背景:血管内皮生长因子(VEGF)是一种重要的促血管生成因子,被认为是口腔鳞状细胞癌(OSCC)抗血管生成治疗的关键靶点。然而,贝伐单抗的临床应用,一种特异性的VEGF抗体,并没有提高OSCC患者的生存率。一个可能的解释是VEGF基因表达不同的同工型,与细胞外囊泡(EV)相关,EV可能导致贝伐单抗对VEGF的耐药。然而,解决这个问题缺乏明确的解决方案。
方法:通过逆转录和聚合酶链反应(RT-PCR)和蛋白质印迹确认OSCC细胞中VEGF亚型的表达。从OSCC细胞条件培养基(CM)中分离的EV通过蛋白质印迹进行表征,透射电子显微镜(TEM)和纳米粒子跟踪分析(NTA)。流式细胞术,应用免疫金标记和蛋白质印迹法研究EVs上的VEGF。管形成测定和基质胶塞血管生成测定用于分析EV-VEGF的血管生成能力。
结果:VEGF基因表达的最流行的同工型是VEGF121、VEGF165和VEGF189。在这项研究中,我们证明了在OSCC细胞中可以不同水平检测到mRNA的所有三种同工型,而仅发现VEGF165和VEGF189蛋白。CM来源于OSCC细胞,可以检测到可溶性和非可溶性形式的VEGF。我们进一步证实了VGEF189以不可溶形式与EV结合。EV结合的VEGF189呈现血管生成活性,贝伐单抗无法中和。发现VEGF189通过硫酸乙酰肝素蛋白聚糖(HSPG)与EV结合。此外,Surfen可以逆转EV-VEGF的血管生成作用,HSPG的一种体外和体内拮抗剂。
结论:靶向HSPG的拮抗剂可能潜在地克服EV-VEGF对贝伐单抗的耐药性,并作为OSCC抗VEGF治疗的替代方案。
BACKGROUND: Vascular endothelial growth factor (VEGF) is an important proangiogenic factor and has been considered as a key target of antiangiogenetic therapy in oral squamous cell carcinoma (OSCC). However, clinical application of bevacizumab, a specific VEGF antibody, didn\'t improve the survival rate of OSCC patients. One possible explanation is that VEGF gene expresses diverse isoforms, which associate with extracellular vesicles (EVs), and EVs potentially contribute to VEGF resistance to bevacizumab. However, clear solution is lacking in addressing this issue.
METHODS: Expression of VEGF isoforms in OSCC cells was confirmed by reverse transcription and polymerase chain reaction (RT-PCR) and western blot. EVs isolated from OSCC cell\'s conditioned medium (CM) were characterized by western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunogold labeling and western blot were applied to study the VEGF on EVs. Tube formation assay and Matrigel plug angiogenesis assay were used for analyzing the angiogenesis capacity of EV-VEGF.
RESULTS: The most popular isoforms expressed by VEGF gene are VEGF121, VEGF165 and VEGF189. In this study, we demonstrated that all three isoforms of mRNA could be detected at varying levels in OSCC cells, while only VEGF165 and VEGF189 proteins were found. CM derived from OSCC cells, both soluble and non-soluble forms of VEGF could be detected. We further confirmed the presence of VGEF189 bound to EVs as a non-soluble form. EV-bound VEGF189 presented angiogenic activity, which could not be neutralized by bevacizumab. It was found that VEGF189 bound to EVs by heparan sulfate proteoglycans (HSPG). In addition, the angiogenic effect of EV-VEGF could be reversed by surfen, a kind of HSPG antagonist both in vitro and in vivo.
CONCLUSIONS: Antagonists targeting HSPG might potentially overcome the resistance of EV-VEGF to bevacizumab and serve as an alternative for anti-VEGF therapy in OSCC.