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

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.

Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury

“…Exosomes play an important role in the cellular microenvironment and are well-studied multi-functional extracellular vesicles. In cancer cells, the exosomes of 5-FU-resistant CCL227-RH cells, are devoid of microRNA-200, and accelerate the formation of circular chemorepellent-induced defects in vascular endothelial cell monolayers as compared to exosomes from naïve CCL227 cells ( 12 ). The paracrine effects of human umbilical vein endothelial cells (HUVECs) improve the generation of endothelial cells from cord blood circulating endothelial progenitor cells and may include the role of exosomes ( 13 ).…”

Tumor-derived exosomes in colorectal cancer progression and their clinical applications

“…Exosomal miR-200c, miR-141 and miR-429 from naïve CRC CCL227 cells decreased expressions of ZEB2 and SNAI with the role of direct transcriptional repressors in CRC metastasis. In addition, these exosomal miRNAs decelerated CCIDs (circular chemorepellent-induced defects) formation in blood endothelial cell (BEC)-barrier as well as LEC (lymphatic endothelial cell)-barrier which are the gates of tumor migration via blood vessels and lymphatics [ 41 ].…”

“…The miRNA-200 family members released from exosomes were confirmed to suppress EMT–regulating transcription factors. The absence of miR-200c, miR-141 and miR-429 in 5-fluorouraclil-resistant colon cancer cells sensitized LECs (lymphendothelial cells) to the migratory signal and accelerated CCIDs formation in BEC-barrier [ 41 , 59 ].…”

Zinc Finger E-Box Binding Protein 2 (ZEB2) Suppress Apoptosis of Vascular Endothelial Cells Induced by High Glucose Through Mitogen-Activated Protein Kinases (MAPK) Pathway Activation

“…Overexpression of ZEB2 decreases the synthesis of ROS induced by hyperglycemia to reduce the oxidative stress response. The possible mechanism might be illuminated that ZEB2 suppresses the expression and activity of Racl gene and reduce the generation of active oxygen clusters, such as H 2 O 2 , to inhibit intracellular oxidation [22,23]. …”