Comment[ArrayExpressAccession] E-GEOD-43254 MAGE-TAB Version 1.1 Public Release Date 2013-01-04 Investigation Title Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils Comment[Submitted Name] Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils Experiment Description The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naive neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-a, IL-1-a/b), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations. Various types of myeloid cells have been shown to promote tumor progression by direct immune suppression and by production of angiogenic factors, matrix-degrading enzymes, or growth factors. In untreated tumors, neutrophils have been reported to produce angiogenic factors and matrix-degrading enzymes, support the acquisition of a metastatic phenotype, and suppress the anti-tumor immune response. Neutrophils, like all other leukocytes, move into tissues from the blood under the influence of specific chemokines (e.g. KC/CXCL-1, MIP-2a/CXCL-2 and GCP-2/CXCL-6), cytokines (e.g. TNFa and IFN-x), and cell adhesion molecules located on their own surface (e.g. CD11b) and on the surface of endothelial cells (e.g. selectins, ICAM-1 and PECAM-1). When they traffic into tumors, they are referred to as TAN. In mice, TAN can be defined by the specific surface markers CD11b and Ly6G with low expression of macrophage markers such as F4/80. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune suppressive cells that are produced excessively in cancer. They comprise at least two subsets -granulocytic (Ly6G+, G-MDSC) and monocytic cells (Ly6C+, M-MDSC), potentially with different immunosuppressive properties. It has been previously shown that MDSC can enter tumors and differentiate to mature macrophages (TAM) or neutrophils (TAN). However, since no definitive markers have been established, it is unknown whether intratumoral N2 neutrophils (N2 TAN) are granulocytic MDSC from spleen that are attracted to the tumor or if they are typical blood-derived neutrophils that are then converted to an N2 phenotype by the tumor microenvironment, specifically by the high local concentrations of TGF-b. The purpose of this study was to use a transcriptomics approach to gain further information about TANs by comparing the RNA profile of these cells to naive bone-marrow neutrophils (NN) and to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC). We examined which pathways and gene-groups varied amongst these 3 populations of neutrophils and performed a detailed analysis on pathways related to the main functions of neutrophils, such as respiratory burst, granule proteins, phagocytosis, apoptosis, structural genes, antigen presentation and specific immune effects. Our data defines TAN as a unique population of neutrophils, quite distinct from both NN and G-MDSC. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Showe Fridlender Sun Mishalian Singhal Cheng Kapoor Horng Fridlender Bayuh Worthen Albelda Person First Name Louise Zvi Jing Inbal Sunil Guanjun Veena Wenhwai Gil Rachel Scott Steven Person Mid Initials C G G M Person Email lshowe@wistar.org Person Affiliation The Wistar Institute Person Phone 215-898-3791 Person Fax 215-898-4521 Person Address The Wistar Institute, 3601 Spruce St, Philadelphia, PA, USA Person Roles submitter Protocol Name P-GSE43254-1 P-GSE43254-5 P-GSE43254-4 P-GSE43254-2 P-GSE43254-3 P-GSE43254-6 Protocol Description ID_REF = VALUE = normalized data Illumina Setting with PMTFactor=2 Hyb on Illumina Mouse Ref-8 v2 Expression BeadChip Kit using Illumina whole-Genome Gene Exprssion with Intellihyb Seal system which detect signal with streptavidin-Cy3. Gene expression was analyzed in neutrophils from 3 different populations M-bM-^@M-^S 7 isolated from bone marrow of naM-CM-/ve mice (NN), 4 isolated from spleens of tumor bearing mice (Granulocytic myeloid derived suppressor cells, G-MDSC), and 4 isolated from flank AB12 tumors (Tumor associated neutrophils, TAN). All neutrophil populations were defined as Ly6G+. Neutrophils were isolated following preparation of single cell suspension, using CD11b-Miltenni beads for enrichment, followed by flow-sorting of the Ly6G+ cells. RNA was processed as previously described and hybridized on Illumina Mouse Ref-8 v2 microarray chips. Biotin Labeling of RNA at 250ng with Ambion IlluminaM-. TotalPrep RNA Amplification Kit (Cat #AMIL1791) Array data were processed by IlluminaM-bM-^@M-^Ys BeadStudio software and expression levels exported for analysis. The gene-wise, median correlation of each array compared to all other arrays was computed to ensure that no outliers existed within the data. The expression levels were then quantile normalized using Matlab software (2010b) and non-informative probes (those with detection p-value >0.05 in all samples) were removed to reduce experimental noise. Ultimately, 16,322 probes were used in our analysis. Protocol Type bioassay_data_transformation image_aquisition hybridization nucleic_acid_extraction labeling feature_extraction Experimental Factor Name GROUP SAMPLE ID CONTROL GROUP Experimental Factor Type group sample id control group Publication Title Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils. Publication Author List Fridlender ZG, Sun J, Mishalian I, Singhal S, Cheng G, Kapoor V, Horng W, Fridlender G, Bayuh R, Worthen GS, Albelda SM PubMed ID 22348096 Publication DOI 10.1371/journal.pone.0031524 Comment[SecondaryAccession] GSE43254 Comment[GEOReleaseDate] 2013-01-04 Comment[ArrayExpressSubmissionDate] 2013-01-02 Comment[GEOLastUpdateDate] 2013-01-05 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-43254.sdrf.txt