Colorectal cancer cell-derived microRNA200 modulates the resistance of adjacent blood endothelial barriers in vitro

Holzner,Silvio; Senfter,Daniel; Stadler,Serena; Staribacher,Anna; Nguyen,Chi  Huu; Gaggl,Anna; Geleff,Silvana; Huttary,Nicole; Krieger,Sigurd; Jäger,Walter; Dolznig,Helmut; Mader,Robert   M.; Krupitza,Georg

doi:10.3892/or.2016.5114

Colorectal cancer cell-derived microRNA200 modulates the resistance of adjacent blood endothelial barriers in vitro

Authors:
- Silvio Holzner
- Daniel Senfter
- Serena Stadler
- Anna Staribacher
- Chi Huu Nguyen
- Anna Gaggl
- Silvana Geleff
- Nicole Huttary
- Sigurd Krieger
- Walter Jäger
- Helmut Dolznig
- Robert M. Mader
- Georg Krupitza
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Affiliations: Clinical Institute of Pathology, Medical University of Vienna, A‑1090 Vienna, Austria, Department of Medicine I, Comprehensive Cancer Centre, Medical University of Vienna, A‑1090 Vienna, Austria, Department of Clinical Pharmacy and Diagnostics, University of Vienna, A‑1090 Vienna, Austria, Institute of Medical Genetics, Medical University of Vienna, A‑1090 Vienna, Austria
Published online on: September 20, 2016 https://doi.org/10.3892/or.2016.5114
Pages: 3065-3071

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Abstract

Since cancer cells, when grown as spheroids, display drug sensitivity and radiation resistance patterns such as seen in vivo we recently established a three‑dimensional (3D) in vitro model recapitulating colorectal cancer (CRC)-triggered lymphatic endothelial cell (LEC)‑barrier breaching to study mechanisms of intra‑/extravasation. CRC metastasizes not only through lymphatics but also through blood vessels and here we extend the 3D model to the interaction of blood endothelial cells (BECs) with naïve and 5‑fluorouracil (5‑FU)‑resistant CRC CCL227 cells. The 3D model enabled quantifying effects of tumour‑derived microRNA200 (miR200) miR200a, miR200b, miR200c, miR141 and miR429 regarding the induction of so-called ‘circular chemorepellent‑induced defects’ (CCIDs) within the BEC‑barrier, which resemble gates for tumour transmigration. For this, miR200 precursors were individually transfected and furthermore, the modulation of ZEB family expression was analysed by western blotting. miR200c, miR141 and miR429, which are contained in exosomes from naïve CCL227 cells, downregulated the expression of ZEB2, SNAI and TWIST in BECs. The exosomes of 5‑FU‑resistant CCL227‑RH cells, which are devoid of miR200, accelerated CCID formation in BEC monolayers as compared to exosomes from naïve CCL227 cells. This confirmed the reported role of ZEB2 and SNAI in CRC metastasis and highlighted the active contribution of the stroma in the metastatic process. CCL227 spheroids affected the integrity of BEC and LEC barriers alike, which was in agreement with the observation that CRC metastasizes via blood stream (into the liver) as well as via lymphatics (into lymph nodes and lungs). This further validated the CRC/LEC and CRC/BEC in vitro model to study mechanisms of CRC spreading through vascular systems. Treatment of CCL227‑RH cells with the HDAC inhibitors mocetinostat and sulforaphane reduced CCID formation to the level triggered by naïve CCL227 spheroids, however, without significantly influencing miR200 expression in CCL227-RH cells.

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