Role of nuclear Met-derived exosomes in hepatocellular carcinoma metastasis and lung premetastatic niche formation

Abstract

INTRODUCTION: Emerging evidences has shown that nuclear Met (nMet) is expressed in some cancerous tissues and cell lines, suggesting Met could have unexplored functions inside nucleus. Our previous study have provided the first evidence about the clinical relevance of nMet in hepatocellular carcinoma (HCC). The present study was aimed to assess the role of exosomes isolated from nMet overexpressing cell line in promoting HCC metastasis as well as formation of lung premetastatic niche. MATERIAL AND METHOD: Exosomes were isolated from metastatic MHCC97L HCC cell in which nMet was overexpressed using ExoQuick solution. The integrity and size of isolated exosomes were examined by both electron microscopy and NanoSight. Immunoblotting was performed to determine the exosomal marker of isolated exosomes. Normal liver and na¨ıve HCC cells were treated with isolated exosomes and functional assays including Transwell migration and invasion assays were performed. The uptake of nMet-exosomes were investigated by fluorescence microscopy. Nude mice were injected with nMet-exosomes to study the HCC metastasis and lung premetastatic niche formation. RESULTS AND DISCUSSION: In our present study, exosomes were isolated from conditioned media of control MHCC97L/Vec and MHCC97L/nMet cells. Transmission electron microscopy images of isolated exosomes revealed typical exosome structure with diameter of approximately 50−80 nm. Immunoblotting showed that the isolated exosomes were positive for exosomal markers (Alix, TSG101 and CD9) while depleted of the cytoskeletal protein alpha-tubulin, cis-Golgi marker GM130 and nucleoporin p62. Further functional assays showed that nMet-exosomes significantly augmented both migratory and invasive properties of normal liver (MIHA) and na¨ıve HCC cells (BEL7402 and MHCC97L). We also labeled exosomes with PKH26 and observed the uptake of exosomes by na¨ıve cells with fluorescence microscopy. Intravenous injection of nMet-exosomes was administered prior to orthotopic liver implantation of tumor xenograft in nude mice. nMet-exosomes treated mice displayed enhanced incidence of distant metastasis from HCC primary tumor to lungs when compared with control mice, suggesting the promoting effect of nMet-exosomes on distant metastasis and lung premetastatic niche formation. CONCLUSION: Our findings will provide useful evidences about tumor-derived exosomes in driving metastasis and yield novel mechanistic insights into liver cancer metastasis.