Introduction
The use of 3D cell culture models, including spheroids and organoids, has proven to be a valuable method to approximate in vivo conditions in basic research. Our recent work on the pathogenesis of spontaneous bacterial peritonitis (SBP) – a life-threatening complication of liver cirrhosis with a 1-year-mortality of 66% – shows that bacteria induce the degradation of important cell-cell contact proteins upon direct contact via various processes. The resulting destabilization of the epithelial barrier enables bacterial translocation and thus represents a mechanism in the development of SBP (Haderer et al., Gut 2022). To evaluate these processes under more physiological conditions, we investigate bacterial-induced effects on cell-cell contact proteins in a 3D spheroid model and subsequently in a patient-based organoid model.
Aims & Methods
To establish the method and create a reference, spheroids were generated from colon carcinoma cell line HCT116 with a concentration of 70,000 cells/ml. Spheroid development and formation of cell-cell contacts were assessed by fluorescence microscopy. Subsequently, bacterial-induced effects on the tight junction proteins Occludin, Claudin4 and Zonulin1 as well as on the adherens junction protein E-Cadherin were investigated after up to 12 hours of bacterial co-culture using qPCR, fluorescence microscopy and Western blot analysis. Bacterial co-culture was performed with the reference strain Escherichia coli (E.coli) O6:Hnt and the E. coli strain Ont:H41, which was isolated from the ascites of a patient with SBP.
In addition, organoids were generated from intestinal biopsies of patients with liver cirrhosis and co-cultured with both bacterial strains. Bacterial-induced effects on cell-cell-contact proteins were investigated using Western blot analysis.
Results
HCT116 cells formed formed spheroids composed of viable cells with well-defined cell-cell contacts. Co-culture with E. coli for up to 12 hours led to a significant degradation of Occludin (up to 60% reduction), E-Cadherin (up to 85% reduction) and Zonulin1 (up to 95% reduction), whereas Claudin4 levels remained mostly constant. Co-culture with heat-inactivated bacteria did not affect cell-cell contacts. The intensity of these effects was dependent on the respective protein, the bacterial strain used, as well as the multiplicity of infection (MOI) and the duration of the co-culture. Degradation of Occludin could also be visualized by fluorescence microscopy.
We were then able to transfer this experimental approach to an organoid setting. Bacterial-induced degradation of the epithelial barrier was confirmed in organoids, showing a clearly visible reduction of E-Cadherin, Occludin and Zonulin1 after 8 hours of bacterial co-culture. The intensity of the effects varied depending on the bacterial strain, with the patient strain showing a slightly higher level of aggressiveness.
Conclusion
Understanding the pathophysiological changes of the intestinal epithelial barrier in patients with liver cirrhosis is essential for advancing therapeutic strategies for SBP. In this context, a representative ex vivo model is crucial to draw reliable conclusions. Our work shows that relevant mechanisms of SBP, such as bacterial-induced degradation of cell-cell contacts, can be successfully modeled in spheroids and organoids. Thus, our study provides an approach for further approximation to in vivo conditions – bridging the gap between basic research and clinical utility.
References
Haderer M, Neubert P, Rinner E, Scholtis A, Broncy L, Gschwendtner H, Kandulski A, Pavel V, Mehrl A, Brochhausen C, Schlosser S, Gülow K, Kunst C, Müller M. Novel pathomechanism for spontaneous bacterial peritonitis: disruption of cell junctions by cellular and bacterial proteases. Gut. 2022 Mar;71(3):580-592. doi: 10.1136/gutjnl-2020-321663 . Epub 2021 Mar 11. PMID: 33707230 ; PMCID: PMC8862089.
