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Levels of vascular endothelial growth factor and matrix metalloproteinase-9 proteins in patients with glioma Chengyuan Ma, Yang Li, Xianfeng Zhang, Gang Zhao and Haiyang Xu Journal of International Medical Research 2014 42: 198 originally published online 7 January 2014 DOI: 10.1177/0300060513481924 The online version of this article can be found at: http://imr.sagepub.com/content/42/1/198
Published by: http://www.sagepublications.com
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Research Note
Levels of vascular endothelial growth factor and matrix metalloproteinase-9 proteins in patients with glioma
Journal of International Medical Research 2014, Vol. 42(1) 198–204 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513481924 imr.sagepub.com
Chengyuan Ma1, Yang Li2, Xianfeng Zhang1, Gang Zhao1 and Haiyang Xu1
Abstract Objective: To investigate the levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) proteins in patients with glioma, in order to determine if either protein has prognostic value. Method: The presence of VEGF and MMP-9 proteins in paraffin-embedded tumour specimens from patients with glioma was detected using immunohistochemistry. The correlation between the levels of VEGF and MMP-9 proteins and tumour grade was analysed. Results: A total of 32 patients with low-grade gliomas (World Health Organization [WHO] grade II) and 48 patients with high-grade gliomas (WHO grades III–IV) participated in the study. Positive immunohistochemical staining of VEGF and MMP-9 proteins was detected in 58/80 (72.5%) and 60/80 (75.0%) of patients, respectively. The level of VEGF immunostaining was significantly positively correlated with the level of MMP-9 immunostaining (r ¼ 0.78). Significantly more highgrade gliomas (grades III–IV) demonstrated positive VEGF and MMP-9 immunostaining compared with the low grade gliomas (grades I–II). Conclusions: These data suggest that VEGF and MMP-9 play an important role in the malignant behaviour of gliomas.
Keywords Glioma, matrix metalloproteinase-9, MMP-9, vascular endothelial growth factor, VEGF, immunohistochemistry Date received: 10 January 2013; accepted: 16 January 2013
1
Department of Neurosurgery, First Hospital of Jilin University, Changchun, Jilin Province, China 2 Department of Respiratory Medicine, First Hospital of Jilin University, Changchun, Jilin Province, China
Corresponding author: Dr Haiyang Xu, Department of Neurosurgery, First Hospital of Jilin University, 71 Xinmin Road, Chaoyang Region, Changchun 130021, Jilin Province, China. Email: [email protected]
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Introduction Gliomas are highly malignant and invasive tumors with tendrils that extend far from the primary tumor site.1 Gliomas are further divided into low-grade gliomas (World Health Organization [WHO] grade II) and high-grade gliomas (WHO grade III–IV) by pathologic evaluation of the tumour.2 Lowgrade gliomas are well differentiated, whereas high-grade gliomas are undifferentiated or anaplastic. The latter are malignant and carry a worse prognosis.3 Although a small portion of low grade gliomas display benign features, most of these tumours are malignant and usually indicate a poor prognosis.4 Despite modern diagnostic methods and treatment strategies, the median survival time does not exceed 15 months.5 This is mainly due to the fact that at the time of surgery, cells from the primary tumour have already invaded normal brain tissue.6 Therefore, it is extremely important to search for sensitive and specific markers that can provide valuable information for the early diagnosis and identification of poorly responsive tumours that may benefit from a modified treatment regimen. Tumour metastasis is a multistep process involving a variety of tumour–host cell interactions. Angiogenesis, one such interaction, is the outgrowth of endothelial cells from pre-existing capillary vessels and their migration from parental vessels under the stimulation of vascular endothelial growth factor (VEGF),7 which is a homodimeric protein identified as a mitogen for endothelial cells in vitro and an angiogenesis promoting factor in vivo.8 Glioma cells produce large amounts of VEGF and high grade gliomas (glioblastoma multiform) produce more VEGF than lower grade astrocytomas.7,8 Clinical studies have demonstrated that circulating VEGF has a negative prognostic value in cancer patients, correlating with tumour grade and vascularity in gliomas.9,10
Matrix metalloproteinases (MMPs) are zinc-binding endopeptidases capable of degrading extracellular matrix (ECM) components such as collagen, fibrinogen and proteoglycans.11 One member of this family, MMP-9, has been assigned an important role in tissue-specific remodelling in normal and pathological processes, most notably during angiogenesis and tumour invasion.12 Elevated levels of MMP-9 have been found in patients suffering from various types of tumours in vivo and a correlation has been established between the secretion of MMP-9 and experimental metastasis.13–16 Furthermore, overexpression of MMP-9 in transfected nonmetastatic tumour cells renders them invasive.13,14, Proliferating endothelial cells subsequently remodel the ECM via MMPs, align into tube-like structures, and eventually form new functional blood vessels.11 In malignant cells, upregulation of angiogenic factors, such as VEGF and MMPs, is closely linked to invasion and metastasis.17 There have been few reports on the role of VEGF and MMPs in gliomas.18 The present study, therefore, investigated the levels of VEGF and MMP-9 protein using immunohistochemical staining in order to determine if either protein has any prognostic value in patients with gliomas
Patients and methods Patients Consecutive patients with gliomas who had been diagnosed, treated and followed-up in the Department of Neurosurgery, First Hospital of Jilin University, Changchun, Jilin Province, China between May 2009 and July 2011, and for whom archival primary tumour material collected at diagnosis, prior to radiotherapy, was available, were enrolled in the study. For all patients, the original diagnosis and tumour grading was peer-reviewed by two experienced pathologists according to the principles laid down in the latest World Health
Downloaded from imr.sagepub.com at LOYOLA UNIV OF CHICAGO on September 1, 2014
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Organization (WHO) classification system.2 The tumours were classified as low grade gliomas (WHO grades II) or high-grade gliomas (grades III–IV). Computed tomography and magnetic resonance imaging data were available in all patients. In each patient, the presence and extent of necrosis (focal/extensive) was also assessed and compared with those in noncancerous adjacent tissues specimens (>3 cm away from the tumor site). There were no other inclusion/exclusion criteria for this study. All patients gave written informed consent to participate in the study. This study was approved by the Ethics Committee of Jilin University, Changchun, Jilin Province, China.
Immunohistochemical staining Primary tumour specimens were fixed in 10% buffered formalin, dehydrated in ethanol, embedded in paraffin wax and cut into 4-mm sections for use in immunohistochemistry. Serial sections were cut from each specimen and mounted on Superfrost slides (Menzel Gla¨ser, Braunschweig, Germany), dewaxed with xylene, gradually rehydrated and left to dry overnight at 37 C. After deparaffinization, immunostaining was performed using the two-step peroxidase technique with a peroxidase-conjugated polymer. Antigen retrieval was achieved by pressure cooking in 0.01 M citrate buffer (pH 6.0) for 5 min. After blocking with 3% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) in 0.01 mM phosphatebuffered saline (PBS; pH 7.2) at 37 C for 1 h, the sections were incubated with mouse monoclonal antibodies against human VEGF or MMP-9 (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500 overnight at 4 C. The slides were then washed three times in 0.01 mM PBS (pH 7.2) for 5 min at room temperature. The sections were then incubated with biotin-labelled rabbit antimouse antibody
(1:500 dilution; ZSJB-BIO, Beijing, China) for 2 h at 37 C. After washing with 0.01 mM PBS (pH 7.2) three times, immunostaining was visualized using a commercial streptavidin–peroxidase reaction system, and then developed with 3% diaminobenzidine–H2O2 (all reagents from Wuhan Boster Biological Technology, Wuhan, Hubei, China). The intensity of the immunostaining was graded using a four-point scoring system as follows: 0 if no immunoreactive cells were observed (negative); 1þ if the proportion of immunoreactive cells was 75% (strong). In cases with variable staining intensities, the most common pattern was recorded. Tissue samples with 0 or 1þ staining were classified as negative, and tissue samples with 2þ or 3þ staining were classified as positive. Five fields were randomly selected from each slide and examined at a magnification of 200 using a Leica DM2700 M light microscope (Leica, Solms, Germany), and were scored independently by two observers who were blinded to the clinicopathological data and clinical outcomes of the patients. Five cases with discordant results were re-evaluated by the two observers to obtain agreement. Negative control sections were incubated with normal mouse serum instead of primary antibody.
Statistical analyses All statistical analyses were performed using the SPSSÕ statistical package, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows and the SAS statistical package, version 9.1 (SAS Institute, Cary, NC, USA). The association between the level of VEGF and MMP-9 protein immunostaining and tumour grade was analysed using 2-test. The correlation between the level of VEGF and MMP-9 protein immunostaining was
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analysed using Spearman’s rank correlation coefficient analysis. A P-value 75% of cells were immunoreactive (strong). b P < 0.01 compared with diffuse astrocytomas (grades I– II); 2-test.
compared with the low-grade gliomas (grades II) (P < 0.01 for both comparisons).
Discussion Malignant glioma is characterized by rapid expansion, invasion of adjacent central nervous system tissues and aberrant vascularization.19,20 A growing understanding of the underlying molecular biology of glioma has identified several molecules pertinent to the pathophysiology. For example, VEGF is a secreted mitogen and the dominant factor that regulates angiogenesis in both normal development and tumour growth.21 During the process of haematogenous metastasis, VEGF is suggested to play a key role by participating in the regulation of angiogenesis.22 VEGF exerts its growthpromoting influence not on the tumour cells themselves but on the vascular endothelial cells, promoting both proliferation and new vessel formation.23 Chaudhry et al.24 demonstrated that the expression of VEGF in primary gliomas correlated with an increase in local microvessel density in the tumour tissue, development of pulmonary metastasis and poor prognosis for patients with glioma. These findings suggest that VEGF secreted by glioma cells stimulates angiogenesis, which critically contributes to the development of the pulmonary metastasis of glioma.25 Moreover, previous research demonstrated that glioma cells produce large amounts of VEGF and high-grade gliomas (glioblastoma multiform) express more VEGF than lower grade astrocytomas.23,24 The results of this present study were in agreement with the earlier findings because a significantly higher proportion of high-grade gliomas (grades III–IV) demonstrated positive VEGF immunostaining compared with the low grade gliomas (grade II) (P < 0.01).23,24 Tumour invasion is a cascade of sequential events that involves detachment of
Downloaded from imr.sagepub.com at LOYOLA UNIV OF CHICAGO on September 1, 2014
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malignant cells from their site of origin and invasion through the surrounding stroma into the lymphovascular channels.25 All of these steps are associated with ECM degradation and the proteolytic breakdown of major ECM components requires specific proteases. MMPs are proteolytic enzymes that degrade ECM components at neutral pH.10,26 Numerous studies have demonstrated that individual MMPs play crucial roles in tumour invasion and metastasis.27,28 In the present study, positive immunohistochemical staining for MMP-9 protein was detected in 60/80 (75.0%) of patients, and noncancerous adjacent tissues were shown to be negative for MMP-9 immunostaining, which was consistent with previous findings in other human tumours.10,29,30 Moreover, the level of VEGF immunostaining was significantly positively correlated with the level of MMP-9 immunostaining (r ¼ 0.78, P < 0.01). The details of the roles of VEGF and MMP-9 in malignant glioma remain unclear and need to be elucidated in further studies. Although our study showed that MMP9 and VEGF expression are related to WHO glioma grade, associations with clinicopathological factors including age, sex, tumour size and overall survival of patients were not investigated as the data were not available. Further studies should be conducted in which this information is collected. In conclusion, the upregulation of VEGF or MMP-9 proteins may be an important feature of malignant glioma. The combined immunohistochemical evaluation of the levels of VEGF and MMP-9 proteins in primary tumours might be of benefit in predicting a poor prognosis of survival in patients with malignant glioma. Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.
References 1. Li Z, Lee JW, Mukherjee D, et al. Immunotherapy targeting glioma stem cells – insights and perspectives. Expert Opin Biol Ther 2012; 12: 165–178. 2. Kleihues P, Burger PC, Scheithauer BW, et al. World Health Organization International Histological Classification of Tumours: Histological Typing of Tumours of the Central Nervous System, 2nd edition. Geneva: WHO, 1997. 3. Vlachostergios PJ, Voutsadakis IA and Papandreou CN. The role of ubiquitin– proteasome system in glioma survival and growth. Growth Factors 2013; 31: 106–113. 4. Ortega-Aznar A, Jimenez-Leon P, Martinez E, et al. Clinico-pathological and molecular aspects of diagnostic and prognostic value in gliomas. Rev Neurol 2013; 56: 161–170. [in Spanish, English abstract]. 5. Piccolo SR and Frey LJ. Clinical and molecular models of glioblastoma multiforme survival. Int J Data Min Bioinform 2013; 7: 245–265. 6. Stupp R, Hegi ME, Mason WP, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009; 10: 459–466. 7. Reijneveld JC, Voest EE and Taphoorn MJ. Angiogenesis in malignant primary and metastatic brain tumors. J Neurol 2000; 247: 597–608. 8. Korkolopoulou P, Patsouris E, Konstantinidou AE, et al. Hypoxiainducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis. Neuropathol Appl Neurobiol 2004; 30: 267–278.
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9. Ferrara N. Vascular endothelial growth factor as a target for anticancer therapy. Oncologist 2004; 9: 2–10. 10. Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004; 10: 145–147. 11. Trojanek J. Matrix metalloproteinases and their tissue inhibitors. Postepy Biochem 2012; 58: 353–362 [in Polish English abstract]. 12. Chambers AF and Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997; 89: 1260–1270. 13. Aoudjit F, Masure S, Opdenakker G, et al. Gelatinase B (MMP-9), but not its inhibitor (TIMP-1), dictates the growth rate of experimental thymic lymphoma. Int J Cancer 1999; 82: 743–747. 14. Bernhard EJ, Gruber SB and Muschel RJ. Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc Natl Acad Sci U S A 1994; 91: 4293–4297. 15. Rao JS, Steck PA, Mohanam S, et al. Elevated levels of M(r) 92,000 type IV collagenase in human brain tumors. Cancer Res 1993; 53: 2208–2211. 16. Zucker S, Lysik RM, Zarrabi MH, et al. M(r) 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res 1993; 53: 140–146. 17. Arico` A, Giantin M, Gelain ME, et al. The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study. BMC Vet Res 2013; 9: 94. 18. Hlobilkova A, Ehrmann J, Knizetova P, et al. Analysis of VEGF, Flt-1, Flk-1, nestin and MMP-9 in relation to astrocytoma pathogenesis and progression. Neoplasma 2009; 56: 284–290. 19. Candolfi M, Curtin JF, Nichols WS, et al. Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression. J Neurooncol 2007; 85: 133–148.
20. Wang Y and Jiang T. Understanding high grade glioma: molecular mechanism, therapy and comprehensive management. Cancer Lett 2013; 331: 139–146. 21. Oshika Y, Nakamura M, Tokunaga T, et al. Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer. Int J Oncol 1998; 12: 541–544. 22. Robles Irizarry L, Hambardzumyan D, Nakano I, et al. Therapeutic targeting of VEGF in the treatment of glioblastoma. Expert Opin Ther Targets 2012; 16: 973–984. 23. Plate KH, Breier G, Weich HA, et al. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992; 359: 845–848. 24. Chaudhry IH, O’Donovan DG, Brenchley PE, et al. Vascular endothelial growth factor expression correlates with tumour grade and vascularity in gliomas. Histopathology 2001; 39: 409–415. 25. Mentlein R, Hattermann K and Held-Feindt J. Lost in disruption: role of proteases in glioma invasion and progression. Biochim Biophys Acta 2012; 1825: 178–185. 26. Salamonsen LA. Matrix metalloproteinases and their tissue inhibitors in endocrinology. Trends Endocrinol Metab 1996; 7: 28–34. 27. Parsons SL, Watson SA, Brown PD, et al. Matrix metalloproteinases. Br J Surg 1997; 84: 160–166. 28. Curran S and Murray GI. Matrix metalloproteinases in tumour invasion and metastasis. J Pathol 1999; 189: 300–308. 29. Ka¨ha¨ri VM and Saarialho-Kere U. Matrix metalloproteinases and their inhibitors in tumour growth and invasion. Ann Med 1999; 31: 34–45. 30. Madigan MC, Kingsley EA, Cozzi PJ, et al. The role of extracellular matrix metalloproteinase inducer protein in prostate cancer progression. Cancer Immunol Immunother 2008; 57: 1367–1379.
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Levels of vascular endothelial growth factor and matrix metalloproteinase-9 proteins in patients with glioma Chengyuan Ma, Yang Li, Xianfeng Zhang, Gang Zhao and Haiyang Xu Journal of International Medical Research 2014 42: 198 originally published online 7 January 2014 DOI: 10.1177/0300060513481924 The online version of this article can be found at: http://imr.sagepub.com/content/42/1/198
Published by: http://www.sagepublications.com
Additional services and information for Journal of International Medical Research can be found at: Email Alerts: http://imr.sagepub.com/cgi/alerts Subscriptions: http://imr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Jan 16, 2014 OnlineFirst Version of Record - Jan 7, 2014 What is This?
Downloaded from imr.sagepub.com at LOYOLA UNIV OF CHICAGO on September 1, 2014
Research Note
Levels of vascular endothelial growth factor and matrix metalloproteinase-9 proteins in patients with glioma
Journal of International Medical Research 2014, Vol. 42(1) 198–204 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513481924 imr.sagepub.com
Chengyuan Ma1, Yang Li2, Xianfeng Zhang1, Gang Zhao1 and Haiyang Xu1
Abstract Objective: To investigate the levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) proteins in patients with glioma, in order to determine if either protein has prognostic value. Method: The presence of VEGF and MMP-9 proteins in paraffin-embedded tumour specimens from patients with glioma was detected using immunohistochemistry. The correlation between the levels of VEGF and MMP-9 proteins and tumour grade was analysed. Results: A total of 32 patients with low-grade gliomas (World Health Organization [WHO] grade II) and 48 patients with high-grade gliomas (WHO grades III–IV) participated in the study. Positive immunohistochemical staining of VEGF and MMP-9 proteins was detected in 58/80 (72.5%) and 60/80 (75.0%) of patients, respectively. The level of VEGF immunostaining was significantly positively correlated with the level of MMP-9 immunostaining (r ¼ 0.78). Significantly more highgrade gliomas (grades III–IV) demonstrated positive VEGF and MMP-9 immunostaining compared with the low grade gliomas (grades I–II). Conclusions: These data suggest that VEGF and MMP-9 play an important role in the malignant behaviour of gliomas.
Keywords Glioma, matrix metalloproteinase-9, MMP-9, vascular endothelial growth factor, VEGF, immunohistochemistry Date received: 10 January 2013; accepted: 16 January 2013
1
Department of Neurosurgery, First Hospital of Jilin University, Changchun, Jilin Province, China 2 Department of Respiratory Medicine, First Hospital of Jilin University, Changchun, Jilin Province, China
Corresponding author: Dr Haiyang Xu, Department of Neurosurgery, First Hospital of Jilin University, 71 Xinmin Road, Chaoyang Region, Changchun 130021, Jilin Province, China. Email: [email protected]
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Introduction Gliomas are highly malignant and invasive tumors with tendrils that extend far from the primary tumor site.1 Gliomas are further divided into low-grade gliomas (World Health Organization [WHO] grade II) and high-grade gliomas (WHO grade III–IV) by pathologic evaluation of the tumour.2 Lowgrade gliomas are well differentiated, whereas high-grade gliomas are undifferentiated or anaplastic. The latter are malignant and carry a worse prognosis.3 Although a small portion of low grade gliomas display benign features, most of these tumours are malignant and usually indicate a poor prognosis.4 Despite modern diagnostic methods and treatment strategies, the median survival time does not exceed 15 months.5 This is mainly due to the fact that at the time of surgery, cells from the primary tumour have already invaded normal brain tissue.6 Therefore, it is extremely important to search for sensitive and specific markers that can provide valuable information for the early diagnosis and identification of poorly responsive tumours that may benefit from a modified treatment regimen. Tumour metastasis is a multistep process involving a variety of tumour–host cell interactions. Angiogenesis, one such interaction, is the outgrowth of endothelial cells from pre-existing capillary vessels and their migration from parental vessels under the stimulation of vascular endothelial growth factor (VEGF),7 which is a homodimeric protein identified as a mitogen for endothelial cells in vitro and an angiogenesis promoting factor in vivo.8 Glioma cells produce large amounts of VEGF and high grade gliomas (glioblastoma multiform) produce more VEGF than lower grade astrocytomas.7,8 Clinical studies have demonstrated that circulating VEGF has a negative prognostic value in cancer patients, correlating with tumour grade and vascularity in gliomas.9,10
Matrix metalloproteinases (MMPs) are zinc-binding endopeptidases capable of degrading extracellular matrix (ECM) components such as collagen, fibrinogen and proteoglycans.11 One member of this family, MMP-9, has been assigned an important role in tissue-specific remodelling in normal and pathological processes, most notably during angiogenesis and tumour invasion.12 Elevated levels of MMP-9 have been found in patients suffering from various types of tumours in vivo and a correlation has been established between the secretion of MMP-9 and experimental metastasis.13–16 Furthermore, overexpression of MMP-9 in transfected nonmetastatic tumour cells renders them invasive.13,14, Proliferating endothelial cells subsequently remodel the ECM via MMPs, align into tube-like structures, and eventually form new functional blood vessels.11 In malignant cells, upregulation of angiogenic factors, such as VEGF and MMPs, is closely linked to invasion and metastasis.17 There have been few reports on the role of VEGF and MMPs in gliomas.18 The present study, therefore, investigated the levels of VEGF and MMP-9 protein using immunohistochemical staining in order to determine if either protein has any prognostic value in patients with gliomas
Patients and methods Patients Consecutive patients with gliomas who had been diagnosed, treated and followed-up in the Department of Neurosurgery, First Hospital of Jilin University, Changchun, Jilin Province, China between May 2009 and July 2011, and for whom archival primary tumour material collected at diagnosis, prior to radiotherapy, was available, were enrolled in the study. For all patients, the original diagnosis and tumour grading was peer-reviewed by two experienced pathologists according to the principles laid down in the latest World Health
Downloaded from imr.sagepub.com at LOYOLA UNIV OF CHICAGO on September 1, 2014
200
Journal of International Medical Research 42(1)
Organization (WHO) classification system.2 The tumours were classified as low grade gliomas (WHO grades II) or high-grade gliomas (grades III–IV). Computed tomography and magnetic resonance imaging data were available in all patients. In each patient, the presence and extent of necrosis (focal/extensive) was also assessed and compared with those in noncancerous adjacent tissues specimens (>3 cm away from the tumor site). There were no other inclusion/exclusion criteria for this study. All patients gave written informed consent to participate in the study. This study was approved by the Ethics Committee of Jilin University, Changchun, Jilin Province, China.
Immunohistochemical staining Primary tumour specimens were fixed in 10% buffered formalin, dehydrated in ethanol, embedded in paraffin wax and cut into 4-mm sections for use in immunohistochemistry. Serial sections were cut from each specimen and mounted on Superfrost slides (Menzel Gla¨ser, Braunschweig, Germany), dewaxed with xylene, gradually rehydrated and left to dry overnight at 37 C. After deparaffinization, immunostaining was performed using the two-step peroxidase technique with a peroxidase-conjugated polymer. Antigen retrieval was achieved by pressure cooking in 0.01 M citrate buffer (pH 6.0) for 5 min. After blocking with 3% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) in 0.01 mM phosphatebuffered saline (PBS; pH 7.2) at 37 C for 1 h, the sections were incubated with mouse monoclonal antibodies against human VEGF or MMP-9 (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500 overnight at 4 C. The slides were then washed three times in 0.01 mM PBS (pH 7.2) for 5 min at room temperature. The sections were then incubated with biotin-labelled rabbit antimouse antibody
(1:500 dilution; ZSJB-BIO, Beijing, China) for 2 h at 37 C. After washing with 0.01 mM PBS (pH 7.2) three times, immunostaining was visualized using a commercial streptavidin–peroxidase reaction system, and then developed with 3% diaminobenzidine–H2O2 (all reagents from Wuhan Boster Biological Technology, Wuhan, Hubei, China). The intensity of the immunostaining was graded using a four-point scoring system as follows: 0 if no immunoreactive cells were observed (negative); 1þ if the proportion of immunoreactive cells was 75% (strong). In cases with variable staining intensities, the most common pattern was recorded. Tissue samples with 0 or 1þ staining were classified as negative, and tissue samples with 2þ or 3þ staining were classified as positive. Five fields were randomly selected from each slide and examined at a magnification of 200 using a Leica DM2700 M light microscope (Leica, Solms, Germany), and were scored independently by two observers who were blinded to the clinicopathological data and clinical outcomes of the patients. Five cases with discordant results were re-evaluated by the two observers to obtain agreement. Negative control sections were incubated with normal mouse serum instead of primary antibody.
Statistical analyses All statistical analyses were performed using the SPSSÕ statistical package, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows and the SAS statistical package, version 9.1 (SAS Institute, Cary, NC, USA). The association between the level of VEGF and MMP-9 protein immunostaining and tumour grade was analysed using 2-test. The correlation between the level of VEGF and MMP-9 protein immunostaining was
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analysed using Spearman’s rank correlation coefficient analysis. A P-value 75% of cells were immunoreactive (strong). b P < 0.01 compared with diffuse astrocytomas (grades I– II); 2-test.
compared with the low-grade gliomas (grades II) (P < 0.01 for both comparisons).
Discussion Malignant glioma is characterized by rapid expansion, invasion of adjacent central nervous system tissues and aberrant vascularization.19,20 A growing understanding of the underlying molecular biology of glioma has identified several molecules pertinent to the pathophysiology. For example, VEGF is a secreted mitogen and the dominant factor that regulates angiogenesis in both normal development and tumour growth.21 During the process of haematogenous metastasis, VEGF is suggested to play a key role by participating in the regulation of angiogenesis.22 VEGF exerts its growthpromoting influence not on the tumour cells themselves but on the vascular endothelial cells, promoting both proliferation and new vessel formation.23 Chaudhry et al.24 demonstrated that the expression of VEGF in primary gliomas correlated with an increase in local microvessel density in the tumour tissue, development of pulmonary metastasis and poor prognosis for patients with glioma. These findings suggest that VEGF secreted by glioma cells stimulates angiogenesis, which critically contributes to the development of the pulmonary metastasis of glioma.25 Moreover, previous research demonstrated that glioma cells produce large amounts of VEGF and high-grade gliomas (glioblastoma multiform) express more VEGF than lower grade astrocytomas.23,24 The results of this present study were in agreement with the earlier findings because a significantly higher proportion of high-grade gliomas (grades III–IV) demonstrated positive VEGF immunostaining compared with the low grade gliomas (grade II) (P < 0.01).23,24 Tumour invasion is a cascade of sequential events that involves detachment of
Downloaded from imr.sagepub.com at LOYOLA UNIV OF CHICAGO on September 1, 2014
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malignant cells from their site of origin and invasion through the surrounding stroma into the lymphovascular channels.25 All of these steps are associated with ECM degradation and the proteolytic breakdown of major ECM components requires specific proteases. MMPs are proteolytic enzymes that degrade ECM components at neutral pH.10,26 Numerous studies have demonstrated that individual MMPs play crucial roles in tumour invasion and metastasis.27,28 In the present study, positive immunohistochemical staining for MMP-9 protein was detected in 60/80 (75.0%) of patients, and noncancerous adjacent tissues were shown to be negative for MMP-9 immunostaining, which was consistent with previous findings in other human tumours.10,29,30 Moreover, the level of VEGF immunostaining was significantly positively correlated with the level of MMP-9 immunostaining (r ¼ 0.78, P < 0.01). The details of the roles of VEGF and MMP-9 in malignant glioma remain unclear and need to be elucidated in further studies. Although our study showed that MMP9 and VEGF expression are related to WHO glioma grade, associations with clinicopathological factors including age, sex, tumour size and overall survival of patients were not investigated as the data were not available. Further studies should be conducted in which this information is collected. In conclusion, the upregulation of VEGF or MMP-9 proteins may be an important feature of malignant glioma. The combined immunohistochemical evaluation of the levels of VEGF and MMP-9 proteins in primary tumours might be of benefit in predicting a poor prognosis of survival in patients with malignant glioma. Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.
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