ONCOLOGY REPORTS 40: 1103-1118, 2018 Systems biology analysis of the lung cancer‑related secretome LIN FENG1*, YIKUN YANG2*, MIN LI1, JIE SONG1, YANNING GAO1, SHUJUN CHENG1 and TING XIAO1 1State Key Laboratory of Molecular Oncology, Beijing Key Laboratory for Carcinogenesis and Cancer Prevention, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 2Department of Thoracic Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, P.R. China Received September 14, 2017; Accepted May 18, 2018 DOI: 10.3892/or.2018.6509 Abstract. Tumorigenesis is closely and highly associated with full-term placenta after delivery. ELISA results demonstrated developmental biology. The present study aimed to discover that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and identify marker proteins strongly associated with the was associated with worse rates of disease-free survival occurrence and development of non-small cell lung cancer (DFS) and overall survival (OS). The biological behaviors of (NSCLC) in humans and to provide new ideas for investi- embryonic implantation are similar to those of tumor inva- gating lung cancer markers by combining biological analyses sion and metastasis, and the information obtained regarding of embryonic development. We established primary cultures developmental biology could facilitate the interpretation of for samples of tumor and control tissues from 9 patients tumor invasion and metastasis. Therefore, similar biological with NSCLC and collected conditioned medium (CM). behaviors combined with analyses at different molecular Subsequently, we used liquid chromatography and linear ion levels from the perspective of systems biology will provide trap (LTQ) mass spectrometry to isolate and identify proteins new ideas for tumor research. in CM samples. Data mining of free proteins was conducted using the analogous analysis strategy in systems biology to Introduction obtain important lung cancer-associated proteins (plasma markers). Proteins with significant plasma enrichment in lung Lung cancer is also known as primary bronchogenic carcinoma cancer patients were detected via enzyme-linked immuno- and is a type of malignancy that originates in the bronchial sorbent assay (ELISA). We identified 987 high‑confidence epithelium. Lung cancer is one of the most common malignant proteins and established a primary database of free proteins tumors and the most deadly cancer worldwide (1). In recent associated with lung cancer. Furthermore, 511 high‑confi- years, due to population aging, smoking, environmental pollu- dence proteins were present in CM from primary tissue tion and other factors, the incidence and mortality rates of lung cultures from at least 2 of the 9 examined cases of lung cancer. cancer have tended to increase across the globe, especially in Analysis using Gene Set Enrichment Analysis (GSEA) soft- China and other developing countries (2). Biomarkers are of ware revealed significant enrichment for 197 proteins from great significance for the diagnosis and treatment of diseases, the CM of lung cancer samples in maternal-placental inter- particularly cancers. With the development of large-scale face expression profiles for a mid‑term placenta with strong proteomic technology, especially biological mass spectrom- invasiveness relative to RNA expression profiles for a human etry (MS), proteomic technology has become a mainstream technological approach in cancer biomarker discovery. Embryos and tumors share great similarities in many respects. In 1829, French scientists, Lobstein and Recamier, first proposed the concept of an embryonic origin of tumors, Correspondence to: Professor Ting Xiao, State Key Laboratory that is, cancer occurs due to the continued proliferation of of Molecular Oncology, Beijing Key Laboratory for Carcinogenesis embryonic cells present in the body (3). In the 1970s, Pierce and Cancer Prevention, National Cancer Center/National Clinical developed the theory of ‘cancer, a problem of developmental Research Center for Cancer/Cancer Hospital, Chinese Academy biology’ and noted that tumorigenesis is closely and strongly of Medical Sciences and Peking Union Medical College, related to developmental biology (4). Due to the similarity 17 Panjiayuannanli, Chaoyang, Beijing 100021, P.R. China E-mail: [email protected] between tumors and embryonic cells during gestation in terms of growth, invasion and immune system suppression, it has *Contributed equally been proposed in recent years that we should think of and study tumors from an evolutionary perspective (5‑7). With Key words: lung cancer, conditioned medium, embryonic development, the development of experimental techniques and the increase systems biology, hypoxanthine phosphoribosyltransferase 1 in research investigations, the early hypothesis that embry- onic development and tumorigenesis are closely related has increasingly been confirmed. 1104 FENG et al: SYSTEMIC ANALYSIS OF EXOSOME PROTEINS IN LUNG CANCER In our preliminary study, to eliminate the interference Supernatants were collected, divided into aliquots and stored of high-abundance proteins in the blood and enrich lung at ‑80˚C until use. Disease‑free survival (DFS) was defined as cancer‑specific markers in body fluid, we established a new the interval between surgery and recurrence; if recurrence was primary organ culture model to detect the free proteins released not diagnosed, the date of death or last follow-up was recorded. by tumor cells into the bloodstream (8). In the present study, Overall survival (OS) was defined as the interval between we used the research system for tumor-associated proteins in surgery and death. After surgery, patients were followed up body fluid that was established in our preliminary study. We for over eight years or until death. At the end of the follow-up also established primary cultures of tumor and control tissue period (11‑99 months, with a mean of 74 months), tumor recur- samples from non-small cell lung cancer (NSCLC) patients rence had been identified in 33 (55.0%) patients; 27 (45.0%) and collected conditioned medium (CM). We then used patients had died at the time of data censorship. liquid chromatography (LC) and linear ion trap (LTQ) MS to isolate and identify the full spectrum of the total proteins Primary tissue culture and CM collection. We chose different in CM samples. Subsequently, we used BRB-ArrayTools control tissues depending on pathological characteristics. For (http://linus.nci.nih.gov/BRB-ArrayTools.html), ArraySVG SCC, the control tissue was normal bronchial tissue from the and other programs and analyzed MS data using the spectral same patient. For ADC and large cell lung cancer (LCLC), the counts produced by label-free quantitative proteomics as the control tissue was normal lung tissue from the same patient. In quantitative parameter. We used the Gene Set Enrichment all cases, the distance between the control and tumor tissues Analysis P (GSEA-P) program to conduct enrichment analysis was >3 cm. Samples of paracancerous bronchial/lung tissues of the free proteins identified in tumor tissue CM based on and lung cancer tissues that were dissected from the body the maternal-placental interface expression profile data at within 30 min were cut into small pieces with volumes of different stages. Data mining of free proteins was conducted approximately 5 mm3 using a scalpel. Tissue pieces were placed to identify important lung cancer-associated plasma proteins. into collagen-coated gridded dishes, and LHC-9 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, Materials and methods USA) was added carefully and slowly in a dropwise manner to prevent disruption of these pieces. The dishes were placed Sample collection. Tissue samples of lung cancer patients for in a culture box, which was then filled with a gas mixture of the present study were all taken from hospitalized patients in 50% N2, 45% O2 and 5% CO2. The culture box was placed the Department of Thoracic Surgery at the Cancer Hospital of on a shaker, with a shaking frequency of 8-10 times/min. the Chinese Academy of Medical Sciences and Peking Union CM was collected after 24 h of incubation in a 36.5˚C incu- Medical College. When the specimens were obtained, none bator. The collected CM was added to an Amicon Ultra tube of the patients had received physical or chemical treatments. (cat no. UFC900596; Millipore; Merck KGaA, Darmstadt, We conducted a comprehensive collection of patient clinical Germany) and then centrifuged for 45 min at 4,000 x g and data. The histopathological types of surgically resected 4˚C. This process was performed to desalinate and concentrate tumor tissues were determined by senior pathologists based the sample. on the World Health Organization (WHO) classification of lung cancer tissue. Tumor staging was determined based on LC‑MS analysis and identification of proteins released into the 7th edition of the Union for International Cancer Control CM by cells (UICC) tumor-node-metastasis (TNM) staging system. Enzyme digestion of proteins in CM. A total of 30 µg of CM During the period from September 2005 to October 2006, we proteins was dissolved in 50 µl of solution containing 8 M urea collected fresh tumor and control tissue samples for primary and 10 mM dithiothreitol (DTT); 100 µl of 50 mM NH4HCO3 culture