Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo 663‑8501, Japan.
Oncol Rep. 2020 Jul;44(1):14-28. doi: 10.3892/or.2020.7580. Epub 2020 Apr 9.
Non‑small cell lung cancer (NSCLC) accounts for over 80% of all diagnosed lung cancer cases. Lung cancer is the leading cause of cancer‑related deaths worldwide. Most NSCLC cells overexpress vascular endothelial growth factor‑A (VEGF‑A) which plays a pivotal role in tumour angiogenesis. Anti‑angiogenic therapies including VEGF‑A neutralisation have significantly improved the response rates, progression‑free survival and overall survival of patients with NSCLC. However, the median survival of these patients is shorter than 18 months, suggesting that NSCLC cells secrete VEGF‑independent angiogenic factors, which remain unknown. We aimed to explore these factors in human NSCLC cell lines, A549, Lu99 and EBC‑1 using serum‑free culture, to which only EBC‑1 cells could adapt. By mass spectrometry, we identified 1,007 proteins in the culture supernatant derived from EBC‑1 cells. Among the identified proteins, interleukin‑8 (IL‑8), macrophage migration inhibitory factor (MIF), galectin‑1, midkine (MK), IL‑18, galectin‑3, VEGF‑A, hepatoma‑derived growth factor (HDGF), osteopontin (OPN), connective tissue growth factor (CTGF) and granulin (GRN) are known to be involved in angiogenesis. Tube formation, neutralisation and RNA interference assays revealed that VEGF‑A and HDGF function as angiogenic factors in EBC‑1 cells. To confirm whether VEGF‑A and HDGF also regulate angiogenesis in the other NSCLC cell lines, we established a novel culture method. NSCLC cells were embedded in collagen gel and cultured three‑dimensionally. Tube formation, neutralisation and RNA interference assays using the three‑dimensional (3D) culture supernatant showed that VEGF‑A and HDGF were not angiogenic factors in Lu99 cells. By gene microarray in EBC‑1 and Lu99 cells, we identified 61 mRNAs expressed only in Lu99 cells. Among these mRNAs, brain‑derived neurotrophic factor (BDNF), fibroblast growth factor‑2 (FGF‑2) and FGF‑5 are known to be involved in angiogenesis. Tube formation and neutralisation assays clarified that FGF‑2 functions as an angiogenic factor in Lu99 cells. These results indicate that HDGF enhances VEGF‑dependent angiogenesis and that FGF‑2 is a VEGF‑independent angiogenic factor in human NSCLC cells.
非小细胞肺癌 (NSCLC) 占所有诊断出的肺癌病例的 80%以上。肺癌是全球癌症相关死亡的主要原因。大多数 NSCLC 细胞过度表达血管内皮生长因子 A (VEGF-A)，后者在肿瘤血管生成中起着关键作用。抗血管生成疗法，包括 VEGF-A 中和，显著提高了 NSCLC 患者的反应率、无进展生存期和总生存期。然而，这些患者的中位生存期短于 18 个月，这表明 NSCLC 细胞分泌 VEGF 独立的血管生成因子，但这些因子尚不清楚。我们旨在使用无血清培养，探索 A549、Lu99 和 EBC-1 人 NSCLC 细胞系中的这些因子，只有 EBC-1 细胞能够适应。通过质谱分析，我们从 EBC-1 细胞的培养上清液中鉴定出 1007 种蛋白质。在鉴定的蛋白质中，白细胞介素-8 (IL-8)、巨噬细胞移动抑制因子 (MIF)、半乳糖凝集素-1、中期因子 (MK)、白细胞介素-18、半乳糖凝集素-3、血管内皮生长因子 A (VEGF-A)、肝癌衍生生长因子 (HDGF)、骨桥蛋白 (OPN)、结缔组织生长因子 (CTGF) 和颗粒蛋白 (GRN) 已知参与血管生成。管形成、中和和 RNA 干扰试验表明，VEGF-A 和 HDGF 在 EBC-1 细胞中作为血管生成因子发挥作用。为了确认 VEGF-A 和 HDGF 是否也调节其他 NSCLC 细胞系中的血管生成，我们建立了一种新的培养方法。将 NSCLC 细胞嵌入胶原凝胶中并进行三维培养。使用三维 (3D) 培养上清液进行的管形成、中和和 RNA 干扰试验表明，VEGF-A 和 HDGF 不是 Lu99 细胞中的血管生成因子。通过 EBC-1 和 Lu99 细胞的基因微阵列，我们鉴定出仅在 Lu99 细胞中表达的 61 种 mRNA。在这些 mRNA 中，脑源性神经营养因子 (BDNF)、成纤维细胞生长因子-2 (FGF-2) 和 FGF-5 已知参与血管生成。管形成和中和试验表明，FGF-2 在 Lu99 细胞中作为血管生成因子发挥作用。这些结果表明，HDGF 增强了 VEGF 依赖性血管生成，而 FGF-2 是人类 NSCLC 细胞中 VEGF 非依赖性的血管生成因子。
