Published OnlineFirst April 21, 2015; DOI: 10.1158/1078-0432.CCR-14-2464 Biology of Human Tumors Clinical Cancer Research Glycodelin: A New Biomarker with Immunomodulatory Functions in Non–Small Cell Lung Cancer Marc A. Schneider1,2, Martin Granzow3, Arne Warth2,4, Philipp A. Schnabel5, Michael Thomas2,6, Felix J.F. Herth2,7, Hendrik Dienemann2,8, Thomas Muley1,2, and Michael Meister1,2 Abstract Purpose: In recent years, immune therapeutic strategies Results: Glycodelin mRNA expression was significantly elevat- against non–smallcelllungcancer(NSCLC)basedontis- ed in tumors (n ¼ 336) compared with matched normal tissue sue-derived biomarkers, for example PD1/PD-L1 (CD274), (P < 0.0001). Overall survival (OS) was significantly reduced in have evolved as novel and promising treatment options. NSCLC with high glycodelin mRNA levels in women but not in However, the crosstalk between tumor and immune cells is men. Glycodelin was detected in the sera of patients, and the levels poorly understood. Glycodelin (gene name PAEP), initially correlated with recurrence and metastatic disease. Knockdown of described in the context of pregnancy and trophoblastic glycodelin with siRNAs in NSCLC cell lines resulted in significant implantation, is a secreted immunosuppressive glycopro- upregulation of immune system modulatory factors such as tein with an as-of-yet largely unknown function in lung PDL1, CXCL5, CXCL16, MICA/B, and CD83 as well as prolifer- cancer. ation stimulators EDN1 and HBEGF. Furthermore, decreased Experimental Design: In this study, we characterized migration of tumor cells was observed. the expression and role of glycodelin in NSCLC through Conclusions: Altogether, the comprehensive characterization mRNA and protein expression analyses, functional knock- of glycodelin in NSCLC provides strong support for its use as a down experiments, and correlations with clinicopathologic biomarker with immune modulatory function. Clin Cancer Res; parameters. 21(15); 3529–40. Ó2015 AACR. Introduction To improve the poor survival of lung cancer patients, chemo- therapeutic treatments are progressively supplemented with pre- With approximately 1.6 million cases worldwide, lung cancer is cision therapy based on genetic analyses. Sequencing of lung the leading cause of cancer-related death (1). Historically, lung carcinoma demonstrated a high somatic mutation rate of TP53, cancer is divided into two groups: non–small cell lung cancer KRAS, and EGFR (3). First-line and second-line tyrosine kinase (NSCLC), which accounts for more than 80% of all cases, and inhibitors that target the EGFR or anaplastic lymphoma kinase small-cell lung cancer (SCLC; ref. 2). (ALK) improve progression-free survival (4–6). Surface markers and secreted chemokines of altered cells attract lymphocytes, natural killer cells (NK) or cytotoxic T cells. The tumor microenvironment is strongly defined by extracellular 1Translational Research Unit, Thoraxklinik at University Hospital Hei- matrix (ECM) reorganization and chemokine secretion of tumor delberg, Heidelberg, Germany. 2Translational Lung Research Center cells that leads to a barrier for lymphocytes (7). Infiltration of Heidelberg (TLRC-H), Member of the German Center for Lung NSCLCs with lymphocytes is associated with better disease-free Research (DZL), Heidelberg, Germany. 3Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany. 4Institute of Patholo- survival (8). Thus, immune therapy has the potential to stimulate gy, University of Heidelberg, Heidelberg, Germany. 5Institut fur€ Allge- the patient's immune system response and attack cancer cells meine und Spezielle Pathologie, University of the Saarland, Homburg/ more effectively. Manipulation of NK cells and natural killer T Saar, Germany. 6Department of Thoracic Oncology, Thoraxklinik at University Hospital Heidelberg, Heidelberg, Germany. 7Department of cells (NKT) is considered to be a useful tool for cancer immuno- Pneumology and Critical Care Medicine, Thoraxklinik at University therapy (9, 10). 8 Hospital Heidelberg, Heidelberg, Germany. Department of Surgery, Glycodelin (gene name PAEP), initially described as placental Thoraxklinik at University Hospital Heidelberg, Heidelberg, Germany. protein 14 (PP14) or progesterone-associated endometrial pro- Note: Supplementary data for this article are available at Clinical Cancer tein (PAEP), is strongly associated with trophoblastic invasiveness Research Online (http://clincancerres.aacrjournals.org/). (11). Downregulation of glycodelin leads to an increased activa- Corresponding Author: Michael Meister, Thoraxklinik at University Hospital Hei- tion of the maternal immune system and can result in abortion delberg, Amalienstrasse 5, Heidelberg 69126, Germany. Phone: 49-6221-396-1650; during the first trimester (12). Glycodelin is differentially glyco- Fax: 49-6221-396-1652; E-mail: [email protected] sylated depending on gender and function (13). Glycodelin A, the doi: 10.1158/1078-0432.CCR-14-2464 immunosuppressive form of glycodelin, functions as a prolifer- Ó2015 American Association for Cancer Research. ation-suppressor and apoptosis inducer of T cells, monocytes, B www.aacrjournals.org 3529 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 21, 2015; DOI: 10.1158/1078-0432.CCR-14-2464 Schneider et al. trol siRNA-transfected and PAEP siRNA pool-transfected samples Translational Relevance per cell line. Detailed descriptions are provided in the Supple- Glycodelin is well described as an immune system mod- mentary Materials and Methods. ulator during pregnancy. In this work, we demonstrate the – expression and secretion of glycodelin in non small cell lung Quantitative Real-Time PCR cancer (NSCLC), its potential role as an immune system Real-time quantitative PCR (qPCR) was performed in accor- modulator, and the feasibility of using glycodelin as a bio- dance with MIQE-guidelines (19) using a LightCycler 480 Real- PAEP marker for NSCLC. Glycodelin mRNA (gene name ) was Time PCR Instrument in a 384-well plate format (Roche). Gene- overexpressed in approximately 80% of all tumors compared specific primers and probes (Universal ProbeLibrary; Roche) were PAEP with normal tissue. Knockdown of resulted in a dereg- used in combination with ABsolute QPCR Mix (Thermo Scien- ulation of immune system modulators, for example, PD-L1, a tific). Ct values were calculated with LightCycler 480 software current immune therapeutic target. We demonstrate the detec- version 1.5 using the second derivative maximum method tion of glycodelin in tissues and sera of NSCLC patients. The (Roche). Detailed descriptions are provided in the Supplementary fact that the glycodelin serum levels correlated with the Materials and Methods. patients' response to treatment suggests its use as a biomarker for follow-up of NSCLC. A diagnostic tool for glycodelin quantification in pregnancy is commercially available and Immunohistochemistry might be conveniently for use in lung cancer diagnoses. To compare the expression of whole glycodelin and glycodelin A, the polyclonal N-20 antibody (sc-12289, Santa Cruz Biotech- nology) and the monoclonal A87-B/D2 (Glycotope) antibody, raised against glycodelin A purified from mid-trimester amniotic fluid, were used. Detailed descriptions are provided in the Sup- cells and NK cells (14). Atypical glycodelin expression is observed plementary Materials and Methods. in hormone-related breast cancer, endometrial cancer, and ovar- ian cancer (15). Recent studies have demonstrated glycodelin Detection of glycodelin in human sera expression in melanoma cells and lung cancer (16, 17). Gene- Human sera were collected before any disease-specific treat- PAEP expression data from our group revealed that is expressed in ment and stored at À80C within 2 hours after venipuncture. The a lung cancer cell line as well (18). NSCLC validation cohort included pretherapeutic sera of 159 To elucidate the role of glycodelin in NSCLC, we comprehen- randomly selected NSCLC patients (clinical stage I–IV). The sively analyzed its expression in resection specimens as well as its chronic obstructive pulmonary disease (COPD) control cohort function in cell culture-based experiments. Furthermore, expres- included 90 randomly selected sera of COPD I–IV patients. The sion data were correlated with clinical data, including patient glycodelin levels of the sera were detected using an enzyme-linked outcomes. We demonstrate that glycodelin is highly expressed in immunosorbent assay kit (ELISA BS-20-30; Bioserv Diagnostics) fi NSCLC. It regulates tumor cell expression pro les to undergo with 50 mL of each serum in two technical replicates. The readout immune system defense and can be used as a biomarker to track and standard curve were performed with ELISA Reader (Tecan tumor progression, recurrence, or metastatic spread. Group Ltd.). The results of ELISA were visualized with GraphPad Prism 5. Detailed descriptions are provided in the Supplementary Materials and Methods Materials and Methods. Tissue sample collection, characterization, and preparation Tissue samples were provided by Lung Biobank Heidelberg, a Statistical analyses member of the accredited Tissue Bank of the National Center for Data of qPCR analyses were statistically analyzed under Tumor Diseases (NCT) Heidelberg, the BioMaterialBank Heidel- REMARK criteria (20) with SPSS 22.0 for Windows (IBM). The berg and the Biobank platform of the German Center for Lung