尼古丁通过刺激内源性胆碱能途径充当血管生成因子。烟碱乙酰胆碱受体（nAChR）的几种亚型已被证明与不同类型癌症的形成和进展密切相关。最近，多项研究发现烟碱型乙酰胆碱受体α9（α9-nAChRs）在乳腺肿瘤中高表达，特别是在晚期诊断患者的肿瘤中。体外研究表明，α9-nAChR 的激活与乳腺癌的增殖和迁移增加有关。为了研究 α9-nAChR 在乳腺癌中的促肿瘤作用，我们生成了一种新型抗 α9-nAChR 和甲氧基聚乙二醇 (mPEG) 双特异性抗体 (α9 BsAb)，用于剖析 α9-nAChR 介导的肿瘤的分子机制进展。出乎意料的是，我们发现了 α9-nAChR 在尼古丁诱导的肿瘤新血管形成中的血管生成作用。结果表明，α9 BsAb 可减少尼古丁诱导的内皮细胞管形成、基质胶塞测定中的血管发育以及微管阵列膜鼠模型 (MTAM) 中的血管生成。为了阐明 α9-nAChR 在尼古丁介导的血管生成中的分子机制，应用 α9 BsAb 揭示了在尼古丁诱导的缺氧诱导因子 2 α (HIF-2α)、血管内皮生长因子 A 的产生中的抑制作用。来自三阴性乳腺癌细胞 (MDA-MB-231) 的 VEGF-A)、磷酸化血管内皮生长因子受体 2 (p-VEGFR2)、血管内皮生长因子受体 2 (VEGFR2) 和基质金属蛋白酶 9 (MMP9)，表明 α9-nAChR 在尼古丁诱导的血管生成中发挥重要作用。为了证实我们的结果，设计了靶向 α9-nAChR 的 shRNA 并用于沉默 α9-nAChR 表达，然后评估 α9-nAChR 的血管生成作用。结果显示α9 shRNA在阻断尼古丁诱导的血管生成信号传导方面也发挥了抑制作用。总而言之，α9-nAChR 在尼古丁诱导的血管生成中发挥着关键作用，这种双特异性抗体 (α9 BsAb) 可以作为治疗 α9 阳性癌症的潜在治疗候选药物。© 2024。作者，拥有独家许可施普林格自然 B.V.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers.© 2024. The Author(s), under exclusive licence to Springer Nature B.V.