Einfluss von TGF-β auf die EMT-Marker-Expression in Ko-Kulturen oraler Karzinomzellen mit mesenchymalen Stromazellen
Influence of TGF-β on EMT marker expression in Co-cultures of oral carcinoma cells with mesenchymal stromal cells
by Fabian Godek
Date of Examination:2021-08-26
Date of issue:2021-07-16
Advisor:PD Dr. Dr. Björn Florian Böhrnsen
Referee:PD Dr. Dr. Björn Florian Böhrnsen
Referee:Prof. Dr. Carsten Gründker
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Abstract
English
Objectives: TGF-β1 signaling modulates epithelial mesenchymal transitions (EMT) of head and neck squamous cell carcinoma (HNSCC). Bone marrow mesenchymal stromal cells (BMSC) are able to exert a regulating influence on the expression of markers of EMT in HNSCC cells. It was thus the aim of this study to test the hypothesis that TGF-β1 modulates the interactions of tumor transition between BMSCs and HNSCC, affecting the expression of E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin. Furthermore, we analyzed alterations in the AKT-signaling of tumor and stroma cells. Materials and methods: BMSCs were isolated from iliac bone marrow aspirates and co-cultured in trans-well permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13. Following the induction with TGF-β1 under serum free conditions the expression of Vimentin and E-Cadherin was assessed via immunofluorescence. A quantitative RT-PCR analysis of tumor transition markers E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin was performed. Changes in AKT-Signaling were identified via protein analysis. Results: In non-induced co-cultures, BMSC were able to suppress Vimentin in PCI-13 as a marker of tumor transition. In TGF-β1 induced co-cultures PCI-13 significantly increased the expression of Vimentin, Twist, Snail, MMP14, GSK3a, PRAS40, 4E-BP1, and AMPKa compared to monolayer controls. TGF-β1 co-cultured BMSC demonstrated a significant increase of Snail, PRAS40, mTOR, GSK3a/b, Bad, PDK1 and 4E-BP1. Conclusions: TGF-β1 was able to attenuate the modulating influence of BMSC in co-culture and drive the co-culture towards a progressive tumor transition, affecting the expression of markers of EMT, AKT-Signaling and proliferative checkpoints.
Keywords: bmsc; co-culture; EMT; HNSCC; TGF-beta; Transition