背景:肺癌是世界上死亡率最高的恶性肿瘤,从而在降低患者死亡率方面提出了重大的全球挑战。在肿瘤学领域,靶向免疫疗法已成为肺癌的一种新的治疗方法。本研究旨在通过分析铁凋亡指数(FPI)和单细胞RNA测序(scRNA-seq)数据,探索肺腺癌(LUAD)免疫治疗的潜在靶标。这项研究的结果可能为改善LUAD免疫治疗策略和指导临床决策提供有价值的见解。
方法:首先,通过FPI分析了LUAD患者的生存率与铁凋亡之间的关系。随后,通过免疫浸润分析和相关统计,探讨了铁凋亡与免疫细胞浸润和调节的关系。最后,通过GSEA和GSVA分析LUAD患者主要浸润免疫细胞群及其相关通路与预后的关系。筛选出调节免疫细胞群浸润的核心基因,下载了LUAD患者的癌症和癌旁组织的scRNA-seq数据,其次是细胞聚类分析,核心亚群的细胞鉴定,伪时间分析,单细胞GSVA和途径富集分析,核心调控基因的鉴定和功能分析。此外,免疫组化法检测LUAD组织芯片中核心功能基因的表达水平,并验证其与LUAD患者预后的关系。最后,我们使用慢病毒与WDFY4转染LUADA549细胞。CCK-8,流式细胞术,凋亡检测,划痕伤口愈合试验,Transwell迁移试验,移植裸鼠模型,采用免疫组织化学分析等实验方法探讨WDFY4对LUAD的体内外生物学效应。
结果:LUAD患者FPI值的生存分析显示,较小的FPI值与较长的总生存期呈正相关。免疫浸润分析及其与FPI值的相关性表明,B细胞与铁死亡的相关性最强。癌细胞的铁凋亡可以促进B细胞群的浸润和活化,而B细胞浸润较多的LUAD患者有较长的长期生存期。scRNA-seq数据分析表明B细胞群是LUAD中免疫细胞浸润的主要细胞群之一。在LUAD的B细胞分化后期,ACAP1、LINC00926、TLR10、MS4A1、WDFY4和TRIM22基因的表达水平下降,而TMEM59,TP53INP1和METTL7A基因的表达水平升高。蛋白质-蛋白质相互作用(PPI)网络分析表明,WDFY4在调节LUAD的B细胞分化中起着至关重要的作用。LUAD组织微阵列的免疫组织化学分析显示WDFY4表达显著下调,与LUAD的发生部位密切相关。此外,低WDFY4表达的LUAD患者表现出较差的预后。此外,实验结果表明,WDFY4的过表达可以抑制A549细胞的增殖和转移,同时促进细胞凋亡。还证实WDFY4可以在体内抑制癌症生长。
结论:结果表明,促进B细胞群的浸润和活化可以提高LUAD患者的长期生存率,从而为LUAD提供了一种潜在的新型免疫治疗方法。此外,已证明促进癌细胞的铁凋亡和WDFY4表达的上调可诱导B细胞群的浸润和活化。此外,WDFY4的过表达能显著抑制肺腺癌的体内外生长,突出了其作为LUAD免疫治疗靶标的潜力。
BACKGROUND: Lung cancer exhibits the world\'s highest mortality rate among malignant cancers worldwide, thereby presenting a significant global challenge in terms of reducing patient mortality. In the field of oncology, targeted immunotherapy has emerged as a novel therapeutic approach for lung cancer. This study aims to explore potential targets for immunotherapy in lung adenocarcinoma (LUAD) through the analysis of Ferroptosis Index (FPI) and Single Cell RNA-Sequencing (scRNA-seq) data. The findings of this research can potentially offer valuable insights for improving LUAD immunotherapy strategies and informing clinical decision-making.
METHODS: Firstly, the relationship between survival and ferroptosis in LUAD patients was analyzed by FPI. Subsequently, the association between ferroptosis and infiltration and regulation of immune cells was explored by immune infiltration analysis and correlation statistics. Lastly, the relationship between major infiltrating immune cell populations and related pathways and prognosis of LUAD patients was analyzed by GSEA and GSVA. To screen out core genes regulating infiltration of immune cell populations, scRNA-seq data of cancer and para-cancerous tissues of LUAD patients were downloaded, followed by cell clustering analysis, cell identification of core subpopulations, pseudotime analysis, single-cell GSVA and pathway enrichment analysis, and identification and functional analysis of core regulatory genes. Moreover, the expression levels of core functional genes in LUAD tissue microarray were detected by immunohistochemistry, and its relationship with the prognosis of LUAD patients was verified. Finally, we used lentivirus with WDFY4 to transfect LUAD A549 cells. CCK-8, flow cytometry apoptosis detection, Scratch wound healing assay, Transwell migration assay, Xenograft nude mice model, immunohistochemical analysis and other experimental methods were used to explore the biological effects of WDFY4 on LUAD in vitro and in vivo.
RESULTS: Survival analysis of FPI values in LUAD patients revealed a positive correlation between smaller FPI values and longer overall survival. Immuno-infiltration analysis and its correlation with FPI values revealed that B cells were most strongly associated with ferroptosis. Ferroptosis of cancer cells could promote infiltration and activation of B cell populations, and LUAD patients with more infiltration of B cell populations had longer long-term survival. scRNA-seq data analysis indicated that the B cell population is one of the major cell populations infiltrated by immune cells in LUAD. During the later phases of B cell differentiation in LUAD, there was a decrease in the expression levels of ACAP1, LINC00926, TLR10, MS4A1, WDFY4, and TRIM22 genes, whereas the expression levels of TMEM59, TP53INP1, and METTL7A genes were elevated. The protein-protein interaction (PPI) network analysis indicated that WDFY4 plays a crucial role in regulating B cell differentiation in LUAD. Immunohistochemical analysis of LUAD tissue microarray revealed a significant downregulation of WDFY4 expression, which was closely related to the occurrence sites of LUAD. Moreover, LUAD patients with a low WDFY4 expression exhibited a poorer prognosis. Additionally, experimental findings demonstrated that the overexpression of WDFY4 could inhibit the proliferation and metastasis of A549 cells while promoting apoptosis. It was also confirmed that WDFY4 could inhibit cancer growth in vivo.
CONCLUSIONS: The results indicate that promoting infiltration and activation of B cell populations could improve the long-term survival of LUAD patients, thereby offering a potential novel immunotherapeutic approach for LUAD. Besides, the promotion of cancer cell ferroptosis and upregulation of WDFY4 expression have been shown to induce the infiltration and activation of B cell populations. Furthermore, the overexpression of WDFY4 can significantly inhibit the growth of lung adenocarcinoma in vitro and in vivo, highlighting its potential as a target for immunotherapy in LUAD.