Published OnlineFirst July 26, 2019; DOI: 10.1158/1055-9965.EPI-18-1122 Research Article Cancer Epidemiology, Biomarkers Potential Markers from Serum-Purified Exosomes & Prevention for Detecting Oral Squamous Cell Carcinoma Metastasis Cuiping Li1, Yang Zhou2, Junjun Liu3, Xiaoping Su1, Hao Qin3, Suhua Huang1, Xuanping Huang1,3, and Nuo Zhou1,3 Abstract Background: Blood contains exosomes that are related to Results: A total of 415 proteins were identified. Compar- tumor cells. Those exosomes can regulate communication ing with HC and OSCC-NLNM, we found 37 proteins and between cells and have a great influence on a variety of 28 proteins in the SEs of OSCC-LNM, respectively. There tumor-associated proceedings through their target cells. There- were significant correlations among the expression of PF4V1 fore, serum exosomes (SE) were supposed to play a crucial role with tumor differentiation level, PF4V1 and F13A1 with in cancer development. the number of positive nodes, and ApoA1 with smoking Methods: This study presented a quantitative proteomics and drinking. ROC curve analysis indicated that the com- analysis to identify the protein content in SEs including 30 binations of the different biomarkers or specimen were subjects from oral squamous cell carcinoma (OSCC) patients obviously superior to single biomarker or specimen for with lymph node metastasis (LNM), OSCC patients with no diagnosing OSCC-LNM. LNM (NLNM), and healthy controls (HC). Differentially Conclusions: We conclude that PF4V1, CXCL7, F13A1, expressed proteins (DEP) were analyzed by bioinformatics, and ApoA1 from SEs may be related to the metastasis and then a total of 30 subjects were used for Western blot of OSCC, which would be helpful in the diagnosis of and 60 subjects for IHC, ELISA, and RT-PCR verifications. OSCC-LNM. The correlations were assessed between DEP expression and Impact: Biomarkers from SEs could help with the diag- clinicopathologic factors. nosis of metastasis in OSCC. Introduction OSCCs have a high rate of lymph node metastasis (LNM; ref. 2). Once patients are diagnosed with OSCC with LNM, their 5-year Oral cancer is a type of head and neck cancer, and the survival rate will fall from 80% to less than 40% (3). Therefore, it is location of oral cancer occurrence includes the lip, tongue, important to understand the mechanisms behind the metastasis floor of mouth, buccal, gingiva, and palate (1). Oral cancer is that is a leading cause in most deaths of OSCC patients and poses a the sixth most common cancer diagnosed worldwide and major challenge in the early detection and timely treatment of causes more than 145,000 deaths annually; it is also the sixth OSCC with LNM. As one of the 10 hallmarks of cancer, the most common cause of cancer death, especially for men who activation of tumor invasion and metastasis is a complex and drink alcohol and smoke. More than 90% of oral cancers are critical multistep process (4, 5). Consequently, there is an area of oral squamous cell carcinomas (OSCC), and more than 50% of intense research on the molecular pathogenesis behind the devel- opment of such metastatic advances and specific markers that can 1Guangxi Key Laboratory of the Rehabilitation and Reconstruction of Oral and be used to screen for metastasis in LNM patients rapidly, nonin- Maxillofacial Research; Guangxi Key Laboratory of Oral and Maxillofacial Surgery vasively, and with high sensitivity. Recent studies have found that Disease Treatment; Guangxi Clinical Research Center for Craniofacial Deformity, extracellular vesicles called exosomes played an important role in College of Stomatology, Guangxi Medical University, Nanning, P.R. China. explaining metastasis (6). 2School of Information and Management, Guangxi Medical University, Nanning, P.R. China. 3Department of Oral and Maxillofacial Surgery, the Affiliated Sto- Exosomes are small, spherical bilayered membrane vesicles, matology Hospital of Guangxi Medical University, Nanning, P.R. China. with an average diameter of 30 to 100 nm, and are secreted from all kinds of cells, including tumor cells, in a range of biological Note: Supplementary data for this article are available at Cancer Epidemiology, fl fl Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). uids, including plasma, saliva, urine, blood, cerebrospinal uid, and cell culture media (6–8). An intercellular communication C. Li, Y. Zhou, and J. Liu contributed equally to this article. system consists of these vesicles, meaning that exosomes are Corresponding Authors: Nuo Zhou, the Affiliated Stomatology Hospital of potentially valuable for biomarker selections as well as disease Guangxi Medical University, Nanning 530021, P.R. China. Phone: 086-771- therapies, such as cancer therapies. Exosomes involve a variety of 2705693; Fax: 086-771-2705693; E-mail: [email protected]; and Xuanping Huang, [email protected] proteins and bioactive lipids, and exosome compositions change with the specific donor cell type. The protein members of exo- Cancer Epidemiol Biomarkers Prev 2019;XX:XX–XX somes include the transmembrane 4 superfamily (CD9, CD63, doi: 10.1158/1055-9965.EPI-18-1122 and CD81) and tumor susceptibility gene 101 (TSG101; ref. 9). Ó2019 American Association for Cancer Research. There is still not a complete understanding of the functional www.aacrjournals.org OF1 Downloaded from cebp.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst July 26, 2019; DOI: 10.1158/1055-9965.EPI-18-1122 Li et al. characteristics of exosomes (10). A new study indicated that graphics were as follows: Male/female ratio was 7/3, 8/2, and 7/3 tumor-derived exosomes could recruit bone marrow–derived for OSCC with LNM, OSCC with NLNM, and HC groups, respec- cells to the premetastatic niche, which is similar to the functions tively; the average age was 43.5, 43.8, and 44.2 for OSCC with of cytokines and soluble factors, thereby contributing to the LNM, OSCC with NLNM, and HC groups, respectively. Serum and production of a permissive microenvironment for tumor metas- whole blood from a total of 60 subjects were used for RT-PCR and tases and spreading by transferring oncoproteins (11). Further- ELISA verification from serum and SEs, and the subject demo- more, tumor-derived exosomes mediate vascular leakiness, which graphics were as follows: For the OSCC with LNM group, the have become a crucial feature of premetastatic niche forma- male/female ratio was 12/8, and the average age was 44.6; for the tion (12, 13). Exosomes are not just simple cellular debris. They OSCC with NLNM group, the male/female ratio was 12/8, and the act as extracellular organelles that play local or distinct roles average age was 44.2; for the HC group, the male/female ratio was in cell–cell communication while promoting the motility of 13/7, and the average age was 44.9. A total of 60 pairs of cancerous recipient cells to proliferate and remodel the extracellular matrix tissues and their corresponding adjacent tissues were used for (ECM) that supports the proliferation and migration of RT-PCR and IHC verification. The demographics of the patients tumor (14). Tumor cell–derived exosomes manifest multiple who provided the tissues were as follows: For the OSCC group, sites of action. Some recent studies confirmed that the tumor the male/female ratio was 33/27, and the average age was 45.6; cell–derived exosomes could induce apoptosis of tumor cells and for the OSCC with LNM group, the male/female ratio was 17/13, enhance antitumor immunity (15, 16). However, other studies and the average age was 45.3; and for the OSCC with NLNM indicated that tumor cell–derived exosomes may promote tumor group, the male/female ratio was 16/14, and the average age was progression by exhibiting immunosuppressive properties, facili- 44.9. The age and sex were matched between all case groups tating tumor invasion and metastasis, stimulating tumor cell and control groups. The whole experimental workflow was proliferation, and inducing drug resistance (17–22). Therefore, shown in Supplementary Fig. S1. exosomes are closely associated with tumor development and could serve as markers and novel therapeutic targets for early Isolation and identification of SEs prognostic and treatment benefit. Up to now, no study has The exosomes of 500 mL serum from each sample were focused on differentially expressed proteins (DEP) of exosomes isolated according to the instructions from the manufacturer purified from serum of OSCC patients, especially OSCC-LNM (SBI System Biosciences). The pellet from every 500 mLserum patients. was resuspended in 500 mL of PBS. The samples were fixed with In this study, we applied proteomic and bioinformatic strate- 3% glutaraldehyde for 3 hours, and soaked and rinsed gies to determine the biological functions of serum exosomes (SE) three times in 0.1 mmol/L sodium cacodylate buffer at room derived from OSCC-LNM. The proteome profiles of SEs from temperature. The samples were fixed with osmium tetroxide for OSCC-LNM patients, OSCC-NLNM patients, and healthy con- 1 hour and soaked and rinsed in 0.1 mmol/L sodium cacodylate trols (HC) were compared using a combination of LC-MS/MS buffer. A Hitachi S7650 scanning electron microscope visualized analyses. Identified DEPs might be associated with tumor cell the exosomes after samples were dried, mounted on specimen growth and adhesion. This study provides the first evidence that stubs, and sputter coated. We also characterized the amount and an initial relationship between the proteins of SEs and OSCC- size of exosomes to confirm this information using nanosight LNM patients may be useful in both the early evaluation of OSCC- analysis with Flow NanoAnalyzer (NanoFCM Inc.). Western LNM and the development of novel diagnostics and therapeutics. blotting was utilized to identify the marker proteins of exo- somes. Fifty micrograms of the protein was used for Western blotting and was then separated on a 12% SDS-PAGE gel and Materials and Methods transferred to membranes (Millipore).