Decoding Lymphangioleiomyomatosis (LAM) Niche Environment via Integrative Analysis of Single Cell Multiomics and Spatial Transcriptomics

Abstract Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease characterized by cystic destruction and metastatic growth of smooth muscle-like cells. Despite advances in understanding its genetic basis, the cellular heterogeneity, regulatory mechanisms, and microenvironmental interactions driving LAM progression remain poorly defined. In this study, we employed an integrative multi-omics approach combining single-cell RNA sequencing (scRNA-seq), single-nucleus ATAC sequencing (snATAC-seq), and high-resolution spatial transcriptomics (Visium, Visium HD, and Xenium) to decode the LAM niche in its native environment. We identified two spatially and functionally distinct LAM subtypes: LAMCORE1 and LAMCORE2. LAMCORE1 cells exhibited a uterine smooth muscle-like phenotype, expressing associated markers (ACTA2, MYH11) and were enriched in MTORC1 signaling and myogenic pathways, supporting a uterine origin. In contrast, LAMCORE2 cells displayed fibroblast-like features, with upregulated extracellular matrix (ECM) remodeling genes (COL1A1, MMP11) and epithelial-to-mesenchymal transition (EMT) pathways, suggesting a role in niche formation. Pseudotime and regulon analyses revealed dynamic transitions between these subtypes, driven by distinct transcriptional networks (e.g., HOX/PBX in LAMCORE1, TWIST/ZEB in LAMCORE2). The presence of the two distinct LAM subtypes was further validated by RNAscope and immunofluorescence microscopy. We identified LAM-associated fibroblasts (LAFs) as activated stromal cells expressing canonical markers (FAP, S100A4, VIM, IGFBP7, SPARC) and localized within LAM lesions. Subpopulations of LAFs, LAF-seed (proximal to LAMCORE1) and LAF-niche (surrounding LAM niches), exhibited unique functional profiles, including ECM deposition, TGF-β signaling, and myofibroblast activation. Regulatory network analysis pinpointed EGR1 as a central hub governing LAF phenotype. Our comprehensive spatial profiling revealed niche structures dominated by LAMCORE1 cells and surrounded by lymphatic endothelial cells (LECs), LAFs, scattered LAMCORE2 cells, macrophages, and reprogrammed alveolar epithelial cells (AT2). ECM remodeling and aberrant organization of cable-like structures (α-smooth muscle actin+) of the LAM niches were further validated by second harmonic generation microscopy. These findings provide a high-resolution blueprint of LAM pathogenesis, highlighting the interplay between uterine-derived LAMCORE cells, activated fibroblasts, and the remodeled lung microenvironment. They significantly enhance our understanding of the LAM niche microenvironment and offer insights into potential therapeutic targets and strategies for managing this complex disease. Competing Interest Statement The authors have declared no competing interest.