SPATIAL TRANSCRIPTOMICS RESOLVES TISSUE-LEVEL MOLECULAR EFFECTS OF DENOSUMAB THERAPY IN A RANDOMIZED CLINICAL TRIAL FOR PERIPROSTHETIC OSTEOLYSIS

In a clinical trial we demonstrated that the human monoclonal antibody denosumab reduces bone resorption activity by ∼90% in patients undergoing revision total hip arthroplasty (THA) for osteolysis (Lancet Rheum, PMID 38279382). Here, we apply spatial transcriptomics to the bone of these trial participants to the resolve molecular events associated with this treatment. Bone biopsies taken from the prosthesis-osteolysis lesion interface at revision surgery (n=6 denosumab treated and n=6 that had received placebo) were fixed in formalin, decalcified in EDTA and embedded in paraffin wax. Spatial transcriptomics was performed using the Cytassist Visium v2 platform (10× Genomics, Pleasanton, CA), and libraries sequenced by Novogene Ltd (Cambridge, UK). The analysis pipeline was optimized using Space Ranger and Loupe Browser (10× Genomics), and Seurat v5 (Satija lab, NYU, NY) in R. The patients in both groups were of similar age. Gene expression analysis identified n=1,047 genes that were differentially expressed between the treatment groups at FDR p<0.05). Subsequent Gene Ontology (GO) analysis identified 5 key themes that were enriched between the groups at padj<1.0e-07. As expected, those involving bone mineral, collagen and actin metabolism were downregulated in the denosumab group. However, leukocyte migration and adhesion was also significantly enriched between groups. A volcano plot for this GO term showed differential regulation of 172 genes. The 5 most upregulated genes in the denosumab group were AZU1 , S100A8 , S100A9 , ITGAX and PRAM1 . All encode pro-inflammatory processes that enhance monocyte, macrophage or neutrophils at inflammatory sites by chemotaxis, increasing vascular permeability, upregulating granulocyte differentiation, and/or enhancing NFκB signalling. ITGAX , that encodes CD11c, was clearly identified on the bone surfaces, whilst the other 4 genes were mainly differentially upregulated within the bone marrow. Denosumab reduces bone resorption activity in periprosthetic osteolysis. However, in osteoporosis, cessation of treatment is associated with a rebound bone loss and fracture. These spatial transcriptomics data indicate that denosumab causes an upregulation of pro-inflammatory signalling within osteolytic bone that may set up a local permissive environment for rebound bone loss upon cessation of therapy.