Enhanced Acid Sphingomyelinase Activity Drives Immune Evasion And

Published OnlineFirst September 7, 2017; DOI: 10.1158/0008-5472.CAN-16-3313 Cancer Microenvironment and Immunology Research

Enhanced Acid Sphingomyelinase Activity Drives Immune Evasion and Tumor Growth in Non–Small Cell Lung Carcinoma Katerina Kachler1, Maximilian Bailer1, Lisanne Heim1, Fabian Schumacher2,3, Martin Reichel4,5, Corinna D. Holzinger1, Sonja Trump1, Susanne Mittler1, Juliana Monti5, Denis I. Trufa6, Ralf J. Rieker7, Arndt Hartmann7, Horia Sirbu6, Burkhard Kleuser2, Johannes Kornhuber5, and Susetta Finotto1

Abstract

The lipid hydrolase enzyme acid sphingomyelinase (ASM) is rendered them resistant to serum starvation–induced apoptosis. required for the conversion of the lipid cell membrane compo- In a murine model of lung adenocarcinoma, ASM deﬁciency nent sphingomyelin into ceramide. In cancer cells, ASM-mediated reduced tumor development in a manner associated with signif- ceramide production is important for apoptosis, cell prolifera- icant enhancement of Th1-mediated and cytotoxic T-cell–medi- tion, and immune modulation, highlighting ASM as a potential ated antitumor immunity. Our ﬁndings indicate that targeting multimodal therapeutic target. In this study, we demonstrate ASM in NSCLC can act by tumor cell–intrinsic and –extrinsic elevated ASM activity in the lung tumor environment and blood mechanisms to suppress tumor cell growth, most notably by serum of patients with non–small cell lung cancer (NSCLC). enabling an effective antitumor immune response by the host. RNAi-mediated attenuation of SMPD1 in human NSCLC cells Cancer Res; 77(21); 1–14. 2017 AACR.

Introduction ation of ceramide through sphingomyelin hydrolyzation is carried out by sphingomyelinases (SMases), enzymes that can be Sphingolipids are bioactive components of the cellular mem- classified into four different types, according to their characteristic brane that are involved in important cellular processes, as for pH-optimum and cofactor dependence (4). One of the best example cell death, proliferation, cell migration, and immune studied SMases is the acid sphingomyelinase (aSMase, ASM) that reactions. The most prevalent lipid in the outer leaflet of the cell is encoded by the SMPD1 (sphingomyelin phosphodiesterase 1) membrane is sphingomyelin that can be converted into ceramide gene (5). by enzyme acid sphingomyelinase (ASM)–mediated hydrolysis The major importance of ASM-mediated ceramide metabolism (1). Ceramide molecules are able to interact with each other, for normal cell function is evidenced by the Niemann–Pick forming isolated lipid "microdomains," which can promote disease (NPD), a rare, recessively inherited genetic disorder that receptor dimerization as well as other protein interactions within is caused by ASM deficiency. Depending on the type of NPD, the the lipid bilayer of the cell membrane (2). The balance between symptoms can range from severe visceral abnormalities to a ceramide and sphingosine-1-phosphate is referred to as the rapidly progressive neurodegeneration, leading to death by the "ceramide/sphingosine-1-phosphate rheostat," which regulates age of 2 to 3 years (6). In order to understand the mechanisms a proper balance between cell death and growth (3). The gener- behind this disease, several groups have studied ASM knockout mice, thereby identifying important functions of ASM for various cellular events. One of the most prominent observations was that 1Department of Molecular Pneumology, University Hospital, Friedrich-Alexan- ASM-deficient cells are resistant to apoptosis, induced by diverse € € 2 der-Universitat Erlangen-Nurnberg, Erlangen, Germany. Institute of Nutritional stimuli, including chemotherapy and TNFa treatment (7, 8). Science, University of Potsdam, Nuthetal, Germany. 3Department of Molecular Other studies have revealed that ceramide, which is generated by Biology, University of Duisburg-Essen, Essen, Germany. 4Department of Nephrology and Hypertension, University Hospital, Friedrich-Alexander- ASM, is required for the induction of cell-cycle arrest (9). In Universitat€ Erlangen-Nurnberg,€ Erlangen, Germany. 5Department of Psychiatry addition to that, it has been shown that ASM has an influence and Psychotherapy, University Hospital, Friedrich-Alexander-Universitat€ on the cells of the immune system. Thus, ASM is necessary for a Erlangen-Nurnberg,€ Erlangen, Germany. 6Department of Thoracic Surgery, correct phagocytosis and can modulate T-cell activation and € € University Hospital, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlan- differentiation as well as the secretion of effector molecules from 7 þ gen, Germany. Institute of Pathology, University Hospital, Friedrich-Alexander- cytotoxic CD8 T-cell granules (10). Furthermore, ASM has been Universitat€ Erlangen-Nurnberg,€ Erlangen, Germany. recently shown to influence regulatory T-cell differentiation (11). Note: Supplementary data for this article are available at Cancer Research These findings have drawn the attention to ASM as a possible Online (http://cancerres.aacrjournals.org/). target for other human diseases in addition to NPD, especially Corresponding Author: Susetta Finotto, University of Erlangen, Hartmannstraße cancer. In fact, it is known that resistance to apoptosis, as it is 14, 91054 Erlangen, Germany. Phone: 0049-09131-8542-454; Fax: 0049-09131- found in ASM-deficient cells, is a typical characteristic of tumor 8535-977; E-mail: [email protected] cells (12). Besides that, ASM has been proven to be involved in the doi: 10.1158/0008-5472.CAN-16-3313 regulation of other biological processes such as the cell cycle and 2017 American Association for Cancer Research. the inactivation of the cells of the immune system resulting in

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tumor cell development and tumor progression. Together, this board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: raised the question, if ASM expression or activity might be DRKS00005376). The patient studies were conducted in accor- dysregulated both in cancer cells and in other cells of the immune dance with the ethical guidelines of the Declaration of Helsinki. system surrounding the tumor and to what extent that could Before undergoing surgery, 61 patients diagnosed with NSCLC contribute to the pathogenesis of the disease. gave their approval to being enrolled in this study. The confi- The hypothesis that ASM could be important for cancer biology dentiality of the patients was maintained. is supported by the observation that ceramide levels are signifi- Lung cancer diagnosis was based on pathologic confirmation. cantly decreased in different kinds of tumors, including colon The NSCLC patients were divided into subgroups according to the cancer, ovarian cancer, and glioma (13–15). However, previous classification of histologic types of lung cancer formulated by the studies that were conducted to clearly define the role of ASM either World Health Organization in 2004. Staging was based on the as a tumor promoter or a tumor suppressor yielded in different Cancer TNM Staging Manual by the International Association for results. For instance, it was found that low levels of ceramide as the Study of Lung Cancer, issued in 2010. well as ASM correlate with increasing malignancy of brain tumors During surgery, lung tissue samples were taken from two and melanoma, respectively (13, 16). Consistently, downregula- different regions: The tumoral area (TU: solid tumor tissue) and tion of ASM in murine melanoma cells triggers their proliferation the tumor-free control area (CTR: >5 cm away from solid tumor). as well as their metastatic capacity, and also the formation of Clinical data such as tumor size, clinical stage, and tumor grading liver metastases is enhanced in ASM-deficient mice (16, 17). were provided by the Department of Thoracic Surgery. This cohort Moreover, it has been shown that ASM-generated ceramide is of patients was already described (22). necessary for drug-induced apoptosis of ovarian carcinoma cells (18). Whereas these findings support the view of ASM as a tumor Determination of ASM activity in blood serum suppressor, a recently published study indicates the opposite. ASM activity was measured in blood serum as previously There, it has been demonstrated that ASM-deficient mice are described (23). In short, samples were incubated with N-(4,4- protected from the development of melanoma cell–derived difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodeca- metastases in the lung. That was explained by the necessity of noyl)-sphingosylphosphocholine (BODIPY FL C12-sphingo- ASM for the interaction between platelets and tumor cells, which myelin; Life Technologies). After separation by thin-layer chro- is a crucial process during an early phase of metastasis (19). In matography, the levels of ceramide and sphingomyelin were conclusion, these previous studies show that ASM can have determined using a Typhoon Trio scanner (GE Healthcare). protumoral as well as antitumoral properties, depending on the site of ASM expression as well as the tumor type. However, Mass spectrometry additional analyses are strongly required in order to understand Ceramides and sphingomyelin were extracted from lung tissue the role of ASM in tumor diseases. samples, blood serum, or pelleted cells and quantified as recently The aim of the present study was to analyze the role of ASM described (24). Briefly, lipid extraction was performed using C17- in non–small cell lung carcinoma (NSCLC) by modulating ceramide and C16-d31-sphingomyelin as internal standards. ASM expression both in tumor cells and in the cells of the Sample analysis was carried out by rapid-resolution liquid chro- immune system of the host. Even though significant advances matography-MS/MS using a Q-TOF 6530 mass spectrometer in prevention, diagnosis, and therapy of lung cancer have been (Agilent Technologies) operating in the positive electrospray made, it remains the cancer type with the highest mortality rate ionization mode. The precursor ions of ceramides [C16-ceramide (20), causing over a million deaths per year worldwide. The (m/z 520.508), C17-ceramide (m/z 534.524), C18-ceramide prognosis for patients diagnosed with NSCLC, the most com- (m/z 548.540), C20-ceramide (m/z 576.571), C22-ceramide mon type of lung cancer, is largely unfavorable, with 5-year (m/z 604.602), C24-ceramide (m/z 632.634), C24:1-ceramide survival rates ranging from 59.2% in localized stages to 4.9% (m/z 630.618)] were cleaved into the fragment ion m/z 264.270. when metastasized (20). Unfortunately, as lung cancer lacks The precursor ions of sphingomyelin [C16-sphingomyelin (m/z specific early symptoms, it is often diagnosed late during the 703.575), C16-d31-sphingomyelin (m/z 734.762), C18-sphin- progression of the disease. NSCLC can be divided into three gomyelin (m/z 731.606), C20-sphingomyelin (m/z 759.638), major histologic subtypes, large cell carcinoma, adenocarcino- C22-sphingomyelin (m/z 787.669), C24-sphingomyelin (m/z ma, and squamous cell carcinoma (SCC), the latter two being 815.700), C24:1-sphingomyelin (m/z 813.684)] were cleaved the most common types as they account for 45% (adenocar- into the fragment ion m/z 184.074. Quantification was per- cinoma) and 23% (SCC) of all lung cancer cases (20). Recent formed with Mass Hunter Software (Agilent Technologies). studies suggest that immunotherapy could be a promising approach to improve the prognosis for this disease (21). Cell lines Despite the evidence that ASM is involved in immune reactions, The human A549 cell line was purchased authenticated, accord- little is known about its role for antitumoral immune ing to tissue type or gene mutation for cancer research, from the responses, especially in the context of lung carcinoma. Unco- ATCC bank circa 5 years ago. Mycoplasma contamination was vering the role of ASM in tumor cells and T cells could improve detected using the Mycoplasma Detection Kit (Absource Diag- current immunotherapy options for NSCLC. nostics GmbH), according to the manufacturers protocol (latest date: August 9, 2016). On average, we perform ten cell passages between thawing and use in the described experiments. Cells were Patients and Methods cultured in F12 K Nut Mix medium (Gibco; Life Technologies), Human subjects and study population supplemented with L-glutamin (L-Glu), 10% of FCS (PAA Labo- This study was performed at the Friedrich-Alexander-University ratories), and 1% of the antibiotics penicillin and streptavidin of Erlangen, Germany, after being approved by the ethics review (Pen/Strep; PAA Laboratories) at 37 C and 5% of CO2.

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The human H520 cell line was kindly provided by Dr. Paolo were normalized by DDCT calculation. A template-free negative Ceppi from the University Hospital Erlangen. They were pur- control was included in all experiments. chased from the ATCC bank in 2009 and checked for authen- tification via short tandem repeat DNA profiling and myco- siRNA transfection of A549 and H520 cells and apoptosis assay 5 plasma contamination in 2015. Number of passages between For siRNA-mediated silencing of ASM, 3 10 cells were thawing and application was similar to A549 cells. H520 cells incubated overnight in 6-well plates in antibiotic-free medium were cultured in RPMI medium, supplemented with 10% of (Gibco F-12 Nut Mix; Thermo Fisher Scientific) containing 10% FCS (PAA Laboratories), 1% Pen/Strep (PAA Laboratories), FCS. For transfection, 25 nmol/L ASM-siRNA (Eurofins-MWG- and 1% of L-Glu (Gibco; Invitrogen) at 37 Cand5%ofCO2. Operon) or 25 nmol/L nontargeting control siRNA (NT-Ctr; GE The murine LL/2-luc-M38 cell line was purchased and authen- Dharmacon) were applied together with 4 mL DharmaFECT ticated from Caliper LifeScience (Bioware cell line, Caliper Life- Transfection reagent 1 (GE Dharmacon) in 2 mL antibiotic-free Science). On average, we perform three cell passages between medium, supplemented with 10% FCS according to the manu- thawing and use in the described experiments. Cells were cultured facturer's instructions. After 24-hour incubation, transfected cells in DMEM medium (Gibco; Invitrogen), supplemented with 10% were washed with PBS and then cultured in 2 mL antibiotic-free of FCS (PAA Laboratories), 1% of the antibiotics Pen/Strep (PAA medium, supplemented with either 10% FCS or 0.4% FCS for SMPD1 Laboratories), and 1% of L-Glu (Gibco; Life Technologies) at 37 C another 24 hours. RNA was then isolated, and -mRNA and 5% of CO2. Mycoplasma contamination was detected using expression was analyzed via qPCR as described. Apoptosis assay the Mycoplasma Detection Kit (Absource Diagnostics GmbH), was performed according to the manufacturer's instructions by according to the manufacturers protocol (latest date: August 9, staining the cells with Annexin V (BD Biosciences) and propidium 2016). iodide (PI), followed by flow cytometry analysis (25). The murine CTLL2 cell line was kindly provided by Qualified Professor Dr. Ulrike Schleicher from the Institute of Microbiology, Analysis of A549 and H520 cell proliferation University Hospital Erlangen. It was cultured in RPMI medium, via Ki-67 staining supplemented with 10% of FCS (PAA Laboratories), 1% Pen/ A549 and H520 lung tumor cells were harvested and pelleted Strep (PAA Laboratories), 1% of L-Glu (Gibco; Invitrogen), and 4 by centrifugation. To each pellet, 500 mL of cold 75% ethanol was ng/mL IL2 (PeproTech GmBH) at 37 C and 5% of CO2. added dropwise while vortexing and incubated for at least 48 hours at 20C. Fixed cells were then washed twice (1,000 rpm, 10 minutes, 4C) with 3 mL wash buffer containing PBS, 1% FCS, RNA isolation and cDNA synthesis and 0.09% NaN and resuspended in 100 mL wash buffer per 1 Total RNA was extracted from lung tissue samples and cell 3 106 cells. Note that 20 mL of FITC mouse anti–Ki-67 antibody (BD suspensions using peqGold RNA Pure (Peqlab) according to the Pharmingen) was added to the cell suspension per 100 mL, mixed manufacturers protocol. Tissue samples were homogenized in gently, and incubated at room temperature for 25 minutes in the Precellys Lysing Kits (Bertin Technologies) using the benchtop dark. The reaction was stopped by adding 1 mL of PBS per 1 106 homogenizer Minilys (Bertin Technologies) as described in the cells, followed by centrifugation and resuspension in 250 mL PBS. manufacturers protocol. RNA was reverse-transcribed into cDNA After adding 3 mL of PI staining solution per 1 106 cells (BD with the RevertAid First Strand cDNA Synthesis Kit (Fermentas) Pharmingen), the samples were analyzed by flow cytometry according to the manufacturers instructions. (FACSCalibur; BD Biosciences).

Quantitative real-time PCR Bioluminescence analysis of LL/2-luc-M38 cell Quantitative real-time PCR of the resulting cDNA was per- For bioluminescence analysis, LL/2-luc-M38 cells (Bioware cell formed using iTaq Universal SYBR Green Supermix (BIO-RAD) in line, Caliper LifeScience) were cultured for 24 and 48 hours at a a total volume of 20 mL. Primers were purchased from Eurofins- cell density of 1 105 to 2 105 cells/mL in DMEM medium MWG-Operon. The following primer sequences were used: (Gibco; Invitrogen), supplemented with 10% of FCS (PAA Lab- 0 0 Human: HPRT (sense 5 -TGA CAC TGG CAA AAC AAT GCA-3 ; oratories), 1% Pen/Strep (PAA Laboratories), and 1% L-Glu 0 0 antisense 5 -GGT CCT TTT CAC CAG CAA GCT-3 ), SMPD1 (sense (Gibco; Life Technologies) at 37 C and 5% of CO2, in the 50-CCT CAG AAT TGG GGG GTT CTA TGC-30; antisense 50-CAC presence and absence of 25 to 33 ng/mL IFNg (BioLegend). ACG GTA ACC AGG ATT AAG G-30), Murine: Hprt (sense 5-GCC After 24 hours, LL/2-luc-M38 cells were treated with 15 mg/mL CCA AAA TGG TTA AGG TT-30; antisense 50-TTG CGC TCA TCT luciferin (Promega) to detect the luminescence intensity using TAG GCT TT-30), Grzb (sense 5-CCC AGG CGC AAT GTC AAT-30; the Centro XS3 LB 960 Microplate Luminometer (Berthold Tech- antisense 50-CCA GAA TAA GAA ACT CGA-30), Prf1 (sense 5-GAT nologies). Respective cell numbers were calculated using a stan- GTG AAC CCT AGG CCA GA-30; antisense 50-GGT TTT TGT ACC dard curve. After 48 hours, LL/2-luc-M38 cells were treated with 30 AGG CGA AA -30). mg/mL luciferin (Promega) to measure the luminescence with the Reactions (50 cycles, initial activation at 98C for 2 minutes, Caliper IVIS Imaging System (Bioware cell line, Caliper Life- denaturation at 95C for 5 minutes, and hybridization and Science) immediately after the treatment. The total flux (photons elongation at 60C for 10 minutes) were performed using the per second) was used as a measure for the cell number. CFX-96 Real-Time PCR Detection System (BIO-RAD) and analyzed with the CFX Manager Software (BIO-RAD). To minimize Mice the effect of variances in RNA quality and the amount of input ASMKO, ASMHet, and wild-type (WT) littermate control mice cDNA, the relative expression level of the specific transcripts was were on a C57BL/6 genetic background and maintained under calculated in relation to an internal standard, hypoxanthine- specific pathogen-free conditions. All experiments in mice were guanine-phosphoribosyltransferase (HPRT). Expression levels done under the licence number IR5520301020 in Erlangen,

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Germany. The animal studies described in this article have been anti-CD3 (10 mg/mL) and anti-CD28 antibody (2 mg/mL). After conducted in accordance with an Institutional Animal Care and 48 hours, RNA was extracted as described above, and IFNg and Use Committee. TNFa were measured in the cell supernatant by ELISA.

Murine model of lung adenocarcinoma and in vivo imaging Th1-in vitro differentiation þ þ For tumor induction, the LL/2-luc-M38 Bioware Cell line Na€ve CD4 CD62L T cells were isolated from the spleens of þ þ (Caliper LifeSciences) was used and cultured in DMEM na€ve mice by magnetic cell sorting using CD4 CD62L T Cell (Thermo Fisher Scientific) supplemented with 10% FCS and Isolation Kit II (Miltenyi Biotec). The purity of the cell isolation 1% Pen/Strep. Note that 1 106 cells suspended in 200 mLof was confirmed by FACS analysis. þ þ DMEM medium (without supplements) were injected in the In order to analyze T-cell differentiation, CD4 CD62L T cells tail vein of ASMKO and WT mice (6–8 weeks old). At the were cultured in RPMI medium (Gibco; Invitrogen), supplemen- describedtimepoints,themicewereweighedandinjectedwith ted with 10% FCS (PAA Laboratories) and 1% Pen/Strep (PAA luciferin (0.15 mg/g body weight) i.p. (Promega). After 20 Laboratories), at 37 C and 5% CO2 in the presence of 5 mg/mL minutes, luciferase activity was measured with the Caliper IVIS plate-bound anti-mouse CD3 antibody (BD Biosciences) and 1 Imaging System (Bioware cell line, Caliper LifeScience) by mg/mL soluble anti-mouse CD28 antibody (BD Biosciences). detecting the luminescence intensity. Mice were anesthetized For Th1 differentiation, the cells were additionally treated with with isoflurane before and during the measurement. 5 mg/mL anti–IL4 antibody (BioLegend) and 20 ng/mL recom- The analysis of the tumor load in the lungs was performed in a binant IL12 (PeproTech). The Th0 condition was stimulated with logarithmic scale mode, and the total flux (photons per second) 5 mg/mL anti–IL4 antibody (BioLegend) and 5 mg/mL anti-IFNg was determined for quantification (21). antibody (XMG 1.2. hybridoma cell line, kindly provided by Professor Edgar Schmitt from the Institute of Immunology at the Hematoxylin and eosin staining on murine paraffin-embedded University of Mainz, Germany). After 3 days in culture, the cells lung sections were restimulated with new medium, containing the initial dif- Lungs were removed, fixed in 10% formalin-PBS solution, ferentiation cocktail as well as 100 ng/mL IL2 (PeproTech). No dehydrated, and embedded in paraffin. Five-micrometer-thick antibodies to CD3 and CD28 were added for the restimulation. lung sections from paraffin blocks were stained with hematoxylin The cells were then cultured for additional 2 days. The cells as well and eosin for visualization of lung tumors. as the supernatants were harvested and analyzed via flow cytometry and qPCR. Flow cytometry analysis Single-cell suspensions from murine lungs were prepared as Cytotoxicity assay 5 previously described (26). Total lung cells (5 10 for surface Single-cell suspensions from murine lungs were prepared as 6 staining, 1 10 for intracellular staining) were washed with 1 mL previously described (26). Note that 1 106 total lung cells were PBS and incubated with the respective master mix of antibodies cocultured with 1 105 LL/2-luc-M38 in DMEM medium (Gibco; used for surface staining dissolved in PBS (adjusted to 80 ml) for Invitrogen), supplemented with 10% of FCS (PAA Laboratories), 30 minutes at 4 C. Afterward the cells were washed again with PBS 1% Pen/Strep (PAA Laboratories), and 1% L-Glu (Gibco; Life fl and either analyzed directly by ow cytometry or used for intra- Technologies) for 24 hours at 37 C and 5% of CO2. For cytotoxic cellular staining. analysis, 7 103 LL/2-luc-M38 cells were incubated with 20% of For intracellular staining, the cells were fixed and permea- the supernatant of the above-described total lung cell coculture for bilized with Fixation/Permeabilization concentrate/diluent 24 hours followed by bioluminescence analysis using the Centro (eBioscience) according to the manufacturer's protocol and XS3 LB 960 Microplate Luminometer (Berthold Technologies) as incubated for 30 minutes at 4 C with the respective antibodies described above. Respective LL/2-luc-M38 cell numbers were for the intracellular proteins dissolved in 1 Permeabilization calculated using a standard curve. Buffer (eBioscience, adjusted to 50 ml). Flow cytometry was performed using FACS Calibur (BD Biosciences). Data were ELISA analyzed by Cell Quest Pro version 4.02 (BD Biosciences) and Cytokines in the supernatants of cultured cells were detected by Flow-Jo v10.2 (FlowJo, LLC). specific sandwich ELISA sets. TNFa (BD Biosciences) and IFNg The following anti-mouse antibodies were used: CD4 FITC (BD ELISA (R&D Systems) were measured according to the manufac- Biosciences), CD4 PerCP (BD), CD8 FITC (BD), CD8 APC (BD), turer's protocol. CD25 PerCP (BD), RORgT PE (BD), Foxp3 APC (Miltenyi Biotec), Tbet PE (BD), Eomes EFluor 660 (eBioscience), CD127 FITC Statistical analysis (eBioscience), CD122 PE (BD), CD119 PE (eBioscience), Differences were evaluated for significance (, P ¼ 0.05; , P ¼ IL12Rb2 Alexa 488 (R&D Systems), and IFNg FITC (BD). 0.01; and , P ¼ 0.001) by the Student one-tailed t test for paired þ þ events or two-tailed t test for independent events (Excel, PC). In vitro analysis of lung CD8 and CD4 T cells þ Graphs were created with GraphPad Prism, Windows. CD8 T cells were isolated from the lungs of na€ve and tumor- bearing mice by magnetic cell separation using anti-CD8 MicroBe- ads (Miltenyi Biotec) according to the manufacturer's protocol. Results þ The CD8-negative fraction was then used to isolate CD4 T cells Increased ASM activity in NSCLC using anti-CD4 MicroBeads (Miltenyi Biotec). The purity of the To investigate the role of ASM in NSCLC, we first measured the þ þ isolated CD8 and CD4 T-cell isolation was confirmed by FACS S-ASM activity in preoperative blood serum samples of patients analysis. The isolated T cells were then stimulated in vitro with with lung adenocarcinoma and SCC as well as of healthy donors

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(HD). Here, we observed significantly increased ASM activity in However, we could not detect any difference in the ratio of C16, patients with adenocarcinoma as well as SCC (Fig. 1A). The C22, and C24:1 ceramide and sphingomyelin species, explain- function of ASM consists of the conversion of sphingomyelin to ing the fact that the total ratio was unchanged as well (Fig. 1B ceramide and phosphorylcholine (5). To further evaluate the and C; Supplementary Fig. S1A and S1B). Moreover, the cer- activity of ASM in NSCLC, we therefore measured the levels of amide-to-sphingomyelin ratio was higher in the TU region of sphingomyelin and ceramide in the blood serum, obtained adenocarcinoma and SCC patients in case of the majority of the from patients with NSCLC and healthy controls (Fig. 1B and C) measured molecular species, at least by tendency (Fig. 1D and as well as in tissue samples taken from the tumoral lung area G; Supplementary Fig. S1C–S1F), and the total ceramide-to- (TU) and the tumor cell–free control lung area (CTR) of the sphingomyelin ratio was significantly increased in lung tissue patients (Fig. 1D–I; Table 1). Ceramides and the corresponding samples from the TU region of patients with adenocarcinoma sphingomyelins can be subdividedintodifferentmolecular and SCC, as compared with the CTR lung area (Fig. 1E, F, H, species based on their carbon chain lengths. There are some and I). These data further support the discovery of increased indications that these different species have different functions. ASMactivityinbothtypesofNSCLC. For instance, it has been reported that Cer18 acts proapoptotic, whereas Cer16 promotes cell survival (27). In accordance with Targeting of SMPD1 mRNA in lung carcinoma cell lines results the upregulated ASM activity, we observed a significantly in decreased tumor cell death and proliferation increased ratio between ceramide and sphingomyelin in case Previous studies indicate that ASM-dependent ceramide of the molecular species C18, C20, and C24 in the serum of metabolism is important for various cellular processes that are patients with adenocarcinoma and SCC as compared with HDs. involved in tumorigenesis, as for example apoptosis, cell

Figure 1. Increased ASM activity and ceramide-to-sphingomyelin ratio in NSCLC. A, ASM activity was measured in blood serum from patients with adenocarcinoma (ADC) and

SCC that was taken before the surgery (presurgery) and compared with the ASM activity in the blood serum of HDs (presurgery: NHD ¼ 8, NADC ¼ 4, NSCC ¼ 9). B and C, Ceramide and sphingomyelin were measured in the blood serum of patients with adenocarcinoma and SCC as well as of HDs using mass spectrometry

(NADC ¼ 33, NSCC ¼ 14, NHD ¼ 7). B, Ratio of different molecular ceramide species to corresponding sphingomyelin species in blood serum. C, Ratio of total ceramide to total sphingomyelin levels in blood serum. D–I, Ceramide and sphingomyelin were measured in lung tissue samples taken from the tumoral region (TU) and the tumor-free control area (CTR) of patients with adenocarcinoma and SCC using mass spectrometry (NADC ¼ 26, NSCC ¼ 20). D and G, Ratio of different molecular ceramide species to corresponding sphingomyelin species in lung samples. E and H, Ratio of total ceramide to total sphingomyelin levels in lung tissue samples. F and I, Paired ceramide to total sphingomyelin levels of the tumoral region and the respective tumor-free control area of each patient. Bar charts indicate mean values SEM using Student one- (F and I) or two-tailed (A, B, C, D, E, G, and H) t test. , P ¼ 0.05; , P ¼ 0.01; and , P ¼ 0.001.

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Table 1. Clinical characteristics of the NSCLC patient cohort analyzed in this study Sample Histologic Maximal tumor Average number Sample ID classiﬁcation diameter (cm) Ta Nb Mc TNM stadium Gender Age smoking (P/Y) 1 3-MP ADC 5 2a 0 0 IB Male 79 60 2 5-MP SCC 10.5 3 0 0 IIB Female 67 50 3 9-MP ADC 2.7 1b 2 0 IIIA Female 84 0 4 13-MP SCC 3 1b 0 0 IA Male 69 50 5 15-MP ADC 2.5 1b 0 0 IA Male 63 100 6 17-MP ADC 2.6 2 0 0 IB Male 74 70 719-MPADC 6.5 2b 0 0 IIA Female 55 30 820-MPADC 2.8 1b 0 0 IA Male 65 60 9 21-MP SCC 2.5 1b 0 0 IA Male 41 10 10 22-MP ADC 7 2b 1 0 IIB Male 68 82 11 23-MP ADC 4.5 2a 0 0 IB Male 73 75 12 26-MP ADC 1.3 1a 0 1 IV Female 52 50 13 27-MP ADC 1.4 1a 0 0 IA Female 70 50 14 28-MP ADC 1.2 1a 0 0 IA Male 76 60 15 29-MP SCC 3.7 1b 0 0 IIB Male 74 100 16 30-MP SCC 1.8 1a 0 0 IA Female 70 30 17 32-MP ADC 4.4 2a 2 0 IIIA Female 60 30 18 34-MP ADC 1.8 1 0 0 IA Female 51 45 19 35-MP ADC 3 1b 0 0 IA Female 72 0 20 36-MP SCC 3.5 2a 1 0 IB Male 74 40 21 37-MP SCC 3.3 2a 1 0 IIA Male 60 45 22 40-MP ADC 1.8 1a 1 0 IIA Male 82 100 23 41-MP SCC 1.3 3 1 0 IIIA Male 70 42 24 42-MP SCC 1.5 2b 1 0 IIB Male 74 40 25 43-MP ADC 4 2a 2 0 IIIA Female 72 0 26 44-MP ADC 1.5 1a 0 0 IA Male 53 70 27 46-MP SCC 9.5 3 1 0 IIIA Male 60 30 28 47-MP SCC 6 2b 0 0 IIA Male 64 80 29 48-MP SCC 5 2b 0 0 IIA Male 55 20 30 50-MP SCC 2.5 3 1 0 IIIA Male 70 0 31 51-MP ADC 2.4 1b 2 1 IV Male 62 90 32 52-MP ADC # ### # # # # 33 53-MP ADC 2.25 1a 0 0 IA Male 62 10 34 54-MP SCC 4.1 3 0 0 IIB Male 72 0 35 55-MP ADC 1.8 1a 2 0 IIIA Female 64 40 36 56-MP ADC 4 2a 0 0 IB Female 67 0 37 57-MP ADC 3.8 2a 0 0 IB Female 35 10 38 58-MP ADC 6.5 3 0 0 IIB Female 69 0 39 59-MP ADC 0.9 4 0 0 IIIA Male 70 # 40 61-MP SCC 1.1 1a 0 0 IA Male 75 # 41 62-MP ADC 3.5 1b 0 0 IA Female 80 # 42 63-MP SCC 9 3 1 0 IIIA Male 69 # 43 64-MP ADC 3.5 1b 0 0 IA Male 55 35 44 65-MP SCC 2.8 1b 0 0 IA Female 76 # 45 68-MP ADC 8 3 0 0 IIB Male 42 22 46 72-MP ADC 3.5 ### # Male 53 # 47 73-MP ADC 4.8 2a 0 0 IB Female 67 22 48 74-MP ADC 3.2 2a 0 0 IB Female 58 # 49 75-MP SCC 4.8 2a 1 0 IIA Male 54 35 50 76-MP SCC 3.1 2a 0 0 IB Male 65 40 51 77-MP ADC 0.9 1a 0 0 IA Female 64 45 52 78-MP ADC 2.1 1b 2 0 IIIA Female 80 0 53 79-MP SCC 10 3 0 0 IIB Female 67 50 54 80-MP ADC 5.4 4 0 0 IIIA Male 62 28 55 81-MP ADC 1.6 1a 0 0 IA Male 61 46 56 82-MP SCC 8.5 3 0 0 IIB Male 82 24 57 83-MP ADC 5.5 3 0 1a IVA Female 60 0 58 84-MP SCC 7.2 2b 0 0 IIA Female 55 45 59 89-MP ADC 3.2 2 2 0 IIIA Female 61 0 60 90-MP SCC 7.4 2b 0 0 IIA Male 66 53 61 91-MP ADC 3.6 2a 2 0 IIIA Male 67 0 NOTE: #, no information available. Abbreviation: ADC, adenocarcinoma. aT-primary tumor: 0, no evidence of primary tumor; 1a, tumor 2 cm or less in greatest dimension; 1b, tumor more than 2 cm but not more than 3 cm in greatest dimension; 2a, tumor more than 3 cm but not more than 5 cm in greatest dimension; 2b, tumor more than 5 cm but not more than 7 cm in greatest dimension; 3, tumor more than 7 cm. bN-regional lymph nodes: 0, no regional lymph node metastasis; 1, metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes, including involvement by direct extension; 2, metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s). cM-distant metastasis: 0, no distant metastasis; 1, distant metastasis.

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The Role of ASM in NSCLC

differentiation, and proliferation (28, 29). We therefore wanted to Interestingly, we observed that serum starvation leads to analyze the influence of ASM on these important processes in lung SMPD1 mRNA upregulation in A549 cells, whereas it has the tumor cells. To this aim, we knocked down the expression of ASM opposite effect in H520 cells (Fig. 2A and B). To obtain a first mRNA in human lung tumor cells using SMPD1-directed siRNA indication on how the loss of ASM in tumor cells influences their (ASM-siRNA). For this method, we chose the human lung ade- growth, we counted the living A549 and H520 cells and found nocarcinoma cell line A549 as well as the squamous carcinoma that ASM knockout reduces the H520 cell numbers but does not cell line H520. To analyze the effects of SMPD1 mRNA knock- affect the overall numbers of living A549 cells (Fig. 2D; Supple- down under different metabolic conditions, we cultured the cells mentary Fig. S2B). To test if SMPD1 siRNA in lung tumor cells either with complete medium (containing 10% FCS) or with affected the induction of programmed cell death (apoptosis), we serum starvation medium (containing 0.4% FCS) after the siRNA performed an Annexin V and PI assay. Here, we found that serum transfection. We achieved specific ASM knockdown and loss of starvation leads to induction of apoptosis in A549 cells, which is ASM activity as compared with nontargeting siRNA (NT-Ctr) in prevented in ASM-siRNA–treated cells (Fig. 2E). In contrast to both media conditions (Fig. 2A and B; Supplementary Fig. S2A). that, we could not observe any changes regarding the induction of We also confirmed the functional effect of siRNA-mediated ASM apoptosis in the SCC cell line H520 in the absence of ASM depletion by measuring sphingomyelin and ceramide levels, (Supplementary Fig. S2C). We also confirmed the functional finding a reduced ceramide-to-sphingomyelin ratio in ASM- effect of siRNA-mediated ASM depletion by measuring ceramide siRNA–treated cells as compared with the nontargeting control and sphingomyelin levels in H520 cells after ASM-siRNA treat- (Fig. 2C). ment, finding reduced ceramide C16 (Supplementary Fig. S2D)

Figure 2. The inﬂuence of siRNA-mediated ASM silencing on human lung tumor cell death and proliferation. A549 and H520 cells were transfected with an SMPD1- directed siRNA (ASM-siRNA) or a NT-Control and cultured in medium containing 10% FCS or 0.4% FCS. A and B, qPCR analysis of SMPD1 mRNA expression in A549 and H520 cells (N ¼ 3). C, Ratio of different ceramide (Cer) and sphingomyelin (SM) levels in H520 cells measured by mass spectrometry (N ¼ 2). D, Cell count of H520 cells (N ¼ 3). E, Flow cytometry analysis of Annexin Vþ and PIþ A549 cells (N ¼ 3). F–G, Flow cytometry analysis of Ki67þ A549 and H520 cells (N ¼ 3). H–I, Cell-cycle analysis using ﬂow cytometry. A549 and H520 cells were stained with PI and an antibody to Ki67. Cells in G0 phase are Ki67 PI . þ þ þ Cells in G1 phase are Ki67 PI . Cells in S, G2, or M phase are Ki67 PI (N ¼ 3). N values are given per group. Bar charts indicate mean values SEM using Student two-tailed t test. , P ¼ 0.05; , P ¼ 0.01; and , P ¼ 0.001.

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and induced sphingomyelin C18, C20, C22 levels (Supplemen- receptor (TCR) signaling and mediate CD28-dependent costi- tary Fig. S2E) in ASM-siRNA–treated cells as compared with the mulation (31–33). Nevertheless, the mechanisms by which nontargeting control–treated cells. ASM can regulate T-cell activation or differentiation are not To verify the influence of ASM on tumor cell proliferation, sufficiently understood. we next performed a Ki67 and PI staining, observing that In order to investigate the role of ASM for T-cell responses in the ASM-siRNA treatment results in a cell-cycle arrest in the context of lung carcinoma, we next analyzed the expression of presence of complete medium in A549 as well as in H520 different activation markers on the surface of lung T cells from cells (Fig. 2F–I). na€ve WT and ASMKO mice. We found increased expression of IL2Ra (CD25), IL2Rb (CD122), and IL7Ra (CD127) on ASM- þ þ Facilitated Th1 differentiation of ASM-deficient na€ve deficient CD4 but not CD8 T cells (Fig. 3A–C; Supplementary þ þ CD4 T cells Fig. S3A and S3B). To further investigate the role of ASM for CD4 þ þ As above indicated, ASM has been demonstrated to be T-cell differentiation, we analyzed na€ve CD4 CD62L T cells involved in the regulation of immune responses. Thus, patients from the spleens of WT and ASM-deficient mice, regarding the with an inherited ASM deficiency show a higher risk for expression of cell surface receptors that are necessary for Th1 cell infections (30). It is known that ASM is able to modulate differentiation. Here, we found that the two Th1-differentiation intracellular signal transduction by generating ceramide. For markers IL12Rb2 and IFNgR1 (CD119) were induced on the þ instance, it has been shown that ASM can influence T-cell surface of na€ve CD4 T cells in the absence of ASM (Fig. 3D

Figure 3. Enhanced Th1 development associates with tumor growth reduction in ASM-deﬁcient mice. A–C, Flow cytometry analyses of whole lung cells derived from na€ve þ þ WT and ASMKO mice. Mean ﬂuorescence intensity (MFI) of IL2Rb and IL7Ra and percentage of IL2Ra T cells gated on CD4 T cells (NWT ¼ 4–9, NASMKO ¼ 5–11). D and E, Flow cytometry analysis of IL12Rb2 and CD119 expression levels on na€ve CD4þ T cells, isolated from the spleens of untreated WT and ASMKO mice (N ¼ 5). F, qPCR analysis of Tbx21 mRNA expression in Th1 cells after in vitro differentiation of na€ve splenic CD4þ T cells from untreated WT and ASMKO mice (N ¼ 5). G, Experimental design for the murine model of lung carcinoma. Tumor growth was induced in WT and ASMKO mice via injection of LL/2-luc-M38 lung carcinoma cells, intravenously. Tumor load was measured on days 10, 13, 17, and 20 using an in vivo imaging system. The experiment was terminated

on day 21. H, ASM activity was measured in blood serum from na€ve and tumor-bearing WT mice taken at the end of the experiment (Nna€ve ¼ 5, NLL2 ¼ 5). I, Measurement of photon ﬂux per second on days 10 to 20 (NWT ¼ 4, NASMKO ¼ 9). J, Representative hematoxylin and eosin staining of the lung of a WT and ASMKO mouse. K, Percentage weight change on day 13 after tumor induction as compared with day 0 (NWT ¼ 15, NASMKO ¼ 19). N values are given per group. Bar charts indicate mean values SEM using Student two-tailed t test. , P ¼ 0.05; , P ¼ 0.01; and , P ¼ 0.001.

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The Role of ASM in NSCLC

þ and E). The differentiation of Th1 cells requires the action of both, Although activated CD25-expressing CD4 T cells were þ þ þ IFNg and IL12. TCR stimulation and the binding of IFNg to its induced, regulatory Foxp3 CD25 CD4 T cells were unchanged receptor lead to the induction of the transcription factor Tbet (Supplementary Fig. S3D). Consistently, we also noted a larger þ þ (T-box expressed in T cells), which controls the transcriptional fraction of CD25 Foxp3 effector CD4 T cells in ASM-deficient program in Th1 cells. Tbet drives the upregulation of different mice (Fig. 4E). In conclusion, the absence of ASM shifted the T-cell Th1-associated factors, including IL12Rb2, increasing the cellular effector/suppressor ratio to the T-cell effector side. responsiveness to IL12. The binding of IL12 to its receptor further Tbet-expressing Th1 cells are considered one of the most strengthens the expression of Tbet and the establishment of the important cellular components of antitumoral immune reac- þ Th1 cell phenotype (34). tions (37). As we demonstrated that ASM-deficient na€ve CD4 þ As ASM-deficient na€ve CD4 T cells show increased expres- T cells express increased levels of Tbx21 mRNA upon Th1 in vitro sion levels of the receptor subunits IL12Rb2andIFNgR1 and differentiation (Fig. 3F), we next asked whether ASM deficiency might thus have a stronger responsiveness to the Th1-inducing also has an influence on Th1-mediated immune responses in cytokines IFNg and IL12, we examined whether the absence of the lung carcinoma model. Because Tbet is known to induce þ ASM results in increased Th1 differentiation. We therefore IFNg (38, 39), we looked at IFNg expression by CD4 Tcellsin þ þ cultured CD4 CD62L T cells from the spleens of WT and the absence and presence of ASM. Flow cytometry of whole ASMKO mice under Th1-polarizing conditions and analyzed lung cells from tumor-bearing mice revealed a significantly the mRNA expression of Tbx21 (encoding Tbet). Indeed, we larger fraction of cells expressing Tbet (Fig. 4G) as well as Tbet found that Tbx21 expression was upregulated in ASM-deficient and IFNg (Fig. 4H). Consistently, significantly higher levels of Th1 cells (Fig. 3F). IFNg (Fig. 4F) and by tendency, TNFa (Supplementary Fig. þ S3E) were found in the supernatants of CD4 T cells, isolated Targeted deletion of ASM in mice results in increased lung from the lungs of tumor-bearing ASMKO mice as compared tumor rejection with those isolated from WT mice. We also analyzed TNFa and We next analyzed WT and ASM-deficient mice in a model of IFNg in the blood serum of WT and ASMKO mice, finding that lung adenocarcinoma. In this model, the tumor induction was IFNg was below detectable levels and TNFa was unchanged, performed by intravenous injection of ASM-expressing LL/2-luc- indicating a local recruitment of Th1/Tc1 cells into the lung M38 cells (Fig. 3G). Thus, this experimental model would allow (Supplementary Fig. S3F). us to verify the role of ASM in cells of the immune system on the IFNg production is known to be one of the most important development of lung tumors. Accordingly to our results in the antitumoral mechanisms of Th1 cells (40). In order to prove human serum, in this model applied in WT littermates, we that IFNg has the ability to inhibit the growth of the lung detected increased ASM activity as well as tendentiously increased carcinoma cell line that we used for tumor induction in our ceramide-to-sphingomyelin ratios in the blood serum of lung murine model, we cultured LL/2-luc-M38 cells in the presence tumor–bearing mice as compared with na€ve mice (Fig. 3H; or absence of IFNg. LL/2-luc-M38 cells express luciferase, which Supplementary Fig. S3C). enabled us to use the bioluminescence as a measure for cell Furthermore, we found that ASM deficiency in mice results in growth. Here, we found significantly reduced luminescence reduced tumor growth as compared with WT littermates (Fig. 3I readout as well as a reduction in tumor cell number upon and J). In contrast to their WT littermates, ASM-deficient mice treatment with IFNg (Fig. 4I and J). We next analyzed the also did not lose body weight during the tumor development cytotoxicity of total lung cells isolated from the lungs of na€ve (Fig. 3K). In conclusion, the differences in tumor development WT, ASMHet (heterozygous), and ASMKO mice after coculture we observed were therefore due to the absence of ASM in the with LL/2-luc-M38 cells to mimic the situation of lung tumor ex cells present in the tumor environment, not in the tumor cells vivo. Supernatants (SN) of this coculture were used to treat themselves. LL/2-luc-M38 cells followed by the analysis of lung tumor cell growth via bioluminescence (Fig. 4K). We could show that SN þ Increased memory and effector CD4 T-cell responses in the from na€ve ASMKO mice significantly reduced LL/2-luc-M38 absence of ASM lung tumor cell number as compared with SN from na€ve WT We were wondering if the reduced tumor growth in ASMKO mice. The cytotoxicity of SN from ASM-Het mice showed the mice could possibly be explained by an enhanced antitumoral same trend, although not statistically significant. Because ASM T-cell–mediated immune response. We therefore analyzed the is known to induce apoptosis, we next performed an Annexin þ þ CD4 T cells in the lungs of LL/2-luc-M38 tumor–bearing WT V/PI staining and found the number of nonapoptotic CD4 T þ and ASMKO mice. Although the total number of CD4 T cells reduced in tumor-bearing as compared with na€ve WT lymphocytes remained unchanged (Fig. 4A), we found signif- mice (Supplementary Fig. S3G). However, no significant dif- þ icant induction of CD4 T-cell subgroups in ASM-deficient ferences could be noted between WT and ASM-deficient mice mice. (Supplementary Fig. S3H and S3I). IL7 has been described to support long-lived memory We furthermore investigated Th17 cells, a lymphocyte subset cells (35), and IL2 is a T-helper cell cytokine known to support with a controversially discussed role in tumor immunology (41), both antitumor effector cells and immunosuppressive cells and found no significant changes in ASM-deficient mice bearing upon binding to its receptor that is composed of different tumor (Supplementary Fig. S3J). combinations of the three subunits IL2Ra (CD25), IL2Rb (CD122), and IL2Rg (CD132; ref. 36). Here, we found that in Induction of lung CTL in the absence of ASM the absence of ASM, there was an induction of activated lung The above-described data indicate that inhibiting ASM in T cells þ CD4 T cells, expressing IL7Ra (CD127), IL2Ra (CD25), and infiltrating the tumor of patients with NSCLC could result in an IL2Rb (CD122; Fig. 4B–D). increased and efficient antitumor immune response. Because

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Figure 4. Increased activation of tumor-associated CD4þ T cells in ASM-deficient mice. A–H, Flow cytometry analyses of whole lung cells derived from LL/2-luc-M38 lung tumor–bearing WT and ASMKO mice according to the protocol described in Fig. 1G. A, Percentage of CD4þ T cells gated on lymphocytes þ (NWT ¼ 4, NASMKO ¼ 9). B, Mean fluorescence intensity (MFI) of CD127 gated on CD4 T cells (NWT ¼ 4, NASMKO ¼ 9). C, Mean fluorescence intensity of þ þ CD122 gated on CD4 T cells (NWT ¼ 4, NASMKO ¼ 9). D, Percentage of CD25-expressing T cells gated on CD4 T cells (NWT ¼ 18, NASMKO ¼ 23). E, Percentage of þ þ þ CD25 and Foxp3 cells gated on CD4 T cells (NWT ¼ 18, NASMKO ¼ 23). F, ELISA analysis of IFNg levels in the supernatants of CD4 T cells, isolated from the lungs of tumor-bearing WT and ASMKO mice and cultured for 40 hours in the presence of aCD3 and aCD28 antibodies (NWT ¼ 5, NASMKO ¼ 3). þ þ G, Tbet-expressing cells gated on CD4 T cells (NWT ¼ 4, NASMKO ¼ 9). H, Tbet and IFNg coexpressing cells gated on CD4 T cells (NWT ¼ 4, NASMKO ¼ 9). I, LL/2-luc-M38 cells were cultured in the presence or absence of IFNg for 48 hours and treated with luciferin prior to luminescence measurement using the IVIS imaging system (N ¼ 3each).J, LL/2-luc-M38 cell number was assessed based on the measured luminescence upon the treatment of LL/2-luc-M38 cells with IFNg or control medium for 24 hours (N ¼ 3each).K, Cytotoxicity assay of LL/2-luc-M38 cells incubated for 24 hours with supernatants obtained after coculture of total lung cells of na€ve WT (NWT ¼ 3), ASMHet (NASMHet ¼ 4), and ASMKO (NASMKO ¼ 4) mice with LL/2-luc-M38. Supernatants of CTLL2 cells were used as a positive control. LL/2-luc-M38 cell number was assessed based on the measured luminescence. N values are given per group. Bar charts indicate mean values SEM using Student two-tailed t test. , P ¼ 0.05; , P ¼ 0.01; and , P ¼ 0.001.

þ effector CD8 T cells have an antitumor function, we next ana- We next reasoned that the memory T-cell repertoire in the lung þ lyzed the number of CD8 tumor-infiltrating lymphocytes in the could be expanded in the absence of ASM. In fact, similarly to þ þ lungs of WT and ASM-deficient mice. We found a comparable CD4 T cells, the CD8 T cells, expressing the long-lived memory þ number of lung CD8 T cells in the absence of ASM (Fig. 5A). markers CD127 and CD122, were found upregulated in the þ Because ASM induces cell apoptosis, we next analyzed CD8 absence of ASM (Fig. 5E and F). T-cell apoptosis in the lungs of tumor-bearing mice. Comparable Because Tbet is the central transcription factor for the expres- þ with CD4 T cells, the tumor inoculation resulted in a reduced sion of IFNg, the cytokine released by antigen-specificantitu- þ number of nonapoptotic CD8 T cells in WT mice (Fig. 5B). mor cytotoxic T cells, we next analyzed the proportion of Tbet- þ þ Congruously, more nonapoptotic CD8 T cells could be found in positive CD8 T cells in the lungs of tumor-bearing mice. Here, þ the lungs of tumor-bearing ASM-deficient mice compared with we found a strong upregulation of Tbet in CD8 T cells in the their WT littermates (Fig. 5C). Furthermore, we found that in the absence of ASM (Fig. 5G). Granzyme B, a protein vital to the þ þ absence of ASM, tumor-infiltrating CD8 T cells exhibit a resis- effector function of CD8 T cells, was found upregulated þ tance to apoptosis (Fig. 5D). in CD8 T cells, isolated from the lungs of ASM-deficient

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The Role of ASM in NSCLC

Figure 5. Increased survival and activation of tumor-associated CD8þ T cells from ASM-deficient mice. A–H, Flow cytometry analyses of whole lung cells derived from na€ve and LL/2-luc-M38 lung tumor–bearing WT and ASMKO mice according to the protocol described in Fig. 1G. A, Percentage of CD8þ T cells gated on þ lymphocytes (NWT ¼ 4, NASMKO ¼ 9). B, Percentage of Annexin V and PI cells gated on CD8 T cells from na€ve and tumor-bearing WT mice (Nna€ve ¼ 5, þ NLL/2 ¼ 4). C, Percentage of Annexin V and PI cells gated on CD8 T cells from tumor-bearing WT and ASMKO mice (NWT ¼ 8, NASMKO ¼ 13). D, Percentage of Annexin þ þ V and CD8 T cells gated on lymphocytes (NWT ¼ 4, NASMKO ¼ 9). E, Mean fluorescence intensity (MFI) of CD127 gated on CD8 T cells (NWT ¼ 4, NASMKO ¼ 9). F, Mean þ þ fluorescence intensity of CD122 gated on CD8 T cells (NWT ¼ 4, NASMKO ¼ 9). G, Tbet-expressing cells gated on CD8 T cells (NWT ¼ 4, NASMKO ¼ 9). H, qPCR analysis of Gzmb mRNA expression in CD8þ T cells, isolated from the lungs of tumor-bearing WT and ASMKO mice and cultured for 40 hours in the presence of aCD3 and aCD28 antibodies (NWT ¼ 4, NASMKO ¼ 7). N values are given per group. Bar charts indicate mean values SEM using Student two-tailed t test. , P ¼ 0.05; , P ¼ 0.01; and , P ¼ 0.001.

tumor-bearing mice (Fig. 5H). Furthermore, ASM deficiency The evasion of apoptosis is one of the most important resulted in increased secretion of TNFa (Supplementary Fig. mechanisms by which cancer progresses (42); it is therefore S3K) and an increased mRNA expression of perforin, by ten- vital to understand how this comes to pass during the course of dency (Supplementary Fig. S3L). All taken together, these data the disease. A universal characteristic of early apoptosis is the indicate a stronger CTL-dependent antitumoral T-cell–mediat- loss of phospholipid asymmetry that can be mediated by ed immune response caused by ASM deficiency in T cells enzymes like ASM (43). Moreover, ceramide triggers p53- surrounding the lung tumor. dependent apoptosis in tumor cells (44). This indicates that ASM could play an important role for tumor-cell apoptosis. This assumption is supported by our finding that the knock- Discussion down of SMPD1 in the adenocarcinoma cell line A549, using Our present study offers insight into the involvement of ASM ASM-siRNA, prevents serum starvation–induced apoptosis. In in NSCLC. We observed an increased ASM activity in the blood contrast to that, the administration of ASM-siRNA had no as well as in the lung tumor tissue of patients with adenocar- effect on the apoptosis of the squamous carcinoma cell line cinoma and SCC as compared with tumor-free control samples H520, suggesting that this mechanism probably does not play and HDs. a role in SCC.

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Besides the ability to evade cell death, another hallmark of A key factor for cancer progression is the ability of the tumor cancer cells is uncontrolled cell proliferation (12). Consistently, in to evade the immune system, which is often mediated by regu- this study, we demonstrated that an in vitro knockdown of ASM in latory T cells (46, 47). Very recently, it has been demonstrated tumor cells leads to cell-cycle arrest in both adenocarcinoma and that ASM can influence regulatory T cell (Treg) differentiation SCC of the lung. in na€ve mice (11, 33). However, this does not appear to be the To test the role of ASM in the hosts antitumoral immune case in the tumor environment of lung adenocarcinoma, because þ þ þ response, we analyzed ASM-deficient mice in which tumor cells we found an unchanged percentage of CD4 CD25 Foxp3 Tregs expressing ASM were injected intravenously. Over the last dec- in the lungs of tumor-bearing ASMKO mice. Conversely, the þ þ ades, a lot of insight has been generated on the antitumoral number of activated T cells (CD4 CD25 ) and effector T cells þ þ capabilities of the immune system. Of fundamental importance (CD4 CD25 Foxp3 ) was increased in ASM-deficient mice, indi- are the recognition of tumor cell antigens and the subsequent cating a shift from a suppressive to an effector phenotype. This is activation of cytotoxic T cells via IFNg (40), with Th1 cells being of particular clinical significance because the prevalence of Tregs is one of the major producers of IFNg. In our study, we were able to associated with poor prognosis in several cancer types (48). show that this pathway is overly active in the lungs of ASM- All taken together, this study revealed that the role of ASM in the deficient mice. Although the overall percentage of lung tumor– pathogenesis of NSCLC is versatile and tissue-dependent. Thus, þ þ infiltrating CD4 and CD8 T cells remained unchanged, we we demonstrated that the expression of ASM in tumor cells can found a larger fraction of Th1 cells as well as Tbet-expressing have tumor-promoting as well as tumor-suppressing properties þ CD8 CTLs in the absence of ASM. Moreover, ASM-deficient due to its ability to induce apoptosis but also to facilitate cell þ þ CD4 and CD8 T cells appeared to be active on a higher proliferation. functional level, marked by increased production of IFNg by On the other hand, we have made the novel discovery of þ þ CD4 T cells as well as of granzyme B by CD8 T cells. Further- a negative regulatory role of ASM in the orchestration of more, we found an induction of CD127 and CD122 in ASM- the organisms antitumor immune response. In agreement with þ þ deficient mice, both in CD4 and CD8 T cells, indicating a larger this hypothesis, our murine model for lung adenocarcinoma memory capability of ASM-deficient immune cells. In addition, revealed a significantly decreased tumor development in ASM- we could show that supernatants from total lung cells from deficient mice. The absence of ASM resulted in an induction of Th1 ASMKO mice that were challenged by LL/2-luc-M38 cells revealed cells, memory T cells, and CTLs as well as a shift of the effector/ a higher cytotoxic potential toward LL/2-luc-M38 cells as com- regulatory T-cell ratio to the effector side, thus generating a pared with SN from lung cells from ASM Het and significantly to proinflammatory tumor microenvironment that significantly lung cells from WT mice, which further supports a stronger ameliorated the tumor rejection by the immune system. This antitumoral immune response caused by ASM deficiency. One makes ASM a promising target for the immunotherapy of NSCLC, explanation for the increased Th1-mediated antitumoral immune where the targeted inhibition of ASM in T cells—possibly achiev- response in ASM-deficient mice is the influence of ASM on the able with a large spectrum of substances (49)—may have poten- expression of T-cell receptors that play a role in T-cell differenti- tial beneficial effects for the patients' outcome. þ ation. Thus, we demonstrated that na€ve CD4 T cells from ASM knockout mice are characterized by increased levels of Th1-induc- Disclosure of Potential Conflicts of Interest ing cytokine receptors for IL12 and IFNg and consequently No potential conflicts of interest were disclosed. increased mRNA expression levels of Tbx21 after Th1 in vitro differentiation. This is of particular interest, since our group has Authors' Contributions previously shown that Tbet-deficient mice are more susceptible to Conception and design: M. Reichel, J. Kornhuber, S. Finotto lung cancer development (21) and that decreased levels of Tbet Development of methodology: C.D. Holzinger, S. Trump, B. Kleuser, S. Finotto can be found in the tumor tissue of NSCLC patients (22). Taken Acquisition of data (provided animals, acquired and managed patients, together, our data suggest that ASM may be an upstream regulator provided facilities, etc.): K. Kachler, M. Bailer, F. Schumacher, M. Reichel, of the Th1 cytokine receptors and therefore a potential target for C.D. Holzinger, S. Trump, S. Mittler, J. Monti, D.I. Trufa, R.J. Rieker, A. Hartmann, H. Sirbu, S. Finotto immunotherapy. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, As ASM possesses a well-known proapoptotic function, we computational analysis): K. Kachler, M. Bailer, M. Reichel, J. Monti, R.J. Rieker, wondered whether the mechanism by which ASM deficiency B. Kleuser, S. Finotto facilitates the CTL-dependent antitumoral immune response Writing, review, and/or revision of the manuscript: K. Kachler, M. Bailer, could involve inhibition of apoptosis. Conclusively, we found F. Schumacher, M. Reichel, R.J. Rieker, A. Hartmann, J. Kornhuber, S. Finotto þ a strong reduction of apoptotic CD8 T cells and an increase of Administrative, technical, or material support (i.e., reporting or organizing þ data, constructing databases): K. Kachler, M. Bailer, C.D. Holzinger, S. Trump, nonapoptotic CD8 T cells in the lungs of tumor-bearing ASMKO S. Mittler, J. Kornhuber, S. Finotto mice. We were also able to demonstrate that significantly fewer Study supervision: A. Hartmann, S. Finotto þ CD8 T cells are in a nonapoptotic state in the lungs of tumor- Others (conducted and analyzed one of the experiments in the paper): bearing as compared with na€ve WT mice. This indicates that in the L. Heim presence of ASM, tumor development acts as a proapoptotic þ signal on CD8 T cells in the tumor environment. This signal Acknowledgments can no longer be processed in the absence of ASM, thus decreasing We thank the team at the Department of Molecular Pneumology, espe- apoptosis of CTL and increasing the CTL-dependent antitumoral cially Adriana Geiger, Bettina Kroß, and Rebekka Springel, as well as the team immune response. Utilizing this new-found mechanism in a at the Department of Psychiatry and Psychotherapy, the Thoracic Surgery Department, and the Institute of Pathology at the University of Erlangen- clinical setting may have very beneficial effects, especially because € þ Nurnberg for their help and support to this work. The present work was in NSCLC, CD8 T-cell infiltration into the stroma surrounding performed in fulfilmentoftherequirementsforobtainingthedegree the tumor is a positive prognostic factor (45). Dr. med. for Maximilian Bailer. Parts of the data and figures will be used

OF12 Cancer Res; 77(21) November 1, 2017 Cancer Research

The Role of ASM in NSCLC

for publication in his dissertation at the Friedrich-Alexander-Universit€at The costs of publication of this article were defrayed in part by the Erlangen-Nurnberg€ payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Grant Support this fact. M. Bailer was supported by a fellowship from the IZKF in Erlangen. This project was supported by an IZKF grant number A59 awarded to S. Finotto in Received December 9, 2016; revised April 26, 2017; accepted August 31, 2017; Erlangen. published OnlineFirst September 7, 2017.

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OF14 Cancer Res; 77(21) November 1, 2017 Cancer Research

Enhanced Acid Sphingomyelinase Activity Drives Immune Evasion and Tumor Growth in Non−Small Cell Lung Carcinoma

Katerina Kachler, Maximilian Bailer, Lisanne Heim, et al.

Cancer Res Published OnlineFirst September 7, 2017.

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