The secretion and biological function of tumor suppressor maspin as an exosome cargo protein

// Ivory Dean 1, 2, 3, 6 , Sijana H. Dzinic 1, 3 , M. Margarida Bernardo 1, 3 , Yi Zou 4 , Vickie Kimler 5, 7 , Xiaohua Li 1, 3, 8 , Alexander Kaplun 1, 3, 9 , James Granneman 3, 4 , Guangzhao Mao 3, 5 , Shijie Sheng 1, 2, 3 1 Department of Pathology, Wayne State University School of Medicine, MI, USA 2 Department of Oncology, Wayne State University School of Medicine, MI, USA 3 The Tumor Biology and Microenvironment Program, Karmanos Cancer Institute, MI, USA 4 Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, MI, USA 5 Department of Chemical Engineering and Materials Science, Wayne State University, MI, USA 6 Current address: Center for Bioengineering and Tissue Regeneration, The University of California San Francisco, San Francisco, CA, USA 7 Current address: Ocular Structure and Imaging Facility, Eye Research Institute, Oakland University, Rochester Hills, MI, USA 8 Current address: Zhangjiagang Aoyang Hospital, Nanjing Medical University, Jiangsu, China 9 Current address: Variantyx, Framingham, MA, USA Correspondence to: Shijie Sheng, email: ssheng@med.wayne.edu Keywords: exosome, tumor progression, exosome cargo, tumor microenvironment, electron microscopy Received: August 15, 2016 Accepted: October 22, 2016 Published: November 11, 2016 ABSTRACT Maspin is an epithelial-specific tumor suppressor shown to exert its biological effects as an intracellular, cell membrane-associated, and secreted free molecule. A recent study suggests that upon DNA-damaging g-irradiation, tumor cells can secrete maspin as an exosome-associated protein. To date, the biological significance of exosomal secretion of maspin is unknown. The current study aims at addressing whether maspin is spontaneously secreted as an exosomal protein to regulate tumor/stromal interactions. We prepared exosomes along with cell extracts and vesicle-depleted conditioned media (VDCM) from normal epithelial (CRL2221, MCF-10A and BEAS-2B) and cancer (LNCaP, PC3 and SUM149) cell lines. Atomic force microscopy and dynamic light scattering analysis revealed similar size distribution patterns and surface zeta potentials between the normal cells-derived and tumor cells-derived exosomes. Electron microscopy revealed that maspin was encapsulated by the exosomal membrane as a cargo protein. While western blotting revealed that the level of exosomal maspin from tumor cell lines was disproportionally lower relative to the levels of corresponding intracellular and VDCM maspin, as compared to that from normal cell lines, maspin knockdown in MCF-10A cells led to maspin-devoid exosomes, which exhibited significantly reduced suppressive effects on the chemotaxis activity of recipient NIH3T3 fibroblast cells. These data are the first to demonstrate the potential of maspin delivered by exosomes to block tumor-induced stromal response, and support the clinical application of exosomal maspin in cancer diagnosis and treatment.