Spatial metabolomics integrated with multi-omics analysis reveals the HK3-lactate axis as a potential mechanism in ligamentum flavum ossification

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Ossification of the ligamentum flavum (OLF) is a degenerative spinal disorder characterized by aberrant endochondral ossification and is a major cause of thoracic spinal stenosis. Current treatment still relies mainly on surgical decompression, which relieves mechanical compression but does not halt the biological processes underlying ossification. Although growing evidence implicates metabolic reprogramming in ectopic ossification and osteochondral differentiation, the key metabolites driving OLF and their proximal upstream regulators remain unclear. Spatial metabolomics was performed on clinical thoracic ligamentum flavum specimens to identify lesion-associated metabolic alterations. Untargeted metabolomics and proteomics were then integrated with bioinformatic and machine-learning analyses to screen for key metabolites and upstream effectors. Public single-cell transcriptomic data were further incorporated to define the cell-state specificity of candidate molecules. Finally, human OLF tissues and an in vitro OLF model were used for functional validation through loss-of-function and rescue experiments, together with molecular assays. Spatial metabolomics clearly separated ossified, non-ossified, and control regions, and identified L-lactate as a core hub metabolite selectively enriched in ossified foci. Lactate-associated proteins and metabolites were enriched in translation, mRNA splicing, secretion, extracellular matrix remodeling, amino acid metabolism, glucose homeostasis, and lipid biosynthesis, indicating a metabolic state characterized by enhanced aerobic glycolysis, active anabolic-secretory programs, and accelerated tissue remodeling. Machine-learning analyses consistently identified HK3 as the glycolytic enzyme most strongly associated with tissue lactate abundance and significantly upregulated in OLF tissue. Single-cell analysis further showed that HK3 was mainly enriched in chondrocyte-like ligamentum flavum cells and closely linked to glycolytic activation and chondrogenic transition. Cross-omics integration highlighted HIF-1 signaling as a convergent upstream pathway activated in ossified lesions. Functionally, HK3 knockdown reduced lactate production and attenuated ossification marker expression, whereas exogenous lactate partially rescued these effects. In addition, HIF-1α inhibition reduced HK3 expression, whereas lactate supplementation partially restored HIF1A expression following HK3 silencing, suggesting a potential reciprocal relationship between HIF-1 signaling and HK3-dependent lactate production. Local lactate accumulation is a key metabolic feature of OLF. The HK3-lactate axis may contribute to OLF progression and may represent a potential metabolic target for future non-surgical strategies.

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