Molecular Mechanisms of Rotator Cuff Degeneration Identified by Spatial Transcriptomics and Multiplex Immunofluorescence.

Rotator cuff tear (RCT) is a prevalent age-related condition whose underlying mechanisms remain poorly understood. This study employed spatial transcriptomics and multiplex immunofluorescence (mIF) to investigate gene expression and spatial heterogeneity in rotator cuff tissues from elderly RCT patients compared to age-matched controls, aiming to uncover key molecular pathways. Tendon samples were collected from RCT patients (n = 10) and controls (n = 10). Five from each group underwent spatial transcriptomic sequencing for differential gene expression, functional enrichment, and cell interaction analyzes. Results were validated with mIF on the remaining samples. Compared to controls, the RCT group showed 1261 downregulated and 2789 upregulated genes. Spatial analysis revealed distinct expression gradients: COMP and CHI3L1 were upregulated in the bone region, CHI3L1 and MT1X in the mid-tendon, and MT1X and FMOD in the tendon area-confirmed by mIF. Biological processes also varied regionally: cartilage development and extracellular matrix (ECM) organization were enriched in the bone, while ECM and collagen fibril organization dominated mid-tendon and tendon regions. The PI3K-AKT and ECM-receptor interaction pathways were central to these processes. Tenogenic progenitor-like cells (TPLCs) were significantly reduced in RCT (p < 0.0001), whereas mesenchymal cells increased in bone and mid-tendon areas (p < 0.001, p < 0.01), consistent with structural gradients. These findings suggest that elderly RCT may arise from chronic inflammation, ECM dysregulation, and failed regeneration. Spatial transcriptomics identified repair-related genes (COMP, CHI3L1, MT1X, FMOD) with region-specific expression, providing new insights into pathology and potential therapeutic targets.