Op0075 Molecular Insights Into Fapi Imaging-Based Activation of Synovial Fibroblasts Preceding the Onset of Arthritis

Background: Synovial fibroblasts play a crucial role in shaping the joint microenvironment and actively contribute to the initiation and progression of inflammatory arthritis 1. Notably, distinct subsets of fibroblast activation protein (FAP)-positive synovial fibroblasts have been implicated in inflammation and bone erosion as well as resolution of inflammation 2. Recent studies using 68Ga-FAPI-04 PET-CT revealed elevated FAPI tracer uptake in psoriasis patients, indicating an increased risk of developing psoriatic arthritis and correlating with disease progression and joint damage 2, 3. Despite these findings, our understanding of FAP activation during the early stages of arthritis and its underlying regulatory mechanisms remains incomplete. Objectives: This study aimed to assess the potential of FAPI imaging in investigating early stromal remodeling during inflammatory arthritis unraveling the regulatory mechanism of early FAP activation and its role in inflammatory arthritis. Methods: We utilized Alexa Fluor 647 (AF647)-conjugated FAPI-04 to evaluate specific cell uptake in vitro via flow cytometry and in vivo using light sheet microscopy (LSFM). In vivo assessments of leukocyte infiltration were conducted with antibody-conjugated fluorophores (CD45, Ly6G). Cellular behavior was monitored over time through LSFM and flow cytometry. FAP+ synovial fibroblasts were sorted before arthritis onset and at peak inflammation, followed by single-cell RNA sequencing (scRNAseq). Results: FAPI-04-AF647 showed selective uptake by FAP-expressing cells both in vitro and in vivo. Early FAP upregulation was detected following STA modeling, prior to leukocyte infiltration. FAPI-04 uptake levels were comparable to those observed at the peak of inflammation. To elucidate whether serologic or cellular components induce early stage FAPI uptake, synovial fibroblasts were incubated with fresh or heat-inactivated KBxN serum. Heat inactivation completely abolished FAPI uptake, indicating that serological components induce fibroblast transition. Subsequently, fibroblasts were stimulated with various cytokines including complement factors, IL-6, TGFβ, and TNFα. As we did not observe unifactorial FAPI-04 uptake, scRNAseq was performed with sorted fibroblasts from joints at an early stage of disease before leukocyte infiltration occurred. Here we observed multifactorially induced FAPI-04 uptake associated with upregulation of the chemokines C-X-C motif chemokine ligand 2 (CXCL2), CXCL12 and CXCL13 and significantly altered subset characteristics distinct from fibroblast subsets at the peak of inflammation. Conclusion: Our findings highlight that FAPI-04 uptake occurs in inflammatory arthritis at early stages before leukocyte infiltration, emphasizing the pivotal role of fibroblasts throughout arthritis progression. Fibroblasts may act as sentinels of inflammation during the early stages, detectable by FAPI imaging. The identification of subtype-specific characteristics suggests the potential for developing drugs targeting specific molecular markers in fibroblast subsets, allowing for the modulation of fibroblasts in a time-course-dependent manner throughout the disease progression. REFERENCES: 1 Croft, A.P., et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature 570, 246-251 (2019). 2 Rauber et al., Nat Immunol. 2024, in press. 3 Fagni F, et al. Fibroblast Activation in Psoriasis Patients Assessed by 68Ga-FAPI-04 PET-CT Is Associated with Progression to Psoriatic Arthritis abstract. Arthritis Rheumatol. 2022; 74 (suppl 9). Acknowledgements: NIL. Disclosure of Interests: None declared.