Abstract 1912: Significant changes in chemokine genes in tumor-associated neutrophils (TANs) using a microarray approach

Abstract

Abstract Background: The role of tumor-associated neutrophils (TAN) in tumor biology is unclear. We have recently published that resident TAN can acquire a protumor phenotype (similar to M2 macrophages), largely driven by TGF-beta to become N2 neutrophils. After TGF-beta blockade, neutrophils acquire an antitumor phenotype (N1 TAN (similar to M1 macrophages). In the current study, we further evaluated the characteristics of TAN using a gene microarray approach. Methods: Balb/C mice (10-20 in each group) bearing large flank tumors of the mesothelioma line AB12, where treated with either control or SM16 (a TGF-β-R1 kinase inhibitor) chow. After one week of treatment, the tumors were harvested, and neutrophils were isolated using microbeads (CD11b) and flow cytometry (Ly6G), into two groups - control TAN (N2) and SM16 TAN (N1). Neutrophils were also isolated from bone marrow of naïve mice (BM) and from spleens harvested from tumor-bearing mice (i.e., the granulocytic fraction of myeloid derived suppressor cells (G-MDSC)). mRNA from each subgroup (4-6 samples) was isolated and subjected to microarray analysis on Illumina chips. Selected results were validated by RT-PCR and neutrophils from a second non Small Cell Lung Cancer tumor, LKR, were studied. Results: After filtering, we found 16,322 informative probes. Hierarchical clustering and PCA analysis showed that the four groups were clearly separated. We found that many more genes were up-regulated than down-regulated in the N2 TAN from untreated mice compared to either BM or G-MDSC. Many of the highly up-regulated genes in TAN were chemokines, and genes encoding for inflammatory and immune responses, especially chemokines. This includes chemoattractants for T-cells (e.g. CXCL-9, CXCL10 and CXCL16), neutrophils (eg. CXCL1, CXCL2 and CCL3), B-cells (CXCL13) and macrophages (e.g. CCL2, CXCL-10 and CCL7). N1 (SM16-treated) and N2 TAN were more similar to each other than to the other groups, suggesting an intra-tumoral polarization between them. In N1 TAN, three different chemokines attracting macrophages were up-regulated - CCL2, CCL6 and CXCL10, and only one major chemokine, the T-regs chemoattractant CCL17, was downregulated. There was no significant difference in most other chemokines. Conclusion: Our data shows the major differences between naïve neutrophils, G-MDSC and N1/N2 TAN. The patterns support the hypothesis that neutrophils enter the tumor, possibly as G-MDSC, and the change between N2 and N1 is intra-tumoral (and not by differential recruitment). Our data further suggests that TAN secrete many chemokines, initiating the recruitment of other inflammatory cells of the immune system. N1 neutrophils appear to up-regulate several macrophage attracting chemokines compared to N2 TAN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1912.