FAP+ activated fibroblasts are detectable in the microenvironment of endometriosis and correlate with stroma composition and infiltrating CD8+ and CD68+ cells.

Study question: Do activated fibroblasts expressing fibroblast activation protein-α (FAP) - which is traceable in positron emission topography/computed topography (PET/CT) - play a role in the microenvironment of endometriosis?

Summary answer: Activated fibroblasts expressing FAP are detectable in endometriotic lesions and correlate with iron and collagen content and infiltrating CD8-positive cytotoxic T cells and CD68-positive macrophages in the microenvironment endometriotic lesions.

What is known already: FAP-positive activated fibroblasts are found in various fibrosis-related pathologies and in the desmoplastic stroma of solid tumours; they can be traced in PET/CT but have not been investigated in the context of endometriosis, a chronic disease involving hormone-mediated repetitive tissue remodelling and fibrosis.

Study design size duration: We analysed a cohort of endometriosis patients (n = 159) who had undergone surgery with removal of endometriotic foci at our University Hospital (tertiary care centre) between 2018 and 2024. All patients provided written informed consent. The median age of the patients was 34 years. In total, 245 samples from different locations were analysed retrospectively.

Participants/materials setting methods: We investigated the expression of FAP and its relation to stroma composition and the immune microenvironment of endometriosis in 245 specimens from peritoneal lesions, ovarian endometriomas, deep infiltrating endometriosis, and extra-abdominal lesions using conventional histology and immunohistochemistry followed by digital image analysis. Tissue within a radius of 500 µm of ectopic endometrium-like epithelium was analysed. To measure FAP expression in the perilesional stroma, a histoscore (H-score) was calculated. Masson trichrome staining was used to determine collagen content. Prussian blue staining for iron was used for age-dating of lesions. The abundance of CD68-positive macrophages and CD8-positive cytotoxic T cells within the microenvironment of ectopic endometriotic glands was analysed. Extra-lesional tissue served as controls.

Main results and the role of chance: Distinct FAP expression (H-score >10) was observed in 84% of endometriotic lesions and in only 4% of extra-lesional controls. FAP expression was significantly higher in endometriotic lesions (mean H-score 61.8) than in extra-lesional tissue (mean H-score 3.8, P < 0.0001). There was a significant (P < 0.05) association with collagen content when comparing samples with low (H-score <100) and high (H-score ≥100) FAP expression, and a significant difference in FAP expression correlating with the tissue iron content when comparing strong staining intensity and negative samples (P < 0.0005) or samples with weak staining intensity (P < 0.005). Moreover, the abundance of CD8-positive and CD68-positive cells was significantly higher (P < 0.0001) in samples with high FAP expression (H-score ≥100).

Large scale data: N/A.

Limitations reasons for caution: This study proves the presence of FAP-positive fibroblasts in endometriosis by immunohistological methods. However, to translate targeting FAP into endometriosis diagnostics, these results have to be compared to imaging data and FAP inhibitor (FAPi) PET/CT has to be validated in a structured way on a large patient cohort. Moreover, we show that FAP expression is intertwined with the immune cell infiltrate in the microenvironment of endometriosis. To explore and understand mechanisms contributing to chronic inflammation, immune evasion, and fibrosis, more studies including more immune cell subtypes and functional experiments are needed.

Wider implications of the findings: FAP-positive activated fibroblasts not only impact the immune microenvironment of endometriosis and are linked to increased macrophage and cytotoxic T-cell infiltration, as we showed, but could also provide new options for non-invasive diagnostic methods and an improvement of the diagnostic workup prior to surgery. FAPi PET/CT should be considered when exploring new diagnostic options in endometriosis.

Study funding/competing interests: This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) 'Clinician Scientist Program in Evolutionary Medicine' (project number 413490537 to F.K.). We acknowledge financial support by Land Schleswig-Holstein within the funding programme 'Open Access Publikationsfonds'. The authors declare that they have no conflicts of interest related to this work.