{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T22:41:51Z","timestamp":1791499311774,"version":"4.3.4"},"reference-count":53,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,11,4]],"date-time":"2021-11-04T00:00:00Z","timestamp":1635984000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>\n                    This study attempted to profile the tumor immune microenvironment (TIME) of non-small cell lung cancer (NSCLC) by multiplex immunofluorescence of 681 NSCLC cases. The number, density, and proportion of 26 types of immune cells in tumor nest and tumor stroma were evaluated, revealing some close interactions particularly between intrastromal neutrophils and intratumoral regulatory T cells (Treg) (\n                    <jats:italic>r<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    = 0.439,\n                    <jats:italic>P<\/jats:italic>\n                    &amp;lt; 0.001), intrastromal CD4+CD38+ T cells and CD20-positive B cells (\n                    <jats:italic>r<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    = 0.539,\n                    <jats:italic>P<\/jats:italic>\n                    &amp;lt; 0.001), and intratumoral CD8-positive T cells and M2 macrophages expressing PD-L1 (\n                    <jats:italic>r<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u00a0= 0.339,\n                    <jats:italic>P<\/jats:italic>\n                    &amp;lt; 0.001). Three immune subtypes correlated with distinct immune characteristics were identified using the unsupervised consensus clustering approach. The immune-activated subtype had the longest disease-free survival (DFS) and demonstrated the highest infiltration of CD4-positive T cells, CD8-positive T cells, and CD20-positive B cells. The immune-defected subtype was rich in cancer stem cells and\u00a0macrophages, and these patients had the worst prognosis. The immune-exempted subtype had the highest levels of neutrophils and Tregs. Intratumoral CD68-positive macrophages, M1 macrophages, and intrastromal CD4+ cells, CD4+FOXP3- cells, CD8+ cells, and PD-L1+ cells were further found to be the most robust prognostic biomarkers for DFS, which were used to construct and validate the immune-related risk score for risk stratification (high\n                    <jats:italic>vs<\/jats:italic>\n                    . median\n                    <jats:italic>vs<\/jats:italic>\n                    . low) and the prediction of 5-year DFS rates (23.2%\n                    <jats:italic>vs<\/jats:italic>\n                    . 37.9%\n                    <jats:italic>vs<\/jats:italic>\n                    . 43.1%,\n                    <jats:italic>P<\/jats:italic>\n                    &amp;lt; 0.001). In conclusion, the intricate and intrinsic structure of TIME in NSCLC was demonstrated, showing potency in subtyping and prognostication.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2021.750046","type":"journal-article","created":{"date-parts":[[2021,11,4]],"date-time":"2021-11-04T05:12:59Z","timestamp":1636002779000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":51,"title":["Profiling Tumor Immune Microenvironment of Non-Small Cell Lung Cancer Using Multiplex Immunofluorescence"],"prefix":"10.3389","volume":"12","author":[{"given":"Haoxin","family":"Peng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangrong","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ran","family":"Zhong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuyu","family":"Cai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaokai","family":"Wen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiyuan","family":"Ao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiana","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yutian","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miao","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caichen","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongbo","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanhui","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenkui","family":"Pan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianxing","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenhua","family":"Liang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,11,4]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.3322\/caac.21660","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J Clin"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra0802714","article-title":"Lung Cancer","volume":"359","author":"Herbst","year":"2008","journal-title":"N Engl J Med"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2019.11058","article-title":"Systemic Therapy for Locally Advanced and Metastatic Non-Small Cell Lung Cancer: A Review","volume":"322","author":"Arbour","year":"2019","journal-title":"JAMA"},{"key":"B4","doi-asserted-by":"publisher","first-page":"4931","DOI":"10.3390\/ijms20194931","article-title":"Prognostic Factors and Biomarkers of Responses to Immune Checkpoint Inhibitors in Lung Cancer","volume":"20","author":"Bianco","year":"2019","journal-title":"Int J Mol Sci"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1093\/annonc\/mdz207","article-title":"Suppressed Immune Microenvironment and Repertoire in Brain Metastases From Patients With Resected Non-Small-Cell Lung Cancer","volume":"30","author":"Kudo","year":"2019","journal-title":"Ann Oncol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-018-0014-x","article-title":"Understanding the Tumor Immune Microenvironment (TIME) for Effective Therapy","volume":"24","author":"Binnewies","year":"2018","journal-title":"Nat Med"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-018-0096-5","article-title":"Phenotype Molding of Stromal Cells in the Lung Tumor Microenvironment","volume":"24","author":"Lambrechts","year":"2018","journal-title":"Nat Med"},{"key":"B8","doi-asserted-by":"publisher","first-page":"339","DOI":"10.1186\/s40425-019-0785-8","article-title":"Tumor Microenvironment Dictates Regulatory T Cell Phenotype: Upregulated Immune Checkpoints Reinforce Suppressive Function","volume":"7","author":"Kim","year":"2019","journal-title":"J Immunother Cancer"},{"key":"B9","doi-asserted-by":"publisher","first-page":"2186","DOI":"10.3390\/ijms18102186","article-title":"Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated With Intravesical Bacille Calmette-Gu\u00e9rin: A Long-Term Follow-Up Study of a Japanese Cohort","volume":"18","author":"Miyake","year":"2017","journal-title":"Int J Mol Sci"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2017.11.052","article-title":"Neutrophils and Snail Orchestrate the Establishment of a Pro-Tumor Microenvironment in Lung Cancer","volume":"21","author":"Faget","year":"2017","journal-title":"Cell Rep"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1038\/nature25183","article-title":"The Biology and Management of Non-Small Cell Lung Cancer","volume":"553","author":"Herbst","year":"2018","journal-title":"Nature"},{"key":"B12","doi-asserted-by":"publisher","first-page":"1904","DOI":"10.1016\/S0140-6736(19)32503-6","article-title":"Quantifying the Benefit of Non-Small-Cell Lung Cancer Immunotherapy","volume":"394","author":"Ludmir","year":"2019","journal-title":"Lancet"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-18-1538","article-title":"Immunotherapy in Non-Small Cell Lung Cancer: Facts and Hopes","volume":"25","author":"Doroshow","year":"2019","journal-title":"Clin Cancer Res"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1016\/j.annonc.2021.02.006","article-title":"High Tumor Mutation Burden Fails to Predict Immune Checkpoint Blockade Response Across All Cancer Types","volume":"32","author":"McGrail","year":"2021","journal-title":"Ann Oncol"},{"key":"B15","doi-asserted-by":"publisher","first-page":"887","DOI":"10.1002\/1878-0261.12891","article-title":"Molecular Profiling of Long-Term Responders to Immune Checkpoint Inhibitors in Advanced Non-Small Cell Lung Cancer","volume":"15","author":"Frigola","year":"2021","journal-title":"Mol Oncol"},{"key":"B16","doi-asserted-by":"publisher","first-page":"e196879","DOI":"10.1001\/jamanetworkopen.2019.6879","article-title":"Association of Survival and Immune-Related Biomarkers With Immunotherapy in Patients With Non-Small Cell Lung Cancer: A Meta-Analysis and Individual Patient-Level Analysis","volume":"2","author":"Yu","year":"2019","journal-title":"JAMA Netw Open"},{"key":"B17","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1186\/s40425-018-0368-0","article-title":"Effect of Neoadjuvant Chemotherapy on the Immune Microenvironment in Non-Small Cell Lung Carcinomas as Determined by Multiplex Immunofluorescence and Image Analysis Approaches","volume":"6","author":"Parra","year":"2018","journal-title":"J Immunother Cancer"},{"key":"B18","doi-asserted-by":"publisher","first-page":"e0247238","DOI":"10.1371\/journal.pone.0247238","article-title":"Developing an Enhanced 7-Color Multiplex IHC Protocol to Dissect Immune Infiltration in Human Cancers","volume":"16","author":"Sun","year":"2021","journal-title":"PloS One"},{"key":"B19","unstructured":"NCCN Clinical Practice Guidelines in Oncology: Non-Small Cell Lung Cancer, V.42019"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1097\/JTO.0b013e3181a0d82e","article-title":"The IASLC Lung Cancer Staging Project: A Proposal for a New International Lymph Node Map in the Forthcoming Seventh Edition of the TNM Classification for Lung Cancer","volume":"4","author":"Rusch","year":"2009","journal-title":"J Thorac Oncol"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2018.03.023","article-title":"The Immune Landscape of Cancer","volume":"48","author":"Thorsson","year":"2018","journal-title":"Immunity"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btq170","article-title":"ConsensusClusterPlus: A Class Discovery Tool With Confidence Assessments and Item Tracking","volume":"26","author":"Wilkerson","year":"2010","journal-title":"Bioinformatics"},{"key":"B23","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1002\/bimj.200900028","article-title":"L1 Penalized Estimation in the Cox Proportional Hazards Model","volume":"52","author":"Goeman","year":"2010","journal-title":"Biom J"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2019.00453","article-title":"Analysis of Survival Curves: Statistical Methods Accounting for the Presence of Long-Term Survivors","volume":"9","author":"Damuzzo","year":"2019","journal-title":"Front Oncol"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-04-0713","article-title":"X-Tile: A New Bio-Informatics Tool for Biomarker Assessment and Outcome-Based Cut-Point Optimization","volume":"10","author":"Camp","year":"2004","journal-title":"Clin Cancer Res"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2013.281053","article-title":"Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects","volume":"310","year":"2013","journal-title":"JAMA"},{"key":"B27","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1007\/s00005-013-0250-1","article-title":"Flow Cytometric Analysis of CD133- and EpCAM-Positive Cells in the Peripheral Blood of Patients With Lung Cancer","volume":"62","author":"Skirecki","year":"2014","journal-title":"Arch Immunol Ther Exp (Warsz)"},{"key":"B28","doi-asserted-by":"publisher","first-page":"e54193","DOI":"10.1371\/journal.pone.0054193","article-title":"Generation and Characterisation of Cisplatin-Resistant non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature","volume":"8","author":"Barr","year":"2013","journal-title":"PloS One"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2011.11.050","article-title":"Glycine Decarboxylase Activity Drives non-Small Cell Lung Cancer Tumor-Initiating Cells and Tumorigenesis","volume":"148","author":"Zhang","year":"2012","journal-title":"Cell"},{"key":"B30","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1186\/s40169-018-0198-1","article-title":"Medicine T. The Role of CD133 in Cancer: A Concise Review","volume":"7","author":"Glumac","year":"2018","journal-title":"Clin Transl Med"},{"key":"B31","doi-asserted-by":"publisher","first-page":"1527","DOI":"10.3390\/cells8121527","article-title":"CD38, a Receptor With Multifunctional Activities: From Modulatory Functions on Regulatory Cell Subsets and Extracellular Vesicles, to a Target for Therapeutic Strategies","volume":"8","author":"Morandi","year":"2019","journal-title":"Cells"},{"key":"B32","doi-asserted-by":"publisher","first-page":"309","DOI":"10.3390\/cancers13020309","article-title":"IFN-\u03b3 and CD38 in Hyperprogressive Cancer Development","volume":"13","author":"Angelicola","year":"2021","journal-title":"Cancers (Basel)"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1016\/j.coi.2012.12.003","article-title":"T Cell Anergy, Exhaustion, Senescence, and Stemness in the Tumor Microenvironment","volume":"25","author":"Crespo","year":"2013","journal-title":"Curr Opin Immunol"},{"key":"B34","doi-asserted-by":"publisher","first-page":"eaau0135","DOI":"10.1126\/science.aau0135","article-title":"T Cell Stemness and Dysfunction in Tumors Are Triggered by a Common Mechanism","volume":"363","author":"Vodnala","year":"2019","journal-title":"Science"},{"key":"B35","doi-asserted-by":"publisher","first-page":"8941260","DOI":"10.1155\/2016\/8941260","article-title":"Effector, Memory, and Dysfunctional CD8(+) T Cell Fates in the Antitumor Immune Response","volume":"2016","author":"Reiser","year":"2016","journal-title":"J Immunol Res"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2016.02.040","article-title":"Tumor-Associated Neutrophils Recruit Macrophages and T-Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and Resistance to Sorafenib","volume":"150","author":"Zhou","year":"2016","journal-title":"Gastroenterology"},{"key":"B37","doi-asserted-by":"publisher","first-page":"898","DOI":"10.1158\/2326-6066.CIR-17-0075","article-title":"Antigen-Presenting Intratumoral B Cells Affect CD4 TIL Phenotypes in Non-Small Cell Lung Cancer Patients","volume":"5","author":"Bruno","year":"2017","journal-title":"Cancer Immunol Res"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-11-1354","article-title":"Tumor-Associated Macrophages Promote Angiogenesis and Melanoma Growth via Adrenomedullin in a Paracrine and Autocrine Manner","volume":"17","author":"Chen","year":"2011","journal-title":"Clin Cancer Res"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1016\/j.neo.2019.01.005","article-title":"Prognostic Value of Macrophage Phenotypes in Resectable Non-Small Cell Lung Cancer Assessed by Multiplex Immunohistochemistry","volume":"21","author":"Rakaee","year":"2019","journal-title":"Neoplasia"},{"key":"B40","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1146\/annurev-pathol-012615-044438","article-title":"Cancer Stem Cells: Basic Concepts and Therapeutic Implications","volume":"11","author":"Nassar","year":"2016","journal-title":"Annu Rev Pathol"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1002\/jcp.27740","article-title":"Cancer Stem Cells (CSCs) in Cancer Progression and Therapy","volume":"234","author":"Najafi","year":"2019","journal-title":"J Cell Physiol"},{"key":"B42","article-title":"Tumor-Associated Macrophages Increase the Proportion of Cancer Stem Cells in Lymphoma by Secreting Pleiotrophin","volume":"11","author":"Wei","year":"2019","journal-title":"Am J Transl Res"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1007\/s10555-014-9529-x","article-title":"Bennewith KJCmr. Emerging Roles of Regulatory T Cells in Tumour Progression and Metastasis","volume":"33","author":"Halvorsen","year":"2014","journal-title":"Cancer Metastasis Rev"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1172\/JCI77053","article-title":"Tumor-Associated Neutrophils Stimulate T Cell Responses in Early-Stage Human Lung Cancer","volume":"124","author":"Eruslanov","year":"2014","journal-title":"J Clin Invest"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2084","article-title":"Neutrophil Elastase-Mediated Degradation of IRS-1 Accelerates Lung Tumor Growth","volume":"16","author":"Houghton","year":"2010","journal-title":"Nat Med"},{"key":"B46","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1126\/science.aaa6204","article-title":"T Cell Exclusion, Immune Privilege, and the Tumor Microenvironment","volume":"348","author":"Joyce","year":"2015","journal-title":"Science"},{"key":"B47","doi-asserted-by":"publisher","first-page":"e000155corr1","DOI":"10.1136\/jitc-2019-000155corr1","article-title":"The Society for Immunotherapy of Cancer Statement on Best Practices for Multiplex Immunohistochemistry (IHC) and Immunofluorescence (IF) Staining and Validation","volume":"8","author":"Taube","year":"2020","journal-title":"J Immunother Cancer"},{"key":"B48","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1093\/annonc\/mdy470","article-title":"Identification and Validation of Novel Microenvironment-Based Immune Molecular Subgroups of Head and Neck Squamous Cell Carcinoma: Implications for Immunotherapy","volume":"30","author":"Chen","year":"2019","journal-title":"Ann Oncol"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1038\/nri3073","article-title":"Wynn TJNrI. Protective and Pathogenic Functions of Macrophage Subsets","volume":"11","author":"Murray","year":"2011","journal-title":"Nat Rev Immunol"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-019-0127-6","article-title":"Ruffell BJNrI. Macrophages as Regulators of Tumour Immunity and Immunotherapy","volume":"19","author":"DeNardo","year":"2019","journal-title":"Nat Rev Immunol"},{"key":"B51","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1186\/s40425-017-0218-5","article-title":"Wolchok JJJfioc. Combination Immunotherapy: A Road Map","volume":"5","author":"Ott","year":"2017","journal-title":"J Immunother Cancer"},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.1093\/jnci\/djx121","article-title":"Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4","volume":"110","author":"Hanley","year":"2018","journal-title":"J Natl Cancer Inst"},{"key":"B53","doi-asserted-by":"publisher","DOI":"10.1038\/nrclinonc.2018.9","article-title":"Kerbel RJNrCo. Improving Immunotherapy Outcomes With Anti-Angiogenic Treatments and Vice Versa","volume":"15","author":"Khan","year":"2018","journal-title":"Nat Rev Clin Oncol"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2021.750046\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,4]],"date-time":"2021-11-04T05:13:05Z","timestamp":1636002785000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2021.750046\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,4]]},"references-count":53,"alternative-id":["10.3389\/fimmu.2021.750046"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2021.750046","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.05.28.446005","asserted-by":"object"}]},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,4]]},"article-number":"750046"}}