Extracellular vesicle-bound VEGF in oral squamous cell carcinoma and its role in resistance to Bevacizumab Therapy.

IF 5.3 2区医学 Q1 ONCOLOGY Cancer Cell International Pub Date : 2024-08-23 DOI:10.1186/s12935-024-03476-1

Jiasheng Zhou, Xue Liu, Qi Dong, Jiao Li, Weidong Niu, Tingjiao Liu

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Abstract

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.

Conclusion: Antagonists targeting HSPG might potentially overcome the resistance of EV-VEGF to bevacizumab and serve as an alternative for anti-VEGF therapy in OSCC.

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口腔鳞状细胞癌中与细胞外囊泡结合的血管内皮生长因子及其在贝伐珠单抗疗法耐药性中的作用。

背景：血管内皮生长因子（VEGF）是一种重要的促血管生成因子，一直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（一种特异性血管内皮生长因子抗体）的临床应用并没有提高 OSCC 患者的生存率。一种可能的解释是血管内皮生长因子（VEGF）基因表达不同的异构体，这些异构体与细胞外囊泡（EVs）相关联，而EVs可能导致血管内皮生长因子对贝伐单抗产生耐药性。然而，在解决这一问题方面还缺乏明确的方案：方法：通过反转录聚合酶链式反应（RT-PCR）和免疫印迹证实了VEGF同工酶在OSCC细胞中的表达。从 OSCC 细胞的条件培养基（CM）中分离出的 EVs 通过 Western 印迹、透射电子显微镜（TEM）和纳米粒子追踪分析（NTA）进行表征。应用流式细胞术、免疫金标记和 Western 印迹法研究了 EVs 上的 VEGF。结果表明，EV-VEGF表达的最多的异构体是EV-VEGF，而EV-VEGF表达的最多的异构体是EV-VEGF：结果：VEGF 基因最常表达的同工型是 VEGF121、VEGF165 和 VEGF189。本研究表明，在 OSCC 细胞中可检测到不同水平的三种同工酶 mRNA，但只发现了 VEGF165 和 VEGF189 蛋白。从 OSCC 细胞中提取的 CM 可以检测到可溶性和非可溶性形式的 VEGF。我们进一步证实，与 EV 结合的 VGEF189 以非可溶性形式存在。与 EV 结合的 VEGF189 具有血管生成活性，贝伐珠单抗无法中和这种活性。研究发现，VEGF189 是通过硫酸肝素蛋白多糖（HSPG）与 EV 结合的。此外，一种 HSPG 拮抗剂 surfen 可以在体外和体内逆转 EV-VEGF 的血管生成效应：结论：靶向 HSPG 的拮抗剂有可能克服 EV-VEGF 对贝伐单抗的耐药性，成为 OSCC 抗血管内皮生长因子疗法的替代疗法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Cancer Cell International ONCOLOGY-

CiteScore

10.90

自引率

1.70%

发文量

360

审稿时长

1 months

期刊介绍： Cancer Cell International publishes articles on all aspects of cancer cell biology, originating largely from, but not limited to, work using cell culture techniques. The journal focuses on novel cancer studies reporting data from biological experiments performed on cells grown in vitro, in two- or three-dimensional systems, and/or in vivo (animal experiments). These types of experiments have provided crucial data in many fields, from cell proliferation and transformation, to epithelial-mesenchymal interaction, to apoptosis, and host immune response to tumors. Cancer Cell International also considers articles that focus on novel technologies or novel pathways in molecular analysis and on epidemiological studies that may affect patient care, as well as articles reporting translational cancer research studies where in vitro discoveries are bridged to the clinic. As such, the journal is interested in laboratory and animal studies reporting on novel biomarkers of tumor progression and response to therapy and on their applicability to human cancers.