Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs. Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen. Targeted antigen showed a surface antigen expression pattern, and the 43–55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs. ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.

中文翻译：

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被鉴定为单克隆抗体12C7抗原的α-烯醇酶（ENO1）通过AMPK / mTOR途径促进肺癌干细胞的自我更新和恶性表型

肿瘤相关抗原（TAA）可以靶向治疗癌症。我们之前鉴定了一种单克隆抗体（mAb）12C7，该抗体在肺癌干细胞（LCSC）中具有抗肿瘤活性。在这里，我们旨在鉴定12C7的靶抗原，并确认其在LCSC中的作用。免疫荧光用于抗原定位。通过电泳和免疫印迹进行靶向抗原纯化后，通过LC-MALDI-TOF / TOF质谱，免疫荧光和免疫沉淀法鉴定抗原。慢病毒转导诱导ENO1的过表达或沉默。LCSC的自我更新，生长和侵袭分别通过球体形成，菌落形成和侵袭分析进行评估。进行高通量转录组测序（RNA-seq）和生物信息学分析，以分析下游靶标和靶向抗原的途径。靶向抗原显示表面抗原表达模式，并且43-55 kDa蛋白带被鉴定为α-烯醇化酶（ENO1）。ENO1下调显着抑制LCSC的自我更新，生长和侵袭能力，而ENO1上调则增强了它们的自我更新，生长和侵袭能力。RNA-seq和生物信息学分析最终筛选了4个自我更新相关和6个入侵相关差异表达的基因。GSEA分析和qRT-PCR证实ENO1调节自我更新，入侵相关基因和途径。KEGG通路分析和免疫印迹表明，ENO1激活了LCSC中的AMPK通路和mTOR通路。ENO1被鉴定为mAb 12C7的靶向抗原，在促进LCSC的自我更新，生长和侵袭中起关键作用。这些发现为肺癌的干细胞治疗提供了有效的治疗靶点，并且具有改善12C7的抗肿瘤活性的潜力。