單細胞與空間轉錄體學揭示肺腺癌中腫瘤相關巨噬細胞的異性及其與癌症相關成纖維細胞之鄰近交互作用

Abstract

肺腺癌（LUAD）是肺癌中上普遍的一種亞型，其特徵是具有高度分子變異性佮病理生長型態的異質性。腫瘤相關巨噬細胞（TAMs）是腫瘤微環境（TME）中重要的細胞組成，會透過代謝重編程、免疫抑制以及細胞外基質重塑，展現促進腫瘤發展的功能，進一步形成纖維化的腫瘤環境佮治療抗性。然而，TAM 的起源多樣性、可塑性，以及佢佇腫瘤進程中如何驅動促腫瘤表型的機制，猶原無完全被釐清。 為著補足這个研究缺口，本研究整合公開資料庫中的單細胞轉錄體資料佮空間轉錄體資料，系統性分析 LUAD 中 TAM 的亞群組成、空間分佈佮細胞間互動，並評估其臨床相關性。 偽時間分析顯示，巨噬細胞主要呈現兩條分化軌跡：一條是佔據穩定分支的組織常駐型肺泡巨噬細胞（TRAMs），另一條則包含可能源自胚胎期巨噬細胞及循環單核球的 TAM 亞群。功能分析結果指出，TRAMs 主要存在佇正常肺組織中，負責維持組織恆定佮免疫監控；相對之下，腫瘤相關的 TAM 亞群則表現出適應低氧環境、促進血管新生佮細胞外基質重塑等促腫瘤特徵，與纖維化活性密切相關。 其中，SPP1 作為一種基質相關蛋白，主要高表現佇腫瘤富集的 TAM 亞群中，並與腫瘤惡性進展相關。進一步的細胞互動分析佮空間定位結果顯示，SPP1⁺TAMs 與癌症相關纖維母細胞（CAFs）會共同聚集佇腫瘤與基質交界區，形成支持促腫瘤發展的訊號網絡。存活分析進一步證實，高 SPP1 表現量以及 SPP1⁺ TAMs 與 CAFs 的共富集程度，皆與 LUAD 病患較差的臨床預後顯著相關。 總結來講，本研究指出 SPP1 可作為 TAM 亞群的重要代表性標記，並參與調控巨噬細胞佇低氧、血管新生及促纖維化腫瘤環境中的適應能力。SPP1⁺ TAMs 與 CAFs 佇腫瘤基質介面的空間共富集，是 LUAD 腫瘤生成過程中關鍵的細胞交互作用，亦與不良預後密切相關。本研究透過分子層級佮空間層級的整合分析，深化對 TAM–CAF 互動機制的理解，並為未來發展以 SPP1 為標的的治療策略，提供重要的研究基礎。

Lung adenocarcinoma (LUAD) is the one of most predominant form of lung cancer, exhibiting by heterogeneity in molecular alterations and pathological growth patterns. Tumor-associated macrophages (TAMs) represent a key cellular component in the tumor microenvironment (TME), exhibiting pro-tumor functions through metabolic reprogramming, immunosuppression, and extracellular matrix (ECM) remodeling, thereby promoting a profibrotic environment and therapeutic resistance. However, the diversity and plasticity of TAM ontogeny and the mechanisms by which they drive pro-tumor phenotypes during tumor progression remain incompletely understood. Addressing this knowledge gap, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) data from the Gene Expression Omnibus (GEO) datasets, aimed to characterize TAM subpopulations, map their spatial distribution and interaction, and evaluate their clinical relevance in LUAD. Pseudotime analysis identified two main differentiation trajectories: Tissue-resident alveolar macrophages (TRAMs) occupying a stable branch, alongside TAM subsets likely derived from both embryonic macrophages and circulating monocytes. Functional profiling revealed that TRAMs predominate in normal lung tissues largely maintain signatures of tissue homeostasis and immune surveillance functions; in contrast, TAM subpopulations display pro-tumorigenic phenotypes via hypoxia adaptation, angiogenesis, and ECM remodeling, consistent with profibrotic activity. Notably, SPP1 (Secreted Phosphoprotein 1), a matricellular protein related to epigenetic changes and reduced drug uptake, was dominantly expressed on tumor-enriched TAM subtypes. Cell-cell communication and spatial mapping detected the colocalization of SPP1+TAMs and cancer-associated fibroblasts (CAFs) at the tumor-stromal boundaries, with ligand–receptor signaling patterns that support pro-tumorigenic networks. Finally, survival analyses further validated that high SPP1 expression and higher inferred SPP1+TAMs-CAFs co-abundance associate with worse survival outcomes in LUAD. In conclusion, this study identified that SPP1 serves as a representative marker on TAM subpopulations, reprogramming the adaptive capability of macrophages in hypoxic, angiogenic, and pro-fibrotic tumor environ ments. The spatial co-enrichment of SPP1+TAMs and CAFs at the tumor-stromal interfaces represents a key cellular interaction in LUAD tumorigenesis linked to worse prognosis. The molecular profiling and advanced spatial analysis expand the insight of TAM-CAF crosstalk,and offer future exploration for SPP1-targeted therapies to counteract therapeutic resistance and enhance survival outcomes. Keywords: lung adenocarcinoma (LUAD), tumor microenvironment (TME), tumor-associated macrophages (TAMs), tissue-resident macrophage (TRAMs), single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST), secreted phosphoprotein 1 (SPP1), cancer-associated fibroblasts (CAFs).