背景:肺腺癌(LUAD)是最常见的肺癌亚型。Xklp2靶向蛋白(TPX2),一个关键的癌基因在包括LUAD在内的各种癌症中表现出高表达水平,可以作为临床干预的潜在目标。此外,肺癌的生长受到肿瘤微环境(TME)的显著影响。然而,目前尚无关于在LUAD的肿瘤浸润免疫细胞(TIIC)中研究TPX2的实验报道.因此,我们在体外和体内验证了TPX2对巨噬细胞极化的影响。
方法:我们在A549细胞中沉默TPX2基因,并收集上清液用于巨噬细胞培养。然后我们使用流式细胞术和蛋白质印迹分析来评估巨噬细胞极化。此外,我们验证了巨噬细胞集落刺激因子(M-CSF)的表达,在LUAD患者的组织标本中通过免疫组织化学(IHC)和CD163。最后,通过全基因组测序分析与TPX2在巨噬细胞极化中的调节功能相关的通路,西方印迹,和免疫荧光(IF)。
结果:沉默TPX2可以影响CD80+M1/CD163+M2的比例,主要通过抑制M-CSF的表达来降低M0巨噬细胞向CD163+M2的分化。在人类LUAD组织中,TPX2、M-CSF和CD163的表达水平随分化程度的增加而增加。沉默TPX2抑制NF-κB信号通路,从而减少M-CSF的表达,并影响巨噬细胞极化。
结论:沉默TPX2可通过阻断NF-κB信号抑制M-CSF的表达,从而降低CD163+M2巨噬细胞极化。TPX2/NF-κB/M-CSF信号轴可能参与调节巨噬细胞极化。
BACKGROUND: Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Xklp2 targeting protein (TPX2), a crucial oncogene exhibits high expression levels in various cancers including LUAD, may serve as a potential target for clinical intervention. Additionally, the growth of lung cancer is significantly influenced by the tumor microenvironment (TME). However, there have been no reports on experiments investigating TPX2 in tumor-infiltrating immune cells (TIICs) in LUAD. Therefore, we verified the effect of TPX2 on macrophage polarization both in vitro and in vivo.
METHODS: We silenced TPX2 the gene in A549 cells and collected supernatants for macrophage culture. We then used flow cytometry and Western blot analysis to assess macrophage polarization. Additionally, we verified the expression of macrophage colony-stimulating factor (M-CSF), and CD163 by immunohistochemistry (IHC) in tissue specimens from LUAD patients. Finally, pathways related to TPX2\'s regulatory function in macrophage polarization were analyzed through whole genome sequencing, Western blotting, and immunofluorescence (IF).
RESULTS: Silencing TPX2 can affect the ratio of CD80+ M1/CD163+ M2 and reduce the polarization of M0 macrophages to CD163+ M2 macrophages mainly by inhibiting the expression of M-CSF. In human LUAD tissues, the expression levels of TPX2, M-CSF and CD163 increased with the degree of differentiation. Silencing TPX2 inhibits the NF-κB signaling pathway, thereby reducing the expression of M-CSF, and affecting macrophage polarization.
CONCLUSIONS: Silencing TPX2 can inhibit the expression of M-CSF by blocking the NF-κB signal, thereby reducing CD163+ M2 macrophage polarization. The TPX2/NF-κB/M-CSF signaling axis may be involved in regulating macrophage polarization.