Semaphorin 3A 通过介导脑肿瘤干细胞的增殖和侵袭促进 EGFRviii 突变型脑胶质瘤的发生。
Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas.
机构信息
Mayo Clinic: College of Medicine, Rochester, MN, 55905, USA.
Department of Neurosurgery, Columbia University Medical Center, 710 W. 168th Street, New York, NY, 10032, USA.
出版信息
BMC Cancer. 2020 Dec 10;20(1):1213. doi: 10.1186/s12885-020-07694-4.
BACKGROUND
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.
METHODS
GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7.
RESULTS
Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD.
CONCLUSIONS
These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.
背景
多形性胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤，中位生存期约为 15 个月。信号素 3A（Sema3A）以其轴突导向和抗血管生成特性而闻名，与 GBM 生长有关。我们假设 Sema3A 通过神经纤毛蛋白 1（Nrp1）和 Plexin A1（PlxnA1）受体直接抑制脑肿瘤干细胞（BTSC）增殖并驱动侵袭。
方法
通过免疫染色和 PCR 检测 Sema3A 及其受体 Nrp1 和 PlxnA1 在 GBM BTSC 细胞系中的水平。进行外源性 Sema3A 处理后，进行定量 BrdU、细胞周期和碘化丙啶标记测定。还显示了定量功能 2-D 和 3-D 侵袭测定以及 Nrp1 和 PlxnA1 的 shRNA 慢病毒敲低。进行了比较敲低与对照 BTSC 肿瘤生长的体内侧翼研究。使用 GraphPad Prism v7 进行统计学分析。
结果
免疫染色和 PCR 分析显示，BTSC 高度表达 Sema3A 及其受体 Nrp1 和 PlxnA1，其中 Nrp1 在 CD133 阳性 BTSC 中表达，而在分化的肿瘤细胞中不存在。在定量 BrdU、细胞周期和碘化丙啶标记测定中，外源性 Sema3A 处理表明 Sema3A 显著抑制 BTSC 增殖而不诱导细胞死亡。定量功能 2-D 和 3-D 侵袭测定表明，Sema3A 处理导致侵袭增加。使用 shRNA 慢病毒，敲低 Nrp1 或 PlxnA1 受体均可消除 Sema3A 的抗增殖和促侵袭作用。有趣的是，受体的缺失模拟了 Sema3A 的作用，抑制了 BTSC 的增殖并驱动了侵袭。此外，比较将敲低和对照感染的 BTSC 植入裸鼠侧翼的肿瘤生长的体内研究证实了受体 KD 时增殖减少。
结论
这些发现表明 Sema3A 信号在 GBM BTSC 增殖和侵袭中的重要性及其作为治疗靶点的潜力。
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