a critical role in cell-cycle control, apoptosis, DNA repair, and metastasis . SPOCK1 encodes a matricellular glycoprotein belonging to a novel Ca2+-binding proteoglycan family. Members of this protein family, which share a similar N-terminus, follistatin-like domain, and C-terminus, are involved in cell proliferation, adhesion, and migration . Other members of this family include SPARC, testican-2, and testican-3. Among these proteins, SPARC has been well studied in various cancers. Increasing evidence has emphasized the importance of SPARC in regulating proliferation, cell-cycle progression, apoptosis, adhesion, and cell-matrix interactions . More interestingly, a number of studies have demonstrated that SPOCK1 plays a critical role in prostate cancer recurrence, glioblastoma invasion, and hepatocellular carcinoma progression [10-12]. However, the underlying mechanism of SPOCK1 overexpression is far from clear. Even less is known about the function and mechanism by which SPOCK1 contributes to cancer development and progression.
Considering the structural similarity between SPOCK1 and SPARC, it is of great interest to investigate the role of SPOCK1 in GBC development and progression. In the present study, we demonstrated a significant correlation between high expression of SPOCK1 and poor prognoses of GBC patients, and its oncogene function was examined further in vitro and in vivo. With a focus on its anti-apoptotic and epithelial-mesenchymal transition (EMT) functions, we demonstrated that SPOCK1 acts as a potential oncogene, which in turn contributes to the initiation and progression of GBC.
Patients, specimens, and cell lines
This study was approved by the ethics committee of Xinhua Hospital, and all patients provided informed consent. Cancer tissue specimens were obtained from 64 GBC patients who underwent radical cholecystectomy without prior radiotherapy or chemotherapy between 2010 and 2013 at the Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, China. In addition, 60 patients with cholelithiasis who underwent simple cholecystectomy were included as controls. All diagnoses of GBC, cholelithiasis, and lymph node metastasis were confirmed by histopathological examination. The tissue specimens had been fixed in 4% formalin immediately after removal and embedded in paraffin for immunohistochemical staining. Fresh GBC tissue samples and paired non-cancerous tissue samples were obtained from 28 GBC patients. These samples were used for quantitative real-time PCR analysis (qRT-PCR). Fresh tissues were processed within 15 min after removal. Each sample was frozen and stored at −80°C. Paired non-cancerous tissues were dissected at least 2 cm away from the tumor border and were confirmed to lack tumor cells by microscopy. Among the 64 GC cases, there were 22 males and 42 females with ages ranging from 44 to 90 years (mean age: 68 years). All specimens and fresh tissue samples had been confirmed by pathological diagnosis and were staged according to the 7th AJCC-TNM Classification of Malignant Tumors. The median follow-up period was 15 months (range, 1–36.5 months).
GBC cell lines GBC-SD, NOZ, SGC-996, OCUG, and EH-GB-1 were obtained from the Health Science Research Resources Bank (Osaka, Japan).
Immunohistochemical analysis and evaluation of SPOCK1 expression
Immunohistochemical staining was performed using a standard immunoperoxidase staining procedure. SPOCK1 expression in benign and malignant specimens was evaluated according to methods described by Pinheiro et al. . Sections were semi-quantitatively scored for the extent of immunoreactions as follows: 0, 0% immunoreactive cells; 1, <5% immunoreactive cells; 2, 5–50% immunoreactive cells; and 3, >50% immunoreactive cells. Additionally, the staining intensity was semi-quantitatively scored as 0 (negative), 1 (weak), 2 (intermediate), or 3 (strong). The final immunoreaction score was defined as the sum of both parameters, and the samples were grouped as negative (0), weak (1–2), moderate (3), and strong (4–6) staining. For statistical purposes, only the final immunoreaction scores of moderate and strong groups were considered as positive, and the other final scores were considered as negative.
Quantitative real-time PCR
Total RNA was extracted from tissue samples or cultured cells with Trizol reagent (Takara, Shiga, Japan). cDNA was synthesized from 2 μg of total RNA using random primers and M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA). RNA expression was measured by qRT-PCR using the SYBR-Green method (Takara) according to the manufacturer’s instructions. The relative expression level of the target gene was calculated by 2-ΔCT (ΔCT = CT target-CT GADPH) and normalized to the relative expression detected in the corresponding control cells, which was defined as 1.0. For the correlation study, the expression level (defined as the fold change) of SPOCK1 was calculated by 2-ΔΔCT (ΔΔCT = ΔCTtumor-ΔCTnontumor). Primer sequences are listed in Additional file 1: Table S1.
Lentivirus-mediated RNA interference
The short hairpin RNAs (shRNAs)  which listed in Additional file 1: Table S1 were used to target SPOCK1. The sequence of the negative control shRNA was 5′-TTCTCCGAACGTGTCACGT-3′. shSPOCK1-1 and negative control shRNA were synthesized and inserted into the pFH1UGW lentivirus core vector containing a cytomegalovirus-driven enhanced green fluorescent protein (EGFP) reporter gene. Expression of the shRNA was driven by the H1 promoter. Recombinant lentiviruses expressing SPOCK1-shRNA or negative control shRNA (Lv-shSPOCK1 and Lv-shNC, respectively) were produced by Genechem (Shanghai, China). GBC-SD and NOZ cells were infected with concentrated virus in serum-free medium. The supernatant was replaced with complete culture medium after 24 h. SPOCK1 expression in the infected cells was validated by qRT-PCR analysis and western blot assays after 120 h.
Construction of plasmids and transfection
The full-length SPOCK1 cDNA (nt 152–1471; GenBank accession number NM_004598) was cloned into the GV143 expression vector (Genechem, Shanghai, China) and transfected into SGC-996 cells. Stable SPOCK1-expressing clones were selected for 2 weeks using neomycin (Genechem), and the expression level of SPOCK1 was determined by qRT-PCR and western blot assays. Empty vector-transfected cells (MOCK) were used as control. Primer sequences for vectors construction are listed in Additional file 1: Table S1.
In vitro tumorigenesis assays
A Cell Counting Kit-8 (CCK-8; Dojindo) cell proliferation assay was performed according to the manufacturer’s instructions. Anchorage-independent growth was assessed by a colony formation assay. Briefly, 500 cells were seeded in 6-well plates. The cells were cultured for approximately 14 days, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Sigma, St. Louis, MO). The total number of colonies (>50 cells/colony) was counted. Edu retention assays were performed to examine the effect of SPOCK1 on DNA replication. Dissociated cells were exposed to 25 μM of 5-ethynyl-2′-deoxyuridine (Edu, RiboBio, Guangzhou, China) for 2 hr at 37°C, and then the cells were fixed in 4% paraformaldehyde. After permeabilization with 0.5% Triton-X, the cells were reacted with 1× Apollo reaction cocktail (RiboBio) for 30 min. Subsequently, the DNA contents of the cells were stained with Hoechst 33342 for 30 min and visualized under a fluorescence microscope. The experiments were performed in triplicate.
In vitro migration and invasion assays
For the in vitro wound-healing assay, a cell-free area of the culture medium was wounded by scratching with a 200-μL pipette tip. Cell migration into the wound area was monitored in serum-free medium and photographed under a fluorescence microscope at 0 and 48 h. Cell migration and invasion were examined using 8-μm transwell filters (BD Biosciences, Franklin Lakes, NJ). GBC-SD (3 × 104), NOZ (4 × 104) cells, and SGC-996 (8 × 104) in 0.5 μL serum-free medium were added to the upper chamber containing an uncoated or Matrigel (BD Biosciences)-coated membrane. The lower chamber was filled with 500 μL basal medium with 10% fetal bovine serum (FBS). After 24 h of incubation at 37°C in a humidified 5% CO2 incubator, cells that migrated to the lower compartment were fixed with methanol and stained with crystal violet. Migrated or invaded cells were counted in five randomly chosen fields in each well. Imaging and cell counting were performed at × 10 magnification under a fluorescence microscope. The experiments were performed in triplicate.
Subcutaneous and peritoneal xenograft models
Nude nu/nu mice, 4–6 weeks old, were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). All mice were housed in specific pathogen-free conditions following the guidelines of the Ethics Committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. To explore the effects of SPOCK1 on tumor growth in vivo, cells were subcutaneously injected into the left axilla of the mice (five mice/group). Tumor growth was monitored every week and measured in two dimensions. The tumor volume was calculated using the following formula: tumor volume = 4π/3 × (width/2)2 × (length/2), where the width and length were the shortest and longest diameters, respectively. After 4 weeks, the mice were sacrificed and the tumors were dissected out and weighed. The proliferative index of Ki-67 was evaluated in xenograft tumors by immunohistochemical staining (IHC). In addition to investigating the effects of SPOCK1 on tumor metastasis in vivo, 1 × 105 NOZ cells (Lv-shNC and Lv-shSPOCK1) were suspended in 1 mL serum-free medium and peritoneally injected into five mice. After 4 weeks, the mice were sacrificed and the peritoneal tumors were collected for IHC.
Cells were seeded in 6-well plates and cultured overnight. Then, the cells were fixed in 3.5% paraformaldehyde and permeabilized in a solution of 0.1% bovine serum albumin (BSA) and 0.5% Triton X-100 at room temperature. After the blocking solution was washed out, the cells were incubated with primary antibodies against SPOCK1, E-cadherin, or vimentin for 60 min at 37°C and then washed with 0.1% BSA twice. After 60 min of incubation at 37°C with Cy3 Goat Anti-Rabbit IgG (Beyotime, Shanghai, China) and then washing with 0.1% BSA, the cells were counterstained with DAPI and imaged under a fluorescence microscope. The experiments were performed in triplicate.
Flow cytometric analysis of cell apoptosis
The extent of apoptosis was measured with an Annexin V-APC Apoptosis Detection kit (BD Biosciences) according to the manufacturer′s instructions. Untransfected and transfected GBC-SD, SGC-996, and NOZ cells were collected, washed with cold PBS twice, gently resuspended in 100 μL of 1× binding buffer containing 2.5 μL APC-conjugated annexin-V and 1 μL of 100 μg/mL PI, and then incubated at room temperature in the dark for 15 min. The stained cells were analyzed by flow cytometry (BD Biosciences). The experiments were performed in triplicate.
Nuclear morphology assay
Untransfected and transfected GBC-SD and NOZ cells were seeded in 6-well culture plates. After 48 h, the cells were washed with PBS, fixed in MeOH-HOAc (3:1, v/v) for 10 min at 4°C, and stained with Hoechst 33342 (5 μg/mL in PBS) for 5 min at room temperature. The stained cells were then examined under a fluorescence microscope. The experiments were performed in triplicate.
Antibodies and western blotting
A rabbit anti-SPOCK1 antibody was purchased from Abcam (MA, USA). Rabbit anti-Snail, anti-vimentin, anti-N-cadherin, anti-E-cadherin, anti-PI3K, anti-phospho-PI3K (Tyr607), anti-Akt, anti-phospho-Akt (Ser473), anti-Bax, anti-Bcl-2, anti-cleaved caspase 3 and 9, anti-poly (adenosine diphosphate-ribose) polymerase (PARP), and anti-GADPH antibodies were obtained from Cell Signaling Technology (Danvers, USA). Briefly, equal quantities of cellular proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and immunoblotted with a primary antibody. After incubation with a secondary antibody, blots were visualized by enhanced chemiluminescence (Millipore, Billerica, MA). GADPH was used as the loading control.
All statistical analyses were performed using SPSS 19.0 software. SPOCK1 mRNA levels in tumor and paired nontumor tissues were compared with the paired Student’s t-test. The independent Student’s t-test was used to compare the means of two groups. The Pearson χ2 test was used to analyze the association of SPOCK1 expression with clinicopathologic parameters. Kaplan-Meier plots and log-rank tests were used for survival analysis. Univariate and multivariate Cox proportional hazard regression models were used to analyze independent prognostic factors. Each experimental value was expressed as the mean ± standard deviation (SD). Differences between groups were considered significant at P < 0.05. All data points represent the mean of triplicate data points.
Clinical significance of SPOCK1 in GBC
Immunohistochemical analysis of SPOCK1 expression in GBC
Weakly stained (1–2)
Moderately stained (3)
Strongly stained (4–6)
Association of SPOCK1 expression with the clinicopathological characteristics of GBC
No. of cases
No. of positive cases (%)
Well or moderate
Lymph node metastasis
Univariate and multivariate analysis of the association of prognosis with clinicopathologic parameters and SPOCK1 expression in GBC patients
HR (95% CI)
HR (95% CI)
Age (<60 vs. ≥60)
Sex (male vs. female)
Jaundice (present vs. absent)
Associated gallstone (present vs. absent)
Histology differentiation (well or moderate vs. poor)
Tumor invasion (AJCC) (Tis-T1 vs. T2-T4)
Lymph node metastasis (present vs. absent)
TNM stage (AJCC) (0-I vs. II-IV)
Type of surgery (curative resection vs. palliative)
Overexpression of SPOCK1 in tumor
(Negative vs. positive)
SPOCK1 expression in GBC cell lines
Effects of SPOCK1 overexpression and knockdown on GBC cell growth in vitro and in vivo
SPOCK1 promotes cell migration and invasion in vitro and in vivo by inducing EMT
SPOCK1 inhibits apoptosis in GBC cells
SPOCK1 exerts an anti-apoptotic effect through the PI3K/Akt pathway
Tumor cells resist cell death through either disruption of apoptotic processes or activation of survival signals. In general, survival signals are mediated by the PI3K/Akt pathway . Deregulation of Akt phosphorylation represents an important anti-apoptotic mechanism in various cancers. Activated Akt can phosphorylate a wide variety of substrate proteins including Bax, a pro-apoptotic member of the Bcl-2 protein family, which is suppressed by phosphorylation. Bax inactivation maintains the integrity of the mitochondrial membrane, which activates caspase-9, caspase-3, and PARP . Therefore, we examined whether SPOCK1 inhibits apoptosis via PI3K and Akt phosphorylation. Compared with control cells, the levels of both phosphorylated PI3K (Tyr607) and Akt (Ser473) were decreased in SPOCK1-transfected cells, while their total protein levels were unaffected. Inactivated Akt subsequently regulates Bcl-2 family proteins. As a result, the subsequent cleavages of caspase-9, caspase-3, and PARP were all increased in SPOCK1 knockdown cells compared with those in control cells (Figure 7C). The PI3K and Akt phosphorylation were reversed when SPOCK1 was overexpressed in SGC-996 cells (Additional file 4: Figure S3B). These results indicated that the PI3K/Akt pathway might participate in the SPOCK1-induced anti-apoptotic effect on GBC cells.
GBC is a highly lethal disease, and most afflicted individuals do not survive because of local tumor spread and invasion. Therefore, efforts are urgently needed to identify reliable tumor markers for early detection and cancer-specific cellular targets for novel therapeutic approaches. SPOCK1 is a proteoglycan that was first isolated in human testes and initially called ‘tesyican’. It is dysregulated in many organs and tissues including the brain, cartilage, vascular endothelium, lymphocytes, and neuromuscular junctions [11,12,17]. Recently, SPOCK1 was also found to be overexpressed in hepatocellular carcinomas . Although SPOCK1 is reported to be overexpressed in several other types of carcinoma, it has not been linked to GBC or any other malignancy of the biliary tract.
Our clinical association study found that SPOCK1 was highly expressed in GBC tissues compared with that in their nontumor counterparts, indicating that SPOCK1 might play a role in GBC development. Moreover, immunohistochemistry showed that overexpression of SPOCK1 was significantly associated with histological differentiation, lymph node metastasis, and a shorter OS time of GBC patients. Cox proportional hazard regression analysis further identified SPOCK1 as an independent factor for poor prognosis. Because SPOCK1 is a secreted protein that can be detected at very low levels in normal tissues, SPOCK1 overexpression in GBC may serve as a biomarker for early detection and precise prognoses.
In this study, we confirmed that SPOCK1 was expressed in GBC by qRT-PCR, western blotting, and immunofluorescence, which represented an ideal model to study the role and molecular mechanisms of SPOCK1. A series of in vitro and in vivo assays showed that cancer cell growth, DNA replication and the colony formation capability were significantly decreased by inhibition of SPOCK1, suggesting its role in cancer cell proliferation and tumor growth. Additionally, we found that SPOCK1 induced GBC cells migration and invasion, indicating that SPOCK1 might undergo metastasis-related genetic alteration in GBC cells. Metastasis is a multistep cellular process and the most common cause of death in GBC patients. This process involves the spread of tumor cells from a primary tumor to a secondary site within the body. It usually involves a variety of complicated molecular and cellular factors related to cell proliferation and migration, degradation of the basement membrane, invasion, adhesion and angiogenesis. At the molecular level, the acquisition of genetic and/or epigenetic alterations, along with the cooperation of stromal cells, contribute to cancer metastasis [19,20].
SPOCK1 promotes cancer cell migration by induction of EMT . EMT is a crucial step in tumor progression and plays a critical role during cancer invasion and metastasis. During this process, epithelial cells lose their properties and acquire mesenchymal phenotypes. Mesenchymal phenotype cells exhibit increased expression of mesenchymal-related markers, such as vimentin, and decreased expression of epithelial-related markers such as E-cadherin [22,23]. In the current study, we showed that suppressed expression of SPOCK1 induced EMT by elevating expression of the epithelial marker E-cadherin and reducing expression of the mesenchymal marker Snail, vimentin and N-cadherin. Our findings indicate that SPOCK1 may drive EMT in cancer cells, resulting in metastasis.
Further experiments revealed that SPOCK1-enhanced tumor cell survival may be attributable to its anti-apoptotic effect. Our data show that SPOCK1 contributes to anti-apoptotic effects through inactivation of the PI3K/Akt pathway, which subsequently activates the caspase 9/caspase 3/PARP pathway. Inhibition of apoptosis is one of the major mechanisms in cancer development and ultimately leads to the expansion of neoplastic cells with deregulated proliferation and accumulation of genetic instability and mutations . Therefore, impaired GBC cell growth and metastasis due to SPOCK1 knockdown can be explained, at least in part, by inactivation of the PI3K/Akt pathway. Previous reports show that PI3K/Akt is a classical signaling pathway [25-27], and its activation induces cell growth [28,29], promotes EMT , and stimulates Bax-mediated signaling for apoptosis progression. Our results suggest that inactivation of PI3K/Akt signaling is responsible for SPOCK1 shRNA-mediated suppression of tumor cell proliferation, migration, invasion, and EMT.
Because SPOCK1 belongs to the Ca2+-binding proteoglycan family, some of these effects may be mediated by the glycan segment of SPOCK1. Increasing evidence has shown that glycan specifically interacts with growth factors, chemokines, and the matrix architecture . Cancer cells usurp these properties to gain a survival advantage and invade tissues throughout the organism. For example, the glycan segment of perlecan protects fibroblast growth factor 2 from proteolytic degradation and potentiates its angiogenic role . In addition to the steady-state properties of glycan, changes in glycan segments affect cancer development, such as glycosylation and depolymerization. Heparanase-induced depolymerization can release fibroblast growth factor 2 from perlecan to facilitate vascular sprouting during angiogenesis . Some of these characteristics of perlecan may be shared by other pericellular proteoglycans such as agrin, collagen type XVIII, and SPOCK1. However, to determine whether SPOCK1 performs its functions by working alone or in concert with other partner molecules, it will be important to identify the portion of the proteoglycan that mediates the interaction.
A better understanding of the oncogenic mechanisms of SPOCK1 during GBC initiation and progression may have implications for future patient treatments.
We have demonstrated that the expression of SPOCK1 is associated with histological differentiation, lymph node metastasis, and the OS time of GBC patients. SPOCK1 promotes GBC cell proliferation and metastasis both in vitro and in vivo. We hypothesize that SPOCK1 might play an important role during the EMT of GBC cells, which results in metastasis. Moreover, SPOCK1 contributes to anti-apoptotic effects through inactivation of the PI3K/Akt pathway. These observations support our belief that SPOCK1 may serve as an oncogene in GBC pathogenesis.
This study was supported by the National Natural Science Foundation of China (No. 81172026, 81272402, 81301816, and 81172029), the National High Technology Research, and Development Program (863 Program) (No. 2012AA022606), the Foundation for Interdisciplinary research of Shanghai Jiao Tong University (No. YG2011ZD07), the Shanghai Science and Technology Commission Inter-governmental International Cooperation Project (No. 12410705900), the Shanghai science and technology commission medical-guiding project (No. 12401905800), the Program for Changjiang Scholars, the Natural Science Research Foundation of Shanghai Jiao Tong University School of Medicine (No. 13XJ10037), and the Leading Talent program of Shanghai and Specialized Research Foundation for the Ph.D Program of Higher Education-Priority Development Field (No. 20130073130014).
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