P065 Determination of the cytokine pattern of human colon organoids derived from Inflammatory Bowel Disease patients

Abstract

Abstract Background Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract which is associated with imbalanced pro- and anti-inflammatory cytokines. Anti-TNF-α therapy is widely used, but 10–30% of the patients are primarily not responding to the treatment or lose response over time. In vitro organoid cultures (OCs) generated from tissue specific adult stem cells can mimic cellular diversity and function of the organ of origin and might be used as a predictive tool for patient specific therapeutic response. However, the reductionist nature of the OCs and lack of knowledge about the proper representation of the disease features currently limit the utilization of the OCs as ex vivo IBD models. Methods Therefore our aims were to determine and compare cytokine profiles of colonic biopsies collected from IBD patients and OCs generated from the same biopsies, and to follow the changes of cytokine expression over time. In this study samples from 20 IBD and 8 non-IBD patients were used. Biopsies were taken during colonoscopy from inflamed part of the colon of IBD patients. Crypts were isolated from biopsy samples to establish colon OCs. Total protein and RNA were isolated from biopsies and OCs. Cytokine Array was used to determine cytokine patterns in our samples. Gene expressions of control and IBD OCs after first passage was compared by qPCR. TNF-α concentrations and cellular expression was determined by EILSA and immunostainings, respectively in control and IBD OCs. To determine therapeutic response OCs were treated with anti-TNF-α therapy. Results Cytokine patterns of colon biopsies and OCs were remarkably similar until first passage. The major pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were detected both in biopsies and OCs. After second passage the cytokine expression decreased or disappeared in OCs. TNF-α was also measurable in the OCs, however a similar decrease was observed, which was also confirmed by immunostaining. Gene expression analysis in IBD OCs showed an increased TNF-α, IL-1β and IL-6 levels compared to healthy control OCs after first passage. ELISA also showed higher TNF-α concentration in organoids from inflamed origin compared to controls. After anti-TNF-α treatment we detected a decrease in the IL-6 gene expression in treated OCs. Conclusion Our results suggest that colon OCs maintain the cytokine expression ex vivo until the 2nd passage and show inflammatory characteristics. Moreover, in vitro treatment induces changes in the cytokine expression. Based on these results the utility of patient-derived organoids to predict the therapeutic response can be investigated.