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Comparative Study
. 2012;7(2):e31524.
doi: 10.1371/journal.pone.0031524. Epub 2012 Feb 14.

Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils

Zvi G Fridlender  1 , Jing Sun, Inbal Mishalian, Sunil Singhal, Guanjun Cheng, Veena Kapoor, Wenhwai Horng, Gil Fridlender, Rachel Bayuh, G Scott Worthen, Steven M Albelda
Affiliations
Comparative Study

Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils

Zvi G Fridlender et al. PLoS One. 2012.

Abstract

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 naïve 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-α, IL-1-α/β), 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Hierarchical clustering (A) and principal component analysis (PCA) (B) of the samples, showing that the three groups of neutrophils are distinct.
□ - Naïve neutrophils (NN); * - Granulocytic fraction of MDSC (G-MDSC). ◊ - Tumor associated neutrophils (TAN).
Figure 2
Figure 2. Heatmaps comparing the most different genes between the 3 groups of neutrophils.
(A) – Heatmap of genes with fold-change between any two groups ≥30. The TAN exhibit a signature that is markedly different than the other two types of neutrophilic cells. (B) Heatmap with all genes with a fold-change ≥8 between NN and G-MDSC, showing a clearly different signature in these two groups of cells. Red – up-regulation; Blue – down-regulation; White – no change from mean.
Figure 3
Figure 3. Validation of array results at the protein level.
(A) – comparison between the secretions of the different cytokines to the supernatant following isolation of each of the neutrophil populations. (B) – An example of TNF-α levels in Ly6G+ neutrophils, comparing TAN (left)to NN (right). A clear up-regulation, similar to the increased m-RNA in the array and in RT-PCR is shown. (C) – An immunoblot, showing the expression of CCL-17 in a protein extract of TAN (left), and the lack of expression in protein extract of NN (right).
Figure 4
Figure 4. Analysis of pathways and gene groups using Genomica, comparing tumor associated neutrophils (TAN) to naïve neutrophils (NN).
Each sample was evaluated for changes in the different pathways, and was marked as positive when ≥3 genes were significantly changed to the same direction. In each panel - Right - Pathways/Groups that had more than 5 samples changed. Left - Pathways/Groups that were significantly changed when the groups were compared to each other (p<0.05, corrected). Red – up-regulation; Blue – down-regulation; Black – no change from mean.
Figure 5
Figure 5. Analysis of pathways and gene groups using Genomica, comparing tumor associated neutrophils (TAN) to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC).
Each sample was evaluated for changes in the different pathways, and was marked as positive when ≥3 genes were significantly changed to the same direction. In each panel - Right - Pathways/Groups that had more than 5 samples changed. Left - Pathways/Groups that were significantly changed when the groups were compared to each other (p<0.05, corrected). Red – up-regulation; Blue – down-regulation; Black – no change from mean.
Figure 6
Figure 6. Analysis of pathways and gene groups using Genomica, comparing naïve neutrophils (NN) to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC).
Each sample was evaluated for changes in the different pathways, and was marked as positive when ≥3 genes were significantly changed to the same direction. In each panel - Right - Pathways/Groups that had more than 5 samples changed. Left - Pathways/Groups that were significantly changed when the groups were compared to each other (p<0.05, corrected). Red – up-regulation; Blue – down-regulation; Black – no change from mean.
Figure 7
Figure 7. Heatmap comparing the expression of chemokines in the three groups of neutrophils - naïve neutrophils (NN), granulocytic fraction of myeloid derived suppressor cells (G-MDSC) and tumor associated neutrophils (TAN), clearly showing that many chemokines were up-regulated in the TAN group.
Red – up-regulation; Blue – down-regulation; White – no change from mean.
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