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. 2022 Sep 20;42(9):1351-1358.
doi: 10.12122/j.issn.1673-4254.2022.09.11.

[Exosomal FZD10 derived from non-small cell lung cancer cells promotes angiogenesis of human umbilical venous endothelial cells in vitro]

[Article in Chinese]
X Wu  1   2 , R Zhan  1   2 , D Cheng  1   3 , L Chen  1   3 , T Wang  1   3 , X Tang  1   3
Affiliations

[Exosomal FZD10 derived from non-small cell lung cancer cells promotes angiogenesis of human umbilical venous endothelial cells in vitro]

[Article in Chinese]
X Wu et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the effect of exosomal FZD10 derived from non-small cell lung cancer (NSCLC) cells on angiogenesis of human umbilical venous endothelial cells (HUVECs) and explore the possible mechanism.

Methods: We analyzed the expression of FZD10 in two NSCLC cell lines (95D and H1299 cells), normal human bronchial epithelial cells (BEAS-2B cells) and their exosomes isolated by ultracentrifugation. Cultured HUVECs were treated with the exosomes derived from NSCLC cells or NSCLC cells transfected with FZD10-siRNA, and the changes in tube formation ability of the cells were analyzed using an in vitro angiogenesis assay. ELISA was performed to determine the concentration of VEGFA and Ang-1 in the conditioned media of HUVECs, and RT-qPCR was used to analyze the mRNA levels of VEGFA and Ang-1 in the HUVECs. The effects of exosomal FZD10 on the activation of PI3K, Erk1/2 and YAP/TAZ signaling pathways were evaluated using Western blotting.

Results: Compared with BEAS-2B cells and their exosomes, 95D and H1299 cells and their exosomes all expressed high levels of FZD10 (P < 0.01). The exosomes derived from 95D and H1299 cells significantly enhanced tube formation ability and increased the expressions of VEGFA and Ang-1 protein and mRNA in HUVECs (P < 0.01), but FZD10 knockdown in 95D and H1299 cells obviously inhibited these effects of the exosomes. Exosomal FZD10 knockdown suppressed the activation of PI3K and Erk1/2 signaling pathways, but had no obvious effect on the activation of YAP/TAZ signaling pathway.

Conclusion: Exosomal FZD10 derived from NSCLC cells promotes HUVEC angiogenesis in vitro, the mechanism of which may involve the activation of PI3K and Erk1/2 signaling pathways.

目的: 探讨非小细胞肺癌（NSCLC）细胞外泌体源性FZD10在血管生成中的作用及其机制。

方法: 采用超速离心法分离外泌体，并利用Western blot和RT-qPCR技术分析NSCLC细胞（95D和H1299）、正常人支气管上皮细胞（BEAS-2B）及其外泌体中FZD10的表达；通过转染FZD10-siRNA敲低FZD10表达，用FZD10未敲低和敲低的NSCLC细胞外泌体分别处理HUVEC细胞，利用体外血管生成实验观察其成管能力，采用ELISA和RT-qPCR技术分析血管生成相关因子VEGFA和Ang-1的表达；进一步利用Western blot分析外泌体源性FZD10对信号通路PI3K、Erk1/2和YAP/TAZ激活的影响。

结果: 同BEAS-2B细胞及其外泌体相比较，FZD10在95D和H1299细胞及其外泌体中高表达（P < 0.01）；95D和H1299细胞来源的外泌体可促进HUVEC的微管形成及VEGFA、Ang-1的蛋白分泌、mRNA的表达（P < 0.01），但在95D和H1299细胞敲低FZD10后这些效果受到抑制。FZD10的敲低可抑制PI3K、Erk1/2信号通路的激活，但对YAP/TAZ信号通路的影响不显著。

结论: NSCLC细胞外泌体源性FZD10可促进体外血管生成，其机制可能与PI3K、Erk1/2信号通路的激活有关。

Keywords: FZD10; angiogenesis; exosomes; non-small cell lung cancer.

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Figures

图 1
图 1
外泌体的鉴定 Identification of the isolated exosomes. A: Results of transmission electron microscopy. B: Results of Western blotting.
图 2
图 2
FZD10在NSCLC细胞及其外泌体中的表达 Expression of FZD10 in NSCLC cells and their exosomes. A, B: Western blotting for analyzing FZD10 protein expression. C: RT-qPCR for analyzing FZD10 mRNAlevels. *P < 0.05, **P < 0.01 vs BEAS-2B group.
图 3
图 3
外泌体内化的结果 Results of exosome internalization (Original magnification×200).
图 4
图 4
NSCLC细胞来源的外泌体对体外血管生成的影响 Effect of the exosomes derived from NSCLC cells on angiogenesis of HUVECs in vitro. A: Tube formation of the HUVECs was observed under a phase-contrast microscope (×200). B: Total tube length measured by Scion Image software. C, D: ELISA for analyzing VEGFA (C) and Ang-1 (D) concentrations in the conditioned media of HUVECs. E: RT-qPCR for analysis of the mRNA levels of VEGFA and Ang-1 in HUVECs. In+95D exo, +H1299 exo and +BEAS-2B exo groups, the HUVECs were treated with the exosomes derived from 95D, H1299 and BEAS-2B cells, respectively. **P < 0.01 vs +BEAS-2B exo group.
图 5
图 5
FZD10在NSCLC细胞来源的外泌体对体外血管生成影响中的作用 Role of FZD10 mediating the effect of the exosomes derived from NSCLC cells on angiogenesis of HUVECs in vitro. A: 95D and H1299 cells were transfected with FZD10-siRNA, and Western blotting was performed to identify the efficiency of knockdown. B, C: Tube formation of HUVECs treated with the exosomes derived from FZD10-siRNA-transfected 95D (B) and H1299 cells (C) was observed under a phase-contrast microscope (×200), and the total tube length was measured by Scion Image software. D, E: ELISA was performed to analyze VEGFA (D) and Ang-1 (E) concentration in the conditioned media of HUVECs. F: RT-qPCR was used to analyze the of VEGFA and Ang-1 mRNA levels in HUVECs. In + 95D exo and + 95D FZD10-KD exo groups, the HUVECs were treated with the exosomes derived from 95D and FZD10-siRNA-transfected 95D cells, respectively; the cells were treated likewise in +H1299 exo and +H1299 FZD10-KD exo groups. **P < 0.01 vs +95D exo or +H1299 exo group.
图 6
图 6
FZD10敲低对PI3K、Erk1/2、YAP/TAZ信号通路激活的影响 Effect of FZD10 knockdown on the activation of PI3K, Erk1/2 and YAP/TAZ signaling pathways. A, C: Western blotting for detecting protein expressions in 95D cells (A) and H1299 cells (C). B, D: Quantitative analysis of the protein expressions. *P < 0.05 vs +95D FZD10-KD exo or +H1299 FZD10-KD exo group.
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国家自然科学基金(81372511); 广东省基础与应用基础研究基金(2019A1515011081); 广东省“扬帆计划”培养高层次人才项目(201635011); 广东省普通高校特色创新类项目(自然科学) (2022KTSCX048)