Targeting inflammatory and angiogenic responses by small-molecule kinase inhibitors in a 3D macrophage-containing spheroid model of rheumatoid arthritis synovial tissue

Abstract Background In rheumatoid arthritis (RA), interactions between macrophages, fibroblast-like synoviocytes (FLS), and endothelial cells (ECs) drive synovial tissue (ST) inflammation and pathological angiogenesis. Preclinical models reflecting this crosstalk are needed to evaluate novel therapies. We developed a human 3D model of RA ST to assess inflammatory mechanisms and responses to small-molecule kinase inhibitors. Methods Polarized M1-like or M2-like macrophages were co-cultured with RAFLS and ECs to generate spheroids embedded in a collagen-based scaffold. Spheroids were unstimulated or exposed to pro-inflammatory stimuli, including tumor necrosis factor-α (TNF) or RA synovial fluid (SF). Treatment effects of tofacitinib (JAK1/3 inhibitor) and an IKKβ inhibitor (IKKβi; targeting NF-κB signaling) were evaluated. Readouts included spheroid outgrowth and macrophage distribution quantified by semi-automated image analysis ( n  = 8), cytokine production measured by ELISA/Luminex ( n  = 8), and RNA sequencing to identify differentially expressed genes ( n  = 4). Results M1-like macrophages exhibited higher core retention, while M2-like macrophages showed increased outward migration ( p  p  p  p  p  p   Conclusions This human 3D multicellular in vitro spheroid model of synovial inflammation recapitulates key RA pathological processes and provides a robust platform for mechanistic studies and therapeutic evaluation.