Published OnlineFirst January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2184 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Surfactant Expression Defines an Inflamed Subtype of Lung Adenocarcinoma Brain Metastases that Correlates with Prolonged Survival Kolja Pocha1, Andreas Mock1,2,3,4, Carmen Rapp1, Steffen Dettling1, Rolf Warta1,4, Christoph Geisenberger1, Christine Jungk1, Leila R. Martins5, Niels Grabe6, David Reuss7,8, Juergen Debus4,9, Andreas von Deimling4,7,8, Amir Abdollahi4,9, Andreas Unterberg1, and Christel C. Herold-Mende1,4 ABSTRACT ◥ Purpose: To provide a better understanding of the interplay of three surfactant metabolism-related genes (SFTPA1, SFTPB, between the immune system and brain metastases to advance and NAPSA) was closely associated with TIL numbers. Their therapeutic options for this life-threatening disease. expression was not only prognostic in brain metastasis but also Experimental Design: Tumor-infiltrating lymphocytes (TIL) in primary lung adenocarcinoma. Correlation with scRNA-seq were quantified by semiautomated whole-slide analysis in data revealed that brain metastases with high expression of brain metastases from 81 lung adenocarcinomas. Multi-color surfactant genes might originate from tumor cells resembling staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) alveolar type 2 cells. Methylome-based estimation of immune cell on a single-cell resolution. Molecular determinants of the fractions in primary lung adenocarcinoma confirmed a positive extent of TILs in brain metastases were analyzed by transcrip- association between lymphocyte infiltration and surfactant tomics in a subset of 63 patients. Findings in lung adenocarci- expression. Tumors with a high surfactant expression displayed noma brain metastases were related to published multi-omic atranscriptomicprofile of an inflammatory microenvironment. primary lung adenocarcinoma The Cancer Genome Atlas data Conclusions: The expression of surfactant metabolism-related (n ¼ 230) and single-cell RNA-sequencing (scRNA-seq) data genes (SFTPA1, SFTPB,andNAPSA)defines an inflamed subtype (n ¼ 52,698). of lung adenocarcinoma brain metastases characterized by high Results: TIL numbers within tumor islands was an independent abundance of TILs in close vicinity to tumor cells, a prolonged prognostic marker in patients with lung adenocarcinoma brain survival, and a tumor microenvironment which might be more metastases. Comparative transcriptomics revealed that expression accessible to immunotherapeutic approaches. Introduction histologically be divided into two groups: small-cell lung cancer and non–small cell lung cancer (NSCLC). NSCLC can be further sub- Lung cancer is the most common type of cancer worldwide and the divided into subtypes, the most frequent being adenocarcinoma (2, 3). most common cause of cancer-related death (1, 2). Lung cancer can Lung adenocarcinoma also accounts for the largest histologic subset of brain metastases (4). Of note, brain metastases are even more frequent than primary brain tumors (5). Patients with brain metastases have a 1Division of Experimental Neurosurgery, Department of Neurosurgery, poor median overall survival of 7–13 months (6). Treatment is usually Heidelberg University Hospital, Heidelberg, Germany. 2Department of Medical multimodal and consists of systemic chemotherapy combined with Oncology, National Center for Tumor Diseases (NCT) Heidelberg, Heidelberg 3 microsurgery, stereotactic radiosurgery, and/or radiotherapy (7). University Hospital, Heidelberg, Germany. Department of Translational Medical There is increasing evidence for the role of the host immune system Oncology, National Center for Tumor Diseases (NCT) Heidelberg, German Cancer Research Center (DKFZ), Heidelberg, Germany. 4German Cancer in cancer development, suppression, and recurrence (8). In colorectal Consortium (DKTK), Heidelberg, Germany. 5Division of Applied Functional cancer for instance, the quantification of tumor-infiltrating lympho- Genomics, German Cancer Research Center (DKFZ) Heidelberg, Heidelberg, cytes (TIL) has become a valid prognostic marker for patient survival Germany. 6Hamamatsu Tissue Imaging and Analysis Center (TIGA), BIOQUANT, and is believed to be superior to the tumor–node–metastasis classi- 7 University of Heidelberg, Heidelberg, Germany. Department of Neuropathol- fication (9). Many studies have since confirmed the prognostic power ogy, Institute of Pathology, Heidelberg University Hospital, Heidelberg, of immune cell infiltrates in a large number of cancer types. The Germany. 8Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Institute of Pathology, Heidelberg University Hospital, Heidel- positive effect of cytotoxic T cells and Th1 T cells on survival has been berg, Germany. 9Department of Radiation Oncology, University of Heidelberg, shown; however, the influence of intratumoral Th2, Th17, and reg- Heidelberg, Germany. ulatory T cells (Tregs) on survival is less clear (10). In patients with Note: Supplementary data for this article are available at Clinical Cancer lung adenocarcinoma with high overall T-cell counts and high cyto- Research Online (http://clincancerres.aacrjournals.org/). toxic T-cell counts, prolonged survival has been observed (11). Tregs, K. Pocha and A. Mock contributed equally to this article. on the other hand, seem to impair prognosis (12). However, in addition to quantity, the spatial distribution of immune Corresponding Author: Christel C. Herold-Mende, Heidelberg University cells and their vicinity to tumor cells in terms of localization within Hospital, INF 400, Heidelberg 69120, Germany. Phone: 4962-2156-6405; Fax: 4962-2156-33979; E-mail: [email protected] tumor islands or retention in the tumor stroma (13, 14) and the differences between primary tumors and subsequent metastases must Clin Cancer Res 2020;26:2231–43 be taken into account. It has been shown that infiltration with cytotoxic doi: 10.1158/1078-0432.CCR-19-2184 T cells decreases from primary lung cancer to metastases (15, 16). Ó2020 American Association for Cancer Research. Nevertheless, data on the impact of T-cell infiltration on survival in AACRJournals.org | 2231 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst January 17, 2020; DOI: 10.1158/1078-0432.CCR-19-2184 Pocha et al. informed consent was obtained from all patients in accordance with Translational Relevance the Declaration of Helsinki and its later amendments. Immunotherapies have become a powerful addition to the established therapies in metastatic non–small cell lung cancer. Immunofluorescence staining Given the dismal prognosis of brain metastases in these patients Tumor tissue was snap-frozen in liquid nitrogen–cooled isopentane it is essential to identify subsets of patients that have a tumor immediately after resection. Frozen tissues were cut into 5–7 mm slices, microenvironment that might be more accessible to immunother- acetone-fixed, and stored at À80C until staining. Immunofluorescent apeutic approaches. We provide evidence that a fraction of lung staining was performed using anti-CD3 (Dako, A0452), anti-CD8 adenocarcinoma brain metastases is indeed characterized by higher (Clone YTC182.20, Abcam, Ab60076), and anti-FOXP3 (Clone 236A/ tumor-infiltrating lymphocyte numbers and is in close association E7, Abcam, Ab20034) as described previously (27). DAPI (Invitrogen, with an inflamed microenvironment and high expression of sur- D1306) was used to counterstain the nuclei. The incubation time was factant genes. 1 hour, and this was followed by three washing steps. Secondary antibodies, including anti-rabbit Alexa Fluor 647 (Life Technologies, A21245), anti-rat Alexa Fluor 488 (Life Technologies, A11006), and anti-mouse Alexa Fluor 555 (Life Technologies, A31570) were then brain metastasis remain controversial, with some studies suggesting a applied for 1 hour, and this was followed by three washing steps. þ þ þ favorable role for CD3 , CD8 , and CD45R0 T cells (17) and other Tumor cells were labeled using anti-pan cytokeratin (Progen, 61835; studies showing no benefit (18, 19). Dako, M0630) staining in combination with an antibody labeling kit Understanding the interplay between the immune system and (Thermo Fisher Scientific, Z25002). Isotype controls were rabbit IgG cancer cells is becoming even more critical in patients with brain (Dako, X0936), IgG2b (eBioscience, 14-4031), and IgG1 (Abcam, metastases as checkpoint inhibitor therapies are on the rise for ab91353). The antibody characteristics are listed in Supplementary treating lung adenocarcinoma (20). Knowledge about the immune Table S1. microenvironment in primary lung cancer cannot be extrapolated to include brain metastases, because of specialized resident immune Semi-automated quantification of T-cell infiltration and stromal cells, including microglia and astrocytes (21–23), and Following immunofluorescent staining, whole-slide images were physical restriction by the blood–brain barrier (24). Genomic generated for all 81 tumor samples using an automated microscope alterations between primary lung cancer and brain metastasis, as (Olympus IX51 equipped with a F-View II camera, both Olympus) at well as their connection to the immune system are the subject of 20-fold magnification with the cellSens Software (Olympus). Tissue- ongoing research (25). Other data suggest that the amount of T-cell Quest Software (version 4.0.1.0137, TissueGnostics GmbH)