Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils

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)

Validation of array results by real time PCR - Ratio of expression in TAN compared to NN.

<p>Confirmation of selected results from the microchip array using real time RT-PCR in isolated tumor associated neutrophils (TAN) from flank tumors of two separate tumor cell lines – the mesothelioma cell line AB12 (Balb/C), and the NSCLC cell line LKR (B6-129/J1). Table shows the gene fold-change of TAN to Naïve neutrophils (NN) using the arrays or by RT-PCR measurements.</p><p>++ - Fold-change >100, +++ - Fold-change >500, ND – Not Done.</p

Validation of array results by real time PCR - Ratio of expression in TAN compared to G-MDSC.

<p>Confirmation of selected results from the microchip array using real time RT-PCR in isolated tumor associated neutrophils (TAN) from flank tumors of two separate tumor cell lines – the mesothelioma cell line AB12 (Balb/C), and the NSCLC cell line LKR (B6-129/J1). Table shows the gene fold-change of TAN to Naïve neutrophils (NN) using the arrays or by RT-PCR measurements.</p><p>++ - Fold-change >100, ND – Not Done.</p

Validation of array results at the protein level.

<p>(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).</p

Summary of the relative changes in the different neutrophil populations.

<p>Pathways and gene-groups were evaluated by the Genomica software, and manually from the literature. The data of each neutrophils-function evaluated for each population of neutrophils is presented.</p><p>(−) - Most genes in the pathway/group were at background levels.</p><p>(+/−) – Some genes of the pathway/group were up-regulated and other down-regulated.</p><p>(+) – A related pathway/group was up-regulated (Genomica), or some (>10%) of the genes in the group were up-regulated (manually).</p><p>(++) – A related pathway/group was up-regulated (Genomica), and/or a significant portion (>30%) of the genes in the group were up-regulated (manually). </p><p>(+++) – A prominent up-regulation of genes in the group/pathway (>50%) was noted.</p

Analysis of pathways and gene groups using Genomica, comparing tumor associated neutrophils (TAN) to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC).

<p>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.</p

Analysis of pathways and gene groups using Genomica, comparing naïve neutrophils (NN) to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC).

<p>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.</p

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.

<p>Red – up-regulation; Blue – down-regulation; White – no change from mean.</p

Heatmaps comparing the most different genes between the 3 groups of neutrophils.

<p>(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.</p