Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 M. is online at: average * The Journal of Immunology published online 12 February 2016 from submission to initial decision 4 weeks from acceptance to publication http://www.jimmunol.org/content/early/2016/02/11/jimmun ol.1502364 Expression Profiling of Macrophages Reveals Multiple Populations with Distinct Biological Roles in an Immunocompetent Orthotopic Model of Lung Cancer Joanna M. Poczobutt, Subhajyoti De, Vinod K. Yadav, Teresa T. Nguyen, Howard Li, Trisha R. Sippel, Mary C. Weiser-Evans and Raphael A. Nemenoff J Immunol Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/02/11/jimmunol.150236 4.DCSupplemental Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 28, 2021. Published February 12, 2016, doi:10.4049/jimmunol.1502364 The Journal of Immunology Expression Profiling of Macrophages Reveals Multiple Populations with Distinct Biological Roles in an Immunocompetent Orthotopic Model of Lung Cancer Joanna M. Poczobutt,* Subhajyoti De,* Vinod K. Yadav,* Teresa T. Nguyen,* Howard Li,*,† Trisha R. Sippel,* Mary C. M. Weiser-Evans,*,‡ and Raphael A. Nemenoff*,‡ Macrophages represent an important component of the tumor microenvironment and play a complex role in cancer progression. These cells are characterized by a high degree of plasticity, and they alter their phenotype in response to local environmental cues. Whereas the M1/M2 classification of macrophages has been widely used, the complexity of macrophage phenotypes has not been well studied, par- ticularly in lung cancer. In this study we employed an orthotopic immunocompetent model of lung adenocarcinoma in which murine lung cancer cells are directly implanted into the left lobe of syngeneic mice. Using multimarker flow cytometry, we defined and recovered several Downloaded from distinct populations of monocytes/macrophages from tumors at different stages of progression. We used RNA-seq transcriptional profiling to define distinct features of each population and determine how they change during tumor progression. We defined an alveolar resident macrophage population that does not change in number and expresses multiple genes related to lipid metabolism and lipid signaling. We also defined a population of tumor-associated macrophages that increase dramatically with tumor and selectively expresses a panel of chemokine genes. A third population, which resembles tumor-associated monocytes, expresses a large number of genes involved in matrix remodeling. By correlating transcriptional profiles with clinically prognostic genes, we show that specific monocyte/macrophage popu- http://www.jimmunol.org/ lations are enriched in genes that predict outcomes in lung adenocarcinoma, implicating these subpopulations as critical determinants of patient survival. Our data underscore the complexity of monocytes/macrophages in the tumor microenvironment, and they suggest that distinct populations play specific roles in tumor progression. The Journal of Immunology, 2016, 196: 000–000. ung cancer remains the leading cause of cancer death in Among the diverse cell types of the TME, macrophages have been men and women, and most patients die of complications of implicated as important participants and are the most abundant non– L metastatic disease (1). Whereas tumor initiation is driven tumor cell type in most cancers (3). Studies in several types of cancer, by somatic mutations in oncogenic drivers and tumor suppressor including lung cancer, have demonstrated that macrophage depletion genes, progression and metastasis involve critical crosstalk be- either through pharmacological or genetic approaches results in by guest on September 28, 2021 tween cancer cells and the tumor microenvironment (TME) (2). slower tumor growth, implicating these cells as mediators of tumor progression (4, 5). In lung cancer, several studies have described *Department of Medicine, University of Colorado Denver, Aurora, CO 80045; correlations in macrophage number or phenotype with clinical out- †Veterans Affairs Medical Center, Denver, CO 80220; and ‡Department of Pharma- comes (6). Many of these studies have employed the model of M1 cology, University of Colorado Denver, Aurora, CO 80045 andM2macrophages.Thismodel,whichisbasedonactivationof ORCIDs: 0000-0002-7211-4176 (T.T.N.); 0000-0002-1230-2941 (H.L.). macrophages in vitro, designates a macrophage phenotype as being Received for publication November 5, 2015. Accepted for publication January 7, either proinflammatory M1, which is proposed to inhibit cancer 2016. progression, or alternatively activated M2, which is proangiogenic, This work was supported by National Institutes of Health/National Cancer Institute Grants R01 CA162226, R01 CA164780, and R01 CA108610 (to R.A.N.); the Colorado immunosuppressive, and promotes cancer progression (7, 8). Al- Lung Specialized Program of Research Excellence (National Cancer Institute Grant P50 though the M1/M2 classification of macrophages serves as a useful CA058187); Ruth L. Kirschstein National Research Service Award T32CA17468 (to T.R.S.); and by Department of Veterans Affairs Grant CDA 1IK2BX001282-01A1 (to H.L.). The starting point, it does not take into account the complexity and Flow Cytometry and the Genomics and Microarray Shared Resources receive support plasticity of these cells. Recent studies have demonstrated that dis- from the National Institutes of Health/National Cancer Institute (University of Colorado tinct populations of macrophages exist in tissues such as the lung, and Cancer Center Support Grant P30 CA046934). S.D. acknowledges support from the Boettcher Foundation and the United Against Lung Cancer Foundation. that these different populations not only express distinct markers but The RNA-seq data presented in this article have been submitted to the National also have different developmental origins (9). For example, the Center for Biotechnology Information Gene Expression Omnibus repository (http:// resident alveolar macrophages in the lung are derived from the www.ncbi.nlm.nih.gov/geo/) under accession number GSE76033. embryonic yolk sac, whereas the recruited monocytes and macro- Address correspondence and reprint requests to Prof. Raphael A. Nemenoff, Univer- sity of Colorado Denver, Department of Medicine, Division of Renal Diseases and phages are derived from the bone marrow. Hypertension C-281, 12700 East 19th Avenue, Aurora, CO 80045. E-mail address: These different populations are likely to play distinct and po- [email protected] tentially opposing roles in cancer progression. In fact, tumors are The online version of this article contains supplemental material. infiltrated by different dynamically changing populations of Abbreviations used in this article: COX, cyclooxygenase; ECM, extracellular matrix; FPKM, monocytes/macrophages (8), and identifying key features of each fragments per kilobase of exon per million fragments mapped; GSEA, gene set enrich- ment analysis; ImmGen, Immunological Genome Project; LLC, Lewis lung carcinoma; population may help to target them therapeutically. LTC4, leukotriene C4; MHC II, MHC class II; -N, from naive mice; PPAR, peroxisome To study the role of the TME in lung cancer, we have devel- proliferator–activated receptor; RNA-seq, RNA sequencing; TME, tumor microenvironment; oped an orthotopic immunocompetent mouse model, in which -2wk, from 2 wk tumor-bearing mice; -3wk, from 3 wk tumor-bearing mice. Lewis lung carcinoma (LLC) cells, which were derived from a Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 spontaneous lung adenocarcinoma in C57BL/6 mice, are directly www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502364 2 EXPRESSION PROFILING OF MACROPHAGES IN LUNG CANCER implanted into the lungs of fully immunocompetent syngeneic RNA extraction and RNA-seq C57BL/6 mice. Within 2–3 wk these cells form a primary tumor Total RNAwas isolated from flow cytometry–sorted cells using QIAshredders that metastasizes to the other lung lobes, mediastinal lymph nodes, and an RNeasy Plus micro kit (Qiagen). RNA quality and quantity were liver, and brain. We have previously shown that tumor progression analyzed using a NanoDrop and Bioanalyzer. RNA-seq library preparation in this model is associated with increases in several monocyte/ and sequencing were conducted at the Genomics and Microarray Core at the macrophage populations (10). However, functional differences University of Colorado Denver–Anschutz Medical Campus. Libraries were constructed using a NuGen Ovation human FFPE RNA-seq multiplex system between these populations have not been identified, and the role of kit customized with mouse specific oligonucleotides for rRNA removal. specific monocyte/macrophage subtypes in tumor progression is Directional mRNA-seq was conducted