A novel non-invasive approach monitoring skeletal stem cell function through ¹⁸F-Pentixafor PET-CT

Fracture nonunion remains a major clinical challenge and is closely associated with dysfunction of osteogenic-lineage cells and their progenitors, skeletal stem cells (SSCs). However, conventional clinical approaches lack the specificity required to assess SSC functional status in vivo. Radiotracer-based positron emission tomography-computed tomography (PET-CT) provides an opportunity for noninvasive functional imaging of SSCs.

We first integrated RNA sequencing with FDA-approved radiotracers, to demonstrate Cxcr4 expression in murine SSCs, sorted by fluorescence-activated cell sorting (FACS), compared to non-stem skeletal cells (NSCs). Furthermore, we isolated SSCs and cultured them in vitro with CXCR4-agonist or antagonist to evaluate the role of CXCR4 in SSC differentiation and mineralization. We utilized ¹⁸F-Pentixafor, a clinically available CXCR4-targeting radiotracer, was used for in vivo PET-CT imaging to monitor SSCs in both murine models and human patients. We then generate bi-cortical femoral fracture models, take advantage of three-point bending tests and combined ¹⁸F-Pentixafor-based PET-CT to assess the correlation of ¹⁸F-Pentixafor uptake and biomechanical tests in bone regeneration. Bone phenotype of murine models was analyzed using micro-CT and histological studies. And we finally involved a case-report of a young patient suffering hamulus fracture to evaluate the role of ¹⁸F-Pentixafor-based PET imaging in assessing bone regeneration and assist in decision on clinical administration to approve the translational potency of ¹⁸F-Pentixafor-based PET imaging.

By integrating SSC RNA sequencing with FDA-approved or radiotracers in III/IV phase clinical trials, we identified CXCR4 as a promising diagnostic target and evaluated SSC activity using ¹⁸F-Pentixafor, a clinically available CXCR4-targeting radiotracer. Bulk RNA sequencing demonstrated markedly enriched Cxcr4 expression in SSCs relative to non-stem skeletal cells (NSCs), a finding supported by CXCR4 co-localization with SSC markers in fracture callus and by ¹⁸F-Pentixafor accumulation in the human growth plate. CXCR4 signaling also reflected SSC function, as CXCR4 agonist stimulation enhanced SSC differentiation and mineralization in vitro, whereas antagonists impaired these processes and delayed fracture healing in vivo. In mice, ¹⁸F-Pentixafor uptake was higher in young than aged femoral metaphyses, consistent with reduced tracer uptake in the femoral heads of aged human subjects. Compared with ¹⁸F-FDG and ¹⁸F-NaF, ¹⁸F-Pentixafor more accurately predicted biomechanical properties of fracture repair. A prospective case further demonstrated clinical feasibility in monitoring hamulus healing.

Together, these findings establish CXCR4-targeted ¹⁸F-Pentixafor PET imaging as a sensitive, non-invasive tool for visualizing SSC enrichment and function to aid in evaluating bone regeneration potential.

This study facilitates the translation of fundamental skeletal stem cell (SSC) biology into clinical decision-making by repurposing the CXCR4-targeting radiotracer ¹⁸F-Pentixafor for functional imaging of bone regeneration. We not only elucidate the role of CXCR4 in SSC function but also establish that CXCR4 as a novel target for in vivo SSC monitoring. These findings suggest that ¹⁸F-Pentixafor-based PET imaging targeting CXCR4-expressing SSCs may enable early risk stratification for fracture nonunion, improve the assessment of age-related or disease-associated impairments in bone regeneration, and support therapeutic decision-making in clinical practice.