PO.BCS01.01 · 生物信息与计算
整合转录组学和免疫克隆演化定义肺腺癌的逐步分子进展
Integrated transcriptomic and immune-clonal evolution defines stepwise molecular progression in lung adenocarcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：进行性免疫重塑是早期肺腺癌（LUAD）演化的一个标志，然而这一过程背后整合的转录和克隆轨迹仍未明确定义。
方法：分析了来自GSE189357和HDAC000630的33个肺样本的单细胞RNA测序（scRNA-seq）数据，涵盖从正常到浸润性腺癌。经质量控制（200<基因数<5,000；线粒体<10%）后，在Seurat中使用Harmony整合、PCA和聚类（分辨率=0.3）处理了187,880个高质量细胞。使用经典标记和CellMarker 2.0注释细胞类型。在scRepertoire中分析匹配的单细胞和批量T细胞受体（TCR）库（来自GSE164789的87个样本），以量化克隆扩增、多样性（Shannon、Simpson、D50）、重叠、CDR3长度、稀有克隆贡献以及TRBV/TRBJ基因使用。
结果：单细胞转录组学解析出15种主要细胞类型，并揭示了进行性微环境重塑。上皮、成纤维细胞和内皮谱系从非典型腺瘤样增生（AAH）到浸润性腺癌（IAC）扩增，而细胞毒性、NK和树突状细胞减少，表明免疫衰减和纤维血管主导。分期特异性转录程序显示细胞外基质组织、血管生成、免疫抑制和氧化代谢的顺序激活，捕捉了浸润的分子演化。整合的单细胞TCR分析揭示了分期特异性免疫分区化和库多样性的进行性丧失。UMAP可视化突显了晚期病变中CD8⁺细胞毒性和FOXP3⁺调节性T细胞克隆及超扩增克隆型的富集。克隆分布从正常组织中的多克隆平衡转变为IAC中的寡克隆主导。批量TCR分析证实了Shannon和逆Simpson多样性指数降低以及高频克隆型的扩增。克隆重叠分析显示病变之间共享极少，提示局部抗原驱动的应答。进行性CDR3缩短和偏向性V-J重组表明沿LUAD连续谱的克隆选择趋同。
结论：整合的单细胞转录组学和TCR分析定义了由转录重编程、基质扩增和进行性免疫限制驱动的LUAD演化统一轨迹。上皮重塑与寡克隆T细胞扩增的趋同勾勒出早期免疫瓶颈，并突显了免疫拦截的潜在治疗窗口。
查看英文原文 English abstract
Background: Progressive immune remodeling is a hallmark of early lung adenocarcinoma (LUAD) evolution, yet the integrated transcriptional and clonal trajectories underlying this process remain poorly defined.
Methods: Single-cell RNA sequencing (scRNA-seq) data from GSE189357 and HDAC000630 were analyzed across 33 lung samples, spanning normal to invasive adenocarcinoma. After quality control (200 < genes < 5,000; mitochondrial < 10%), 187,880 high-quality cells were processed in Seurat with Harmony integration, PCA, and clustering (resolution = 0.3). Cell types were annotated using canonical markers and CellMarker 2.0 . Matched single-cell and bulk T-cell receptor (TCR) repertoires (87 samples from GSE164789 ) were analyzed in scRepertoire to quantify clonal expansion, diversity (Shannon, Simpson, D50), overlap, CDR3 length, rare-clone contribution, and TRBV/TRBJ gene usage.
Results: Single-cell transcriptomics resolved 15 major cell types and revealed progressive microenvironmental remodeling. Epithelial, fibroblast, and endothelial lineages expanded from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma (IAC), whereas cytotoxic, NK, and dendritic cells declined, indicating immune attenuation and fibrovascular dominance. Stage-specific transcriptional programs showed sequential activation of extracellular-matrix organization, angiogenesis, immune suppression, and oxidative metabolism, capturing the molecular evolution of invasion. Integrated single-cell TCR analysis revealed stage-specific immune compartmentalization and progressive loss of repertoire diversity. UMAP visualization highlighted enrichment of CD8⁺ cytotoxic and FOXP3⁺ regulatory T-cell clones with hyperexpanded clonotypes in advanced lesions. Clonal distributions shifted from polyclonal equilibrium in normal tissue to oligoclonal dominance in IAC. Bulk TCR profiling confirmed reduced Shannon and inverse Simpson diversity indices and expansion of high-frequency clonotypes. Clonal overlap analysis demonstrated minimal sharing across lesions, suggesting localized antigen-driven responses. Progressive CDR3 shortening and biased V-J recombination indicated convergent clonal selection along the LUAD continuum.
Conclusions: Integrated single-cell transcriptomic and TCR analyses define a unified trajectory of LUAD evolution driven by transcriptional reprogramming, stromal expansion, and progressive immune restriction. The convergence of epithelial remodeling with oligoclonal T-cell expansion delineates early immunologic bottlenecks and highlights potential therapeutic windows for immune interception.
利益披露 Disclosure
K. Qin, None..
A. Qin, None.