Exosomes as donor of sphingosine-1 phosphate to promote a microenvironment for metastatic lung cancer

Abstract

Statement of Purpose: Exosomes, a part of extracellular vesicles (EVs) with 30~150 nm sizes, have been reported as organelles fostering microenvironment for the progress of cancers to metastatic states. The roles of exosomes in cancer metastasis has not been fully understood, but cellto-cell or tissue-to-tissue signaling mediated by exosome cargos is adopted as a responsible mechanism of exosomes in cancer progress. In this study, sphingosine-1 phosphate (S1P) in cancer exosomes is proposed for a regulatory molecule to promote cancer microenvironment in metastatic lung cancer, induced by drug-resistant mutation in epidermal growth factor receptor (EGFR). There are several types of primary mutations identified in EGFR, such as Del19 (deletion of AA 746-750 of EGFR), and secondary mutation such as T790M evolved from the continuous treatment of anticancer drugs to treat primary EGFR mutation. In this study, we used four types of cells, normal BEAS-2B lung cells, lung adenocarcinoma A549 cells with wild type EGFR, non-small cell lung cancer PC9 cells with Del19 EGFR mutation, and PC9GR with Del19 and T790M mutation of EGFR produced by longtime exposure of anticancer drug to PC9. We identified significant membrane components in exosomes from PC9GR cells compared to those from other cells, to show the role of exosomes from metastatic lung cancer cells. Methods: Exosomes sized in 30~150 nm were isolated from BEAS-2B, A549, C9, and PC9GR cells, using ultrafiltration (100K) and ultracentrifugation (100,000  g, 1 h). Exosome size and morphology were analyzed by nanoparticle tracking analysis (NTA) and TEM analysis. The purity of exosomes was analyzed by western botting of exosome-specific markers, such as CD63, TSG101. To identify the difference of lipid composition in PC9GR from other cells, LC/MS/MS and subsequent principal component analysis (PCA) was performed to select important molecules. The expression of metabolic enzymes of sphingomyelin was investigated in the cell and exosome fractions. The final metabolic product of sphingomyelins, S1P, was analyzed by ELISA. Results: Fig. 1 shows the EGFR mutation states of cells used in this study (Fig. 1A) and isolated exosomes from each cell type (Fig. 1B). The lipid profiles of cells in different state of EGFR mutation shows discrete patterns in PCA (Fig. 2). One of the notable differences was the expression pattern of sphingomyelins in PC9GR exosomes. The metabolic end product of sphingomyelins is S1P, which is recently reported as a regulator of tumor microenvironment.1 The metabolic enzymes of sphingomyelins were increased in PC9GR and then seemed to elevate the level of S1P in PC9GR exosomes. To investigate the role of sphingomyelins in PC9GR exosomes, PC9GR exosomes were treated to BEAS-2B cells and then measure the S1P concentration. As shown in Fig. 3, the exosomes from PC9GR could elevate the S1P level in BEAS-2B cells, likewise the free S1P. Moreover, an inhibitor of S1P formation, PF-543, showed enhanced cytotoxic effect to PC9GR when co-treated with osimertinib, an inhibitor of secondary mutation of EGFR. Conclusions: Exosomes have been proposed as biomaterials for drug vehicles or therapeutic agents, but the details of their functions depending on the membrane composition should be verified more. In this study, the exosome membrane composition was related to the regulatory role of exosomes, which could be applied to develop an anticancer drug to control cancer microenvironment to progress metastasis.