Abstract 907: Uptake of lung cancer exosomes induces migratory and invasive phenotypic changes in lung epithelial cells in an oncogene context dependent manner
Authors: Shimada Yoshihisa , Yenerall Paul , Avila Kimberly , Park Hyunsil , Timmons Brenda , Huffman Kenneth , Gao Boning , Deb Dhruba , Dalvi Maithili , Minna John D. 
Published: July 2017

Abstract
Cancer-derived exosomes are intracellular signaling organelles that can act via inter-cellular communication to help cancer cells invade neighboring tissues and prime metastatic sites for cancer cell spread. Lung cancer is a highly metastatic disease, with metastases often occurring when tumors are in a clinically early stage. Little is known of how lung cancer exosomes influence the migratory/ invasive phenotype of lung cancer cells. We studied migration and invasion mechanisms by transferring cancer-derived exosomes to recipient cells including cancer cells, human bronchial epithelial cells (HBECs) cells, and HBECs that we had modified with common lung cancer oncogenic changes. Exosomes isolated from lung cancer line H1299 cells (p53null, NRAS mutant) and non-malignant, immortalized HBEC3-KT cells were characterized for exosome content and type by nanoparticle tracking analysis, electron microscopy, western blotting with antibodies directed at HSP70 and CD63, and generation of H1299 and HBEC3-KT derivatives stably transfected with CD63-GFP, releasing green fluorescence exosomes for tracking purposes. Exosomes were isolated using a variety of published methods and the amount quantified: 106 H1299 and HBEC-3KT cells produced 9 x 109 and 7 x 109 exosomes of similar size (125.4nm and 129.8nm). Effect of exosomes from cancer and HBECs were tested on: 1. cell motility and invasiveness of HBECs and their oncogenic derivatives determined by transwell chamber migration assay and scratch assay; 2. vascular leakiness properties in the lung by evaluating mouse lung endothelial permeability after exosomes were injected (an early event in metastatic spread for the migrated cells); and 3. xenograft tumor formation. We found CD63-GFP exosomes from H1299 and HBEC3-KT are actively incorporated by all of the cell types. However, only H1299 exosomes (but not HBEC3-KT exosomes) induced migratory/invasive phenotypic and morphologic changes and they did this in a concentration-dependent manner in lung cancer cells and HBECs with oncogenic changes (HBEC sh-p53+KRASv12 and sh-p53+KRASv12+c-MYC manipulated HBECs) but not in the parental HBEC3-KT cells. Also, H1299 exosomes but not HBEC3-KT exosomes enhanced the lung endothelial permeability as evaluated by the extravasated dextran leaking. NSCLC H1299 cells when injected with its own exosomes produced better xenograft takes compared to H1299 cells alone. Lung cancer-derived exosomes compared to exosomes derived from immortalized but non-malignant human bronchial epithelial cells induced an increased migratory/invasive phenotype with lung vascular leakiness and higher xenograft tumor take rates. However, they induced these changes only in the context of key oncogenic changes. These results suggest understanding and targeting the mechanism of such lung cancer derived exosome behavior could lead to novel therapeutic strategies.
Citation Format: Yoshihisa Shimada, Paul Yenerall, Kimberly Avila, Hyunsil Park, Brenda Timmons, Kenneth Huffman, Boning Gao, Dhruba Deb, Maithili Dalvi, John D. Minna. Uptake of lung cancer exosomes induces migratory and invasive phenotypic changes in lung epithelial cells in an oncogene context dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2017-907