A MULTIMODALITY BASED IMAGING APPROACH USING A NOVEL RADIOTRACER, [18F]-PYGAL TO DETECT SENESCENCE IN SMALL AND LARGE ANIMAL MODELS
2023-01-01

Cellular senescence has been implicated in age-related pathophysiologies, including osteoarthritis (OA). The targeted removal of senescent cells can ameliorate the development of OA. To advance our understanding of the role of senescent cells in OA and monitor novel senolytic therapies, there is a pressing need for imaging biomarkers that can detect senescence in arthritic joints. Senescent cells overexpress β-galactosidase. Our objective was to detect senescent cells in knee joints of small (mice) and large (pigs) animal models using a novel β-galactosidase-based radiotracer, [18F]-PyGal, using PET imaging. We hypothesized that senescent cells will demonstrate increased [18F]-PyGal radiotracer uptake compared to viable cells, in vitro and in vivo. Triplicate samples of murine primary chondrocytes incubated with 400 nM doxorubicin (doxo) to induce senescence, followed by incubation with 20 μM [18F]-PyGal radiotracer for one hour and PET-CT imaging. Non-doxo-exposed cells served as controls. Next, senescence was induced in the left knee joint of 30 C57BL/6 mice (5, 12, and 23-month-old mice, both sexes) by intra-articular doxorubicin injection. The contralateral knee served as control. All knees underwent PET/CT and PET/MRI imaging at 1 hour after intravenous injection of 250uCi [18F]-PyGal. The radiotracer signal of senescent and viable joints was measured using operator defined regions of interest (ROI; %ID/g) and compared with a student's t-test. Next, senescent mesenchymal stromal cells (MSC) or viable controls were implanted into twelve cartilage defects of six knees of three Yucatan pigs. 24 hours later, 250uCi [18F]-PyGal was injected into the knee joint, followed by integrated PET-MRI. The standardized uptake value (SUV) of senescent and viable cell implants was compared with a t-test. IL-6, CXCL5 and β-galactosidase immunostains served as reference standards for all experiments. Compared to untreated controls, doxorubicin-exposed chondrocytes demonstrated significantly increased expression of IL6 (Control: 18.39±5.10 pg/mL; senescence 58.85±1.43pg/mL, p<0.005), CXCL5 (Control 153.8162±38.14pg/mL, senescence:1163.24±169.09pg/mL p<0.005) and beta-gal (Control 9.16±0.7%, Senescence 90±2%, p<0.0005). Senescent cells exhibited a 2.28-fold higher uptake of [18F]-PyGal compared to controls (Control 1754±247.22, Senescence 3704.23±570.76, p=0.005). In 12-month-old mice, senescent knees demonstrated significantly higher retention of [18F]-PyGal (0.47±0.18 %ID/g, 0.47±0.12 %ID/g), compared to control knees (0.22±0.03%ID/g, 0.29±0.08, p=0.02, p=0.03 female and male respectively). Accordingly senescent knees demonstrated significantly increased p-16 staining, p-21 staining, and β-gal staining compared to normal controls. In the large animal model, cartilage defects implanted with senescent cells showed significantly higher [18F]-PyGal uptake (39.835±7.3 SUVmax) compared to control knees (19.69±0.69 SUVmax, p<0.05). Histopathologic correlations of large animal studies are ongoing. [18F]-PyGal can detect senescent cells in knee joints of small (mice) and large (pigs) animal models using a novel β-galactosidase-based radiotracer, [18F]-PyGal and PET imaging. Senescent cells demonstrated significantly increased [18F]-PyGal radiotracer uptake compared to viable cells, in vitro and in vivo, in small and large animal models of OA.