0026: Spatial Proteomic-based Phenotyping of Muscle Stem Cells and their Niches in Myositis

Background/Purpose: Immune-mediated inflammatory myopathies (IIMs) are a heterogeneous group of diseases characterized by chronic inflammation, damage and impaired regeneration of the skeletal muscle leading to progressive muscle weakness. Muscle stem cells (MuSCs) play a key role in muscle regeneration, together with other myogenic and non-myogenic stromal cells. However, the exact mechanisms of impaired skeletal muscle regeneration in IIMs are incompletely understood.

Methods: We applied imaging mass cytometry (IMC) to explore the heterogeneity of MuSCs at the single-cell level and characterize cellular alterations of MuSC niches in situ in muscle biopsies of IIM patients. Muscle biopsies of patients with dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), and control individuals (CO) were analyzed.

Results: We analyzed a total of 69288 cells, including 57623 stromal cells, of which 14595 MuSCs, and identified 17 stromal cell subpopulations, including four subpopulations of MuSCs. We observed IIM-subtype-specific changes in the frequencies of MuSC subpopulations. CD56^hi;CD90^hi;PAX7^low MuSCs were particularly upregulated in PM and DM, while CD82^hi;PAX7^low MuSCs were mostly upregulated in IBM muscle biopsies. GLI1^hi;PAX7^hi MuSCs were upregulated only in PM patients and CD82^neg;PAX7^low MuSCs were not changed in frequency compared with CO. Each MuSC subpopulation was characterized by a unique spatial niche. Two MuSC niches were enriched for distinct stromal cells (CD82^hi;PAX7^low and CD82^neg;PAX7^low MuSC niches) and the other two (CD56^hi;CD90^hi;PAX7^low and GLI1^hi;PAX7^hi MuSC niches) for distinct subsets of immune cells. The CD82^hi;PAX7^low MuSC niche was composed of LECs, while the CD82^neg;PAX7^low MuSC niche was composed of ECs and muscle fibers. The CD56^hi;CD90^hi;PAX7^low MuSC cellular neighborhood (CN) was enriched in NK cells, HLA-DR^- M2 macrophages, and regenerating muscle fibers, while the GLI1^hi;PAX7^hi MuSC CN was enriched in HLA-DR⁺ M2 macrophages, HLA-DR⁺ CD20⁺ B cells, HLA-DR⁺ CD28^- T cells, and helper T cells. Furthermore, we identified IIM-subtype-specific perturbations in the cellular interaction network of MuSC populations. The frequencies of MuSC subpopulations correlated with standard histopathological measures of muscle regeneration.

Conclusion: Using spatial proteomics, we unraveled the heterogeneity of MuSCs in DM, PM and IBM patients and identified IIM-subtype-specific changes in the frequencies of the distinct MuSC subpopulations together with unique perturbations in their local niches and cellular interactions. These data might offer potential for therapeutic modulation of individual MuSC subpopulations in IIM, aiming to improve skeletal muscle regeneration and thus combat disabling muscle weakness.