Macrophage-derived Spp1 promotes intramuscular fat in dystrophic muscle

JCI Insight · 2025-07-07 · 10 citations

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder involving cycles of muscle degeneration and regeneration, leading to accumulation of intramuscular fibrosis and fat. Ablation of Osteopontin/Spp1 in a murine model of DMD (mdx) improves the dystrophic phenotype, but the source of Spp1 and its impact on target cells in dystrophic muscles remain unknown. In dystrophic muscles, macrophages are the predominate infiltrating leukocyte and express high levels of Spp1. We used macrophage-specific ablation combined with single-cell transcriptional profiling to uncover the impact of macrophage-derived Spp1 on cell-cell interactions in mdx muscles. Ablation of macrophage-specific Spp1 (cKO) correlated with reduction of 2 PDGFRa+ stromal cell populations, expressing Lifr+ and Procr+. Sorting and transcriptional profiling of these populations confirmed that they are enriched in adipogenesis genes and are highly related to fibroadipogenic precursors (FAPS). These adipogenic stromal cells (ASC) displayed more adipogenic potential in vitro compared with FAPS, likely due to a more differentiated state. Reduction of ASCs correlated with reduced intramuscular diaphragmatic fat and improved diaphragm function. These data suggest a role for myeloid-derived Spp1 in the differentiation of stromal cells towards an adipogenic fate, leading to accumulation of intramuscular fat in dystrophic muscles.

Preclinical evaluation of high-resolution CT, 18F-FDG, and 18F-NaF PET imaging for longitudinal monitoring of atherosclerosis

European Journal of Nuclear Medicine and Molecular Imaging · 2025-04-27 · 3 citations

Abstract Rationale Detection of atherosclerosis is essential to the management and prevention of life-threatening cardiovascular events. Although non-invasive imaging modalities, such as 18 F-sodium fluoride ( 18 F-NaF), 18 F-fluorodeoxyglucose ( 18 F-FDG) PET, and CT, visualize distinct hallmarks of atherosclerosis, there has yet to be a singular multi-cohort interrogation of their strengths and limitations. Thus, we focused on identifying the optimal approach for visualizing atherosclerosis at different stages of disease progression. Methods In this study, 6-week-old, male, ApoE deficient mice ( Apoe −/− ) were placed on a high cholesterol diet for 12–20 weeks to induce calcific atherosclerotic disease. Age-matched, male, wildtype (WT) C57BL/6 mice fed with regular chow served as the control group. Mice were imaged at 12, 15, 18, and 20 weeks after starting their respective diets. To follow the progression of calcified atherosclerotic lesions, at each time point, in vivo , 18 F-NaF microPET/CT images were acquired 1 h and 3 h post tracer i.v. injection. In a separate cohort, in vivo 18 F-FDG PET/CT images were acquired at 3 and 5 h post tracer i.v. injection to follow inflammation as a result of progressive atherosclerotic lesion formation. High-resolution microCT images were acquired for all mice to visualize aorta calcification. After each imaging session, a subset ( n = 3) was euthanized from each group and histological analysis of the aorta was performed to confirm disease progression. Results In this comparative study, within the same cohort, 18 F-NaF PET detected atherosclerotic calcification earlier than microCT. At both 1 and 3 h post-injection (p.i.), calcified lesions were clearly detected by 18 F-NaF with a six-fold higher signal in Apoe -/- compared to WT mice. Interestingly, 18 F-NaF signal peaked at week 18, whereas aortic CT signal progressively increased with a 13-, 16-, and 29-fold at 15, 18, and 20 weeks, respectively. 18 F-FDG arortic accumulation at weeks 12 and 15, were significantly greater in Apoe −/− mice than WT control when images were acquired at 5 h but not at 3 h p.i.. In contrast to histological analysis, at ≥ 16 weeks where inflammation is significantly elevated, 18 F-FDG was equivalent in Apoe −/− and WT control mice and significantly reduced with disease progression. Conclusions Our results show that 18 F-NaF PET and 18 F-FDG PET are sensitive imaging modalities for the early detection of atherosclerotic lesions. However, both 18 F-NaF PET and high-resolution microCT prove to be effective methods for monitoring late-stage and progressive disease.

Advancing adeno-associated virus for Duchenne muscular dystrophy treatment: Moving beyond rodent models

Molecular Therapy — Methods & Clinical Development · 2025-04-25

Supplemental Tables from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<p>Supplemental Tables</p>

Figure S3 from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<p>S3. EMP2 expression in cancer cell lines. A. Based on public repositories, upregulation of EMP2 occurs in breast, brain, and ovarian cancers, while downregulation is predicted in hematopoietic cancers. B, C. Flow cytometry studies using a panel of human (B) and murine (C) cancer cell lines to validate differential EMP2 expression.</p>

Figure S5 from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<p>S5. Binding characteristics. Analysis of the various anti-EMP2−DFO conjugates using flow cytometry binding on U87MG/EMP2FLuc. N=3, with the EC50 determined using nonlinear regression. B. 10:1 DFO:mAb ratio retains specific binding to EMP2 high (4T1FLuc, CT26) murine cell lines compared to low cells (Panc02). N=3, with a representative experiment shown.</p>

Data from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<div>Abstract<p>Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for mAb-based therapy. In the current study, image-guided therapy for an anti-EMP2 mAb was evaluated by PET in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2-expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with <sup>89</sup>Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2-positive tumors within 24 hours after injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, and U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02 and Ramos). Biodistribution at 5 days after injection revealed that the tumor uptake ranged from 2 to approximately 16%ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 Ab fragments may be useful to track EMP2-positive tumors for subsequent therapeutic interventions.</p></div>

Figure S3 from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<p>S3. EMP2 expression in cancer cell lines. A. Based on public repositories, upregulation of EMP2 occurs in breast, brain, and ovarian cancers, while downregulation is predicted in hematopoietic cancers. B, C. Flow cytometry studies using a panel of human (B) and murine (C) cancer cell lines to validate differential EMP2 expression.</p>

Figure S6 from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<p>S6. Pharmacokinetics and in vivo tumor expression. A. Tumor growth of syngeneic 4T1FLuc, CT26, and Panc02 tumors over the imaging period. B. Correlation of the average EMP2 histological score and mean %ID/cc in 4T1FLuc, CT26 and Panc02 tumors. C. Representative staining of EMP2 in 4T1FLuc, CT26 and Panc02 tumors. Magnification: 100m. D. Pharmacokinetics of the anti-EMP2 mAb as measured by western blot detection of the anti-Fc portion of the mAb. Mice were injected with 200 g or 20 g of the anti-EMP2 mAb. Cmax was defined by quantitation at t=2 hrs with the PK estimated at 140 hrs.</p>

Supplementary Data from <sup>89</sup>Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors

2024-06-04

<p>Supplemental Figure Legends</p>