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

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. Overall design: 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.

髓系细胞（myeloid cells）在支持肿瘤生长中的作用已得到充分证实。既往多数研究聚焦于荷瘤动物中聚集的髓系来源抑制细胞（myeloid-derived suppressor cells, MDSC），但已知肿瘤相关中性粒细胞（tumor-associated neutrophils, TAN）同样可促进肿瘤生长。然而，目前对其演化过程、表型特征，以及与未致敏中性粒细胞（naive neutrophils, NN）和粒性髓系来源抑制细胞（granulocytic fraction of MDSC, G-MDSC）的关联尚不清楚。

本研究以小鼠为模型，采用转录组学方法对上述细胞类型进行比较分析。结果显示，三类中性粒细胞的mRNA表达谱存在显著差异：未致敏中性粒细胞（NN）与粒性髓系来源抑制细胞（G-MDSC）的亲缘关系较二者与肿瘤相关中性粒细胞（TAN）更为接近。肿瘤相关中性粒细胞（TAN）中，结构基因与细胞毒性相关基因（如呼吸爆发相关基因）的表达显著下调。与之相反，粒性髓系来源抑制细胞（G-MDSC）中多种免疫相关基因及通路出现上调，而肿瘤相关中性粒细胞（TAN）中此类基因的表达进一步升高，包括抗原呈递复合物相关基因（如全部6种MHC-II复合物基因）以及细胞因子（如TNF-α、IL-1α/β）。相较于未致敏中性粒细胞（NN），本研究检测的25种趋化因子中有13种在肿瘤相关中性粒细胞（TAN）中显著上调，其中包括T/B细胞、中性粒细胞及巨噬细胞趋化因子的显著上调。本研究对与肿瘤相关的各类中性粒细胞亚群进行了表征，明确了肿瘤相关中性粒细胞（TAN）与其他两类中性粒细胞群体间的显著差异。

整体设计：已有研究表明，各类髓系细胞可通过直接免疫抑制、分泌血管生成因子、基质降解酶或生长因子促进肿瘤进展。在未接受治疗的肿瘤中，中性粒细胞可分泌血管生成因子与基质降解酶，辅助肿瘤获得转移表型，并抑制抗肿瘤免疫应答。与其他白细胞类似，中性粒细胞可在特定趋化因子（如KC/CXCL-1、MIP-2α/CXCL-2及GCP-2/CXCL-6）、细胞因子（如TNF-α与IFN-x），以及表达于自身表面（如CD11b）和内皮细胞表面（如选择素、ICAM-1、PECAM-1）的细胞黏附分子的调控下，从血液迁移至组织。当中性粒细胞迁移进入肿瘤时，即被称为肿瘤相关中性粒细胞（TAN）。

在小鼠模型中，肿瘤相关中性粒细胞（TAN）可通过特异性表面标志物CD11b与Ly6G，以及低表达巨噬细胞标志物（如F4/80）进行鉴定。髓系来源抑制细胞（MDSC）是一类在癌症中过度产生的异质性免疫抑制细胞群体，至少包含两个亚群：粒性亚群（Ly6G+，即G-MDSC）与单核细胞亚群（Ly6C+，即M-MDSC），二者可能具有不同的免疫抑制特性。既往研究表明，MDSC可进入肿瘤并分化为成熟巨噬细胞（肿瘤相关巨噬细胞, TAM）或中性粒细胞（TAN）。然而，由于尚未建立明确的鉴定标志物，目前尚不清楚瘤内N2中性粒细胞（N2 TAN）究竟是由脾脏来源的粒性MDSC招募至肿瘤，还是由血液来源的典型中性粒细胞在肿瘤微环境（尤其是局部高浓度TGF-β）的作用下转化为N2表型。

本研究的目的在于采用转录组学方法，通过比较肿瘤相关中性粒细胞（TAN）、未致敏骨髓中性粒细胞（NN）以及粒性髓系来源抑制细胞（G-MDSC）的RNA表达谱，进一步解析肿瘤相关中性粒细胞（TAN）的相关信息。我们分析了这三类中性粒细胞群体间存在差异的通路与基因簇，并针对中性粒细胞主要功能相关通路进行了详细分析，包括呼吸爆发、颗粒蛋白、吞噬作用、细胞凋亡、结构基因、抗原呈递及特异性免疫效应等。本研究结果明确了肿瘤相关中性粒细胞（TAN）是一类独特的中性粒细胞群体，与未致敏中性粒细胞（NN）及粒性髓系来源抑制细胞（G-MDSC）均存在显著差异。