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Introduction

Osteosarcoma (OS) is one of the most prevalent types of malignant bone tumors, which predominantly occurs in adolescents and young adults; in addition, OS has a morbidity rate of ~5 cases per million (1). The conventional treatment of OS consists of surgery and radiotherapy; however, additional radiotherapy may not be used due to the risk of radiation-induced necrosis of surrounding structures (2). In addition, there is a high risk of relapse or metastasis for OS patients, even following curative resection; therefore, a substantial portion of patients with OS respond poorly to chemotherapy (3). Thus, elucidating the mechanisms underlying the metastasis of OS may lead to the development of novel therapeutic strategies for the treatment of OS.

Twist is a member of a highly conserved basic helix-loop-helix transcription factor family and is located on human chromosome 7 (4). It has been reported that Twist mediates cell migration and differentiation under various physiological conditions (5). In addition, Twist has been demonstrated to significantly enhance tumor malignancies, including breast cancer (6), hepatocellular carcinoma (HCC) (7) and prostate cancer (8). It was reported that the role of Twist in tumor cell invasion and metastasis may be associated with the regulation of cancer-associated functions, such as angiogenesis (9). Furthermore, Twist was found to regulate apoptosis and angiogenesis under a variety of pathological conditions (10). Therefore, it was hypothesized that Twist was closely correlated with malignant potential, progression and survival in patients with OS. However, to the best of our knowledge, there is limited information regarding the pathological roles of Twist expression in human OS tissues.

Angiogenesis is a key factor that mediates tumor metastasis; in addition, correlations between angiogenesis and patient survival in OS have been previously reported (11). Microvessel density (MVD) was demonstrated to be increased in histopathologically aggressive cancers, including esophageal squamous cell carcinoma (12) and mammary carcinoma (13). High MVD was also reported to be correlated with metastasis and poor survival in various types of cancer, such as OS (14). Increasing evidence has suggested that MVD may be considered an indirect marker of neoangiogenesis, which is commonly labeled by CD31 or CD34 (14).

Vascular endothelial growth factor (VEGF), the most important mediator of vascular formation, is essential for the initiation of immature vessel formation (15). VEGF has been reported to be directly involved in the angiogenesis process, tumor growth and metastasis (16). Neovascularization, promoted by VEGF, is known to reflect the aggressiveness and the metastatic potential of OS (17). However, current understanding of the correlation of Twist, VEGF and MVD in human OS tissues is limited.

The present study aimed to investigate Twist, VEGF and CD34 expression levels in OS tissues, using immunohistochemical (IHC) staining, in order to illustrate correlations among these components in OS.

Materials and methods

Tissues

A total of 32 cases of different phase OS and 10 cases of osteochondroma (OC), a benign tumor of the bone, were obtained from patients who underwent surgery between June 2011 and March 2013 at the Department of Orthopedics, Xiangya Hospital of Central South University (Changsha, China). Patients with OS underwent amputation surgery, while patients with OC underwent curettage. All OS tumor tissues were formalin-fixed and paraffin-embedded following resection and then pathologically diagnosed (18): Phase I OS, 3 cases; phase II OS, 17 cases; and phase III OS, 12 cases. Ages of patients range from 9 to 54 years old (mean, 23.21±8.73), including 18 males and 14 females. In 20 cases OS was identified in the femur, in 8 cases the malignancy was located in the tibia and the remaining 4 cases were located in other bone regions. OC tissues were used as the control. All tissues were collected prior to any chemoradiotherapy. Written informed consent was obtained from the patients. The study was approved by the Ethics Committee of the Xiangya Hospital of Central South University.

Immunohistochemical staining

Immunohistochemical staining was performed using the streptavidin-peroxidase (SP) method (SP kit; ZSGB-Bio, Beijing, China), according to the manufacturer's instructions. Slides were deparaffinized with xylene (BaiYi, Co. Ltd., Jining, China) twice for 30 min each, dehydrated three times in a gradient series of ethanol (100, 95, 90, 80 and 70%) and rinsed with phosphate-buffered saline (PBS). Following 15 min of treatment with 3% H2O2, slides were blocked using normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA) for 20 min. Slides were incubated with the following primary antibodies overnight at 4°C: Rabbit polyclonal anti-Twist (1:50; cat. no. ab50581; Abcam, Cambridge, UK), mouse monoclonal anti-VEGF (1:150; cat. no. ab1316; Abcam) or mouse monoclonal anti-CD34 (1:100; cat. no. ab8536; Abcam). Slides were subsequently washed three times with PBS for 15 min. Slides were treated with SP reagent for 20 min and then incubated with a secondary antibody for 90 min at 37°C, using an SP rabbit and mouse horseradish peroxidase kit (cat. no. CW0120; CWBiotech Co. Ltd., Wuhan, China) according to the manufacturer's instructions. Subsequently, the slides were washed twice with PBS for 15 min per wash, and visualized using 3,3′-diaminobenzidine for 5 min and then counterstained with hematoxylin (Solarbio Science ﹠ Technology Co., Ltd., Beijing, China). The slides were mounted and dried. Images were captured using an Olympus microscope (C-7070; Olympus Corporation, Tokyo, Japan).

Evaluation of staining

Slides were evaluated by two investigators, who were blinded to experiments, under a light microscope (BX43; Olympus Corporation). Twist and VEGF staining intensity were scored as follows: 0, negative; 1, weak; 2, medium; and 3, strong. Extent of staining was scored as: 0, 0%; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The final staining score (0–7) was calculated as the sum of the intensity score and extent score. Staining scores of 0–1 were considered to be negative, scores of 2–3 were considered as low expression and score of >3 were considered as high expression.

Measurement of MVD

Slides were examined at low-power magnification (x40; microscope, BX43) to identify the areas with the highest density of microvessels (labeled by CD34). In each case, the most vascularized area was selected and the microvessels within a high-power magnification (x200) field of vision were counted three times. Macrovascular structures with smooth muscle cells were excluded. The mean of the three highest counts per tumor was used for analysis.

Statistical analysis

SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. The ratio of high expression of Twist and VEGF in different phases of OS was compared using chi-squared tests. MVD in different phases of OS was compared using Student's t-tests. Associations among Twist, VEGF and MVD were assessed using the Spearman's rank correlation test. P<0.05 was considered to indicate a statistically significant difference between values.

Results

Expression of Twist, VEGF and CD34 in OS and OC tissues

Immunohistochemical staining data revealed that Twist was located in the nuclei and cytoplasm, VEGF was identified in the cytoplasm and CD34 (for MVD) in the microvessels (Fig. 1A–F). The positive expression of Twist was detected in 18 of the total 32 OS tissues, but in only 1 of the 10 OC tissues; therefore, the positive rate of Twist in OS tissues was significantly increased compared with that in OC tissues (χ2=6.579, P=0.01) (Table I). In addition, the positive expression of VEGF was detected in 23 out of 32 OS tissues and in only 2 of the 10 OC tissues, demonstrating that the positive rate of VEGF in OS tissues was significantly higher compared with that of OC tissues (χ2 =8.510, P=0.004) (Table II). Furthermore, MVD in OS tissues was significantly elevated compared with that in OC tissues (t=17.086, P=0.008) (Table III).

Figure 1. Representative images of immunohistochemical staining for Twist, VEGF and CD34 protein expression. Twist expression in (A) osteosarcoma and (B) osteochondroma tissues; VEGF expression in (C) osteosarcoma and (D) osteochondroma tissues; and CD34 expression in (E) osteosarcoma and (F) osteochondroma tissues. Magnification, x400.

Table I. Twist expression in osteochondroma and osteosarcoma.

Table I.

Twist expression in osteochondroma and osteosarcoma.

Tumor	Cases	Negative cases	Positive cases	Positive rate, %	χ2	P-value
Osteochondroma	10	9	1	10.00	6.579	0.01
Osteosarcoma	32	14	18	56.25

Table II. Vascular endothelial growth factor expression in osteochondroma and osteosarcoma.

Table II.

Vascular endothelial growth factor expression in osteochondroma and osteosarcoma.

Tumor	Cases	Negative cases	Positive cases	Positive rate, %	χ2	P-value
Osteochondroma	10	8	2	20.00	8.510	0.004
Osteosarcoma	32	9	23	71.88

Table III. MVD values in osteochondroma and osteosarcoma.

Table III.

MVD values in osteochondroma and osteosarcoma.

Tumor	Cases	MVDa	χ2	P-value
Osteochondroma	10	7.97±3.67	17.086	0.008
Osteosarcoma	32	37.38±7.20

a Values are presented as the mean ± standard deviation of three counts of MVD within a high-power magnification (x200) field. MVD, microvessel density.

Expression of Twist, VEGF and CD34 in different phases of OS

The positive expression of Twist was detected in 10 of the total 12 phase III OS tissues and in 8 of the 20 phase I/II OS tissues; therefore, the positive rate of Twist in phase III OS was significantly higher compared with that in phase I/II OS (χ2=5.732, P=0.018) (Table IV). The positive expression of VEGF was detected in all 12 phase III OS tissues and in 11 out of 20 phase I/II OS tissues; thus, the positive rate of VEGF in phase III OS was significantly increased compared with that in phase I/II OS (χ2=7.513, P=0.006) (Table V). The MVD in phase III OS tissues (41.2±4.17 per field) was significantly increased compared with that of phase I/II OS tissues (31.08±3.36 per field) (t=7.536, P<0.001) (Table VI).

Table IV. Twist expression in different phase of osteosarcoma.

Table IV.

Twist expression in different phase of osteosarcoma.

Phase	Cases	Negative cases	Positive cases	Positive rate, %	χ2	P-value
I	3	3	0	0.00
II	17	9	8	47.06	5.732a	0.018a
III	12	2	10	83.33

a Phase I and II vs. phase III.

Table V. Vascular endothelial growth factor expression in different phases of osteosarcoma.

Table V.

Vascular endothelial growth factor expression in different phases of osteosarcoma.

Phase	Cases	Negative cases	Positive cases	Positive rate, %	χ2	P-value
I	3	3	0	0.00
II	17	6	11	64.71	7.513a	0.006a
III	12	0	12	100.00

a Phase I and II vs. phase III.

Table VI. MVD values in different phase of osteosarcoma.

Table VI.

MVD values in different phase of osteosarcoma.

Phase	Cases	MVDa	t-value	P-value
I	3	23.33±4.36
II	17	37.15±3.74
I and II	20	31.08±3.36	7.536b	<0.0001b
III	12	41.20±4.17

a Values are presented as the mean ± standard deviation of three counts of MVD within a high-power magnification (x200) field

b phase I and II vs. phase III. MVD, microvessel density.

Associations among Twist, VEGF and MVD expression in correlation analysis

Spearman's rank correlation analysis revealed that Twist expression was positively associated with VEGF expression (r=0.371, P=0.002) and with MVD (r=0.393, P=0.001) in OS; in addition, VEGF expression was demonstrated to have a positive correlation with MVD (r=0.469, P=0.001).

Discussion

OS is an aggressive type of cancer that affects the skeletal system. Although investigated by numerous previous studies, the molecular mechanisms of the etiology and pathogenesis underlying OS remain to be elucidated (19,20). Therefore developing effective therapeutic strategies for the treatment of OS is challenging. The present study demonstrated that Twist expression was positively correlated with tumor phase and metastasis in patients with OS. It has previously been reported that Twist expression was significantly higher in cancer cells of sarcomatoid renal cell carcinoma compared with those at the edge of the tumors; in addition, Twist was associated with tumor aggressiveness and poor prognosis in patients with renal cell carcinoma (21). Oncogenic activation of Twist was reported to be essential for the epithelial-mesenchymal transition (EMT) that initializes invasion and metastasis (22). Twist has been demonstrated to couple aberrant signals from EMT to senescence and was found to be an important candidate biomarker for cervical cancer prognosis (23). In addition, the downregulation of Twist expression was suggested to facilitate apoptosis and recover the sensitivity of chemoresistance induced by cisplatin in ovarian cancer (24). Furthermore, the enhanced production of Twist resulted in VEGF secretion that promotes tumor angiogenesis in breast cancer cells, which in turn enhances cancer invasion and metastasis (9).

VEGF, as a prime mediator of angiogenesis, has been implicated in carcinogenesis and metastasis in human and murine OS cells (25). It was reported that the survival and proliferation of highly aggressive OS cells was dependent on autocrine VEGF/VEGF receptor 1 signaling in vitro (17). Numerous studies have suggested that VEGF expression may act as a biomarker of prognosis in OS patients (26–28). A previous study reported that patients with high VEGF expression had significantly reduced disease-free survival and overall survival rates compared with OS patients with low or negative VEGF expression (28). In line with previous studies, the present study determined that the positive rate of VEGF in OS tissues was significantly increased compared with that in OC tissues with increased grade and metastasis, suggesting that VEGF expression may be an efficient biomarker for predicting the prognosis of OS patients. However, further large-scale prospective trials are required in order to confirm the prognostic value of VEGF for survival in OS patients.

The present study determined that Twist expression was positively correlated with VEGF expression in OS. Niu et al (7) reported that increased Twist messenger RNA and protein expression levels were positively associated with the upregulation of VEGF expression in HCC patients with a poor prognosis, suggesting that Twist may have a critical role in the angiogenesis and metastasis of HCC (7). In cases of supraglottic carcinoma, Twist and VEGF expression levels in lymph node metastasis patients were significantly increased compared with those in patients without metastasis; in addition, the levels of VEGF were reported to be positively correlated with those of Twist (29). One study reported that stable overexpression of Twist in the MCF-7 breast cancer cell line resulted in increased VEGF synthesis in vitro and xenograft experiments with MCF-7 cells overexpressing Twist produced tumors with a higher vascular volume and vascular permeability in vivo (6). Overall, these previous studies indicated that Twist overexpression may enhance cancer invasion and metastasis through increasing VEGF expression, resulting in the induction of angiogenesis, which is pivotal for the transformation into an aggressive phenotype.

In the present study, the staining of endothelial cells for CD34 was used to evaluate the MVD in OS tissues. The results indicated that the MVD in OS tissues was significantly increased compared with that of OC tissues; in addition, this increase was correlated with OS phase. Spearman's rank correlation analysis revealed that Twist expression was positively correlated with MVD, while VEGF expression also exhibited a positive correlation with MVD. Positive Twist expression in HCC specimens was previously demonstrated to have an elevated MVD compared with specimens with negative Twist expression (7). Another study demonstrated that the MVD in paraffin sections from 97 patients with HCC was correlated with the upregulation of Twist expression (30). In addition, Twist expression was reported to be positively associated with MVD in cancer cells of sarcomatoid renal cell carcinoma (21). It has been demonstrated that VEGF regulated the development of microvessels and was significantly correlated with MVD in Ewing's sarcoma family of tumors (31). Serum VEGF levels were identified to be significantly elevated in OS patients with pulmonary metastasis compared with patients without detectable disease relapse; in addition, these VEGF levels were positively correlated with the MVD, suggesting that the pre-therapeutic serum VEGF levels reflected the angiogenic property of OS (32,33). However, Ek et al (14) demonstrated that the degree of MVD and VEGF expression did not provide prognostic information for OS, as determined through a small-scale (25 cases) investigation. Thus, the association between VEGF expression and MVD in OS requires further large-scale investigations, in addition to further studies regarding Twist and VEGF expression.

In conclusion, the present study investigated the expression of Twist, VEGF and CD34 (MVD) in 32 cases of OS at different phases and analyzed the associations among Twist, VEGF and MVD. The results revealed that OS tissues exhibited elevated expression levels of Twist and VEGF as well as high MVD values. In addition, it was indicated that Twist overexpression in OS may enhance cancer invasion and metastasis in OS through increasing VEGF expression, which in turn may result in increased MVD. Therefore, inhibition of Twist expression may have potential therapeutic use for the treatment of OS.

Acknowledgements

This study was supported by grants from the 2014 Hunan Provincial Innovation Foundation For Postgraduates (no. CX2014) and the Open-End Fund for the Valuable and Precision Instruments of Central South University (no. CSUZC2014046).

References