diagnostics Article CD5L as an Extracellular Vesicle-Derived Biomarker for Liquid Biopsy of Lung Cancer Eun-Sook Choi 1,† , Hasan Al Faruque 1,† , Jung-Hee Kim 2 , Kook Jin Kim 3 , Jin Eun Choi 4 , Bo A. Kim 3 , Bora Kim 3 , Ye Jin Kim 3 , Min Hee Woo 3 , Jae Yong Park 5,6 , Keun Hur 4 , Mi-Young Lee 7 , Dong Su Kim 3 , Shin Yup Lee 5,6, * and Eunjoo Kim 2, *   Citation: Choi, E.-S.; Faruque, H.A.; Kim, J.-H.; Kim, K.J.; Choi, J.E.; Kim, B.A.; Kim, B.; Kim, Y.J.; Woo, M.H.; Park, J.Y.; et al. CD5L as an Extracellular Vesicle-Derived Biomarker for Liquid Biopsy of Lung Cancer. Diagnostics 2021, 11, 620. https://doi.org/10.3390/ diagnostics11040620 Academic Editor: Paola Gazzaniga Received: 4 February 2021 Accepted: 25 March 2021 Published: 30 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Division of Bi-Fusion Research, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Techno-jungangdaero 333, Dague 42988, Korea; stom96@dgist.ac.kr (E.-S.C.); hasan@dgist.ac.kr (H.A.F.) 2 Division of Electronic Information System Research, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Techno-Jungangdaero 333, Dague 42988, Korea; cell84@dgist.ac.kr 3 Genomine Research Division, Genomine Inc., Pohang Technopark, Pohang 37668, Korea; gjkim@genomine.com (K.J.K.); boa88@genomine.com (B.A.K.); pupybr@genomine.com (B.K.); yejini1@genomine.com (Y.J.K.); wmh0929@genomine.com (M.H.W.); dskimi@genomine.com (D.S.K.) 4 Department of Biochemistry, School of Medicine, Kyungpook National University, Daegu 41944, Korea; jechoi.9711@gmail.com (J.E.C.); KeunHur@knu.ac.kr (K.H.) 5 Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Korea; jaeyong@knu.ac.kr 6 Lung Cancer Center, Kyungpook National University Chilgok Hospital, Daegu 41404, Korea 7 Department of Medical Science, Soonchunhyang University, Asan 31538, Korea; umma7008@gmail.com * Correspondence: shinyup@knu.ac.kr (S.Y.L.); ejkim@dgist.ac.kr (E.K.); Tel.: +82-53-200-2632 (S.Y.L.); +82-53-785-2530 (E.K.) † These authors contributed equally to this work. Abstract: Cancer screening and diagnosis can be achieved by analyzing specific molecules within serum-derived extracellular vesicles (EVs). This study sought to profile EV-derived proteins to identify potential lung cancer biomarkers. EVs were isolated from 80 serum samples from healthy individuals and cancer patients via polyethylene glycol (PEG)-based precipitation and immunoaffin- ity separation using antibodies against CD9, CD63, CD81, and EpCAM. Proteomic analysis was performed using 2-D gel electrophoresis and matrix-assisted laser desorption ionization–time-of- flight mass spectrometry (MALDI–TOF MS). The expression of proteins that were differentially upregulated in the EVs or tissue of lung cancer samples was validated by Western blotting. The area under the curve (AUC) was calculated to assess the predictability of each differentially expressed protein (DEP) for lung cancer. A total of 55 upregulated protein spots were selected, seven of which (CD5L, CLEC3B, ITIH4, SERFINF1, SAA4, SERFINC1, and C20ORF3) were found to be expressed at high levels in patient-derived EVs by Western blotting. Meanwhile, only the expression of EV CD5L correlated with that in cancer tissues. CD5L also demonstrated the highest AUC value (0.943) and was found to be the core regulator in a pathway related to cell dysfunction. Cumulatively, these results show that EV-derived CD5L may represent a potential biomarker—detected via a liquid biopsy—for the noninvasive diagnosis of lung cancer. Keywords: lung cancer; extracellular vesicle; exosome; CD5L; 2-D gel electrophoresis; proteomics; biomarker; liquid biopsy 1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide, with a five- year survival rate below 21% [1]. It is categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which constitute 15% and 85% of the lung cancer cases, respectively [2]. Generally, the initial identification of lung cancer can be occurred by X-ray and computed tomography scans (CT), but there can be significant diagnostic Diagnostics 2021, 11, 620. https://doi.org/10.3390/diagnostics11040620 https://www.mdpi.com/journal/diagnostics