MHC-II+ macrophage differentiation is impaired in metastasized lungs via PGE2 receptor EP2

Monocytes differentiate into macrophages (Mφs) to facilitate lung metastasis, but the monocyte-to-Mφ transition during this process is not well understood. To investigate, we performed bulk RNA sequencing on Mφs isolated from the lungs of mice bearing Lewis lung carcinoma tumors and from naive lungs. Our results showed impaired differentiation of monocytes into MHC-II+ Mφ, with upregulation of PGE2-inducible genes, including Arg1, in tumor-associated Mφs (TAMs). In vitro experiments confirmed that PGE2 inhibits the differentiation of MHC-II+ Mφ while promoting Arg1+ Mφ via the E prostanoid 2 (EP2) receptor, accompanied by DNA methylation. Whole genome bisulfite sequencing revealed that PGE2-EP2 signaling drives hypermethylation and downregulation of gene sets related to myeloid cells in non-neoplastic tissues. Our study highlights PGE2-EP2-driven DNA methylation in the monocyte-to-TAM transition, suggesting potential therapeutic avenues for lung metastasis., NC and LLC Mφs RNA-seq

mRNA-seq profiling of FACS-sorted lung macrophages (CD45+Ly6G-Ly6C-MerTK+CD64+SiglecF-CD11b+) from wild-type C57BL/6 male mice and Lewis Lung Carcinoma (LLC)-intravenously injected mice.
RNA was harvested using Rneasy mini plus kit (Qiagen). 10ng of total RNA was used for the construction of sequencing libraries. 
RNA libraries for RNA-seq were prepared using SMARTer Ultra Low RNA Kit (Takara) following manufacturer's protocols.

In vitro Control, PGE2-treated EP2 WT and EP2 KO BM cells RNA-Seq

mRNA-seq profiling of GM-CSF-grown + IFNg+LPS 24 hours stimulated cells (Control), PGE2-treated EP2 WT and EP2 KO Bone marrow cells.
RNA was harvested using Rneasy mini plus kit (Qiagen). 1ug of total RNA was used for the construction of sequencing libraries. 
RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA Library Prep Kit (Illumina) following manufacturer's protocols.

In vitro Control, PGE2-treated EP2 WT and EP2 KO BM cells Whole genome bi..., , # MHC-II+ macrophage differentiation is impaired in metastasized lungs via PGE2 receptor EP2

[https://doi.org/10.5061/dryad.jm63xsjjc](https://doi.org/10.5061/dryad.jm63xsjjc)

**ALL of the RAW data were processed and prepared by Macrogen ([https://dna.macrogen.com/](https://dna.macrogen.com/))**

**For macrophages sorted from NC (negative control/naive) and LLC lungs RNA-seq**

* **Naive-IM1-3.fastq.gz / LLC-IM1-3.fastq.gz**
* Samples were obtained by these procedures:
* mRNA-seq profiling of FACS-sorted lung macrophages (CD45+Ly6G-Ly6C-MerTK+CD64+SiglecF-CD11b+) from wild-type C57BL/6 male mice and Lewis Lung Carcinoma (LLC)-intravenously injected mice.
* RNA was harvested using Rneasy mini plus kit (Qiagen). 10ng of total RNA was used for the construction of sequencing libraries. 
* RNA libraries for RNA-seq were prepared using SMARTer Ultra Low RNA Kit (Takara) following manufacturer's protocols.

***In vitro*** **Control, PGE2-treated EP2 WT and EP2 KO BM cells RNA-Seq**

* ...,

单核细胞可分化为巨噬细胞（macrophages, Mφs）以促进肺转移，但该过程中单核细胞向巨噬细胞的转化机制尚未完全明晰。为探究该机制，我们对荷路易斯肺癌（Lewis Lung Carcinoma, LLC）小鼠肺部及正常野生型小鼠肺部分离的巨噬细胞进行了批量RNA测序（bulk RNA sequencing）。结果显示，肿瘤相关巨噬细胞（tumor-associated Mφs, TAMs）中，单核细胞向MHC-II+巨噬细胞的分化受损，且前列腺素E2（Prostaglandin E2, PGE2）诱导的基因（包括Arg1）表达显著上调。体外实验证实，PGE2可通过前列腺素E2受体2（E prostanoid 2, EP2）抑制MHC-II+巨噬细胞分化，同时促进Arg1+巨噬细胞的生成，该调控过程伴随DNA甲基化修饰。全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing）结果显示，PGE2-EP2信号通路可驱动非肿瘤组织中髓系细胞相关基因的高甲基化与表达下调。本研究揭示了PGE2-EP2介导的DNA甲基化在单核细胞向肿瘤相关巨噬细胞转化过程中的关键作用，为肺转移的临床治疗提供了潜在靶点。
# 转移灶肺部中MHC-II+巨噬细胞分化受损通过PGE2受体EP2介导
## 正常对照（NC）与LLC巨噬细胞RNA测序
mRNA测序分析对象为野生型C57BL/6雄性小鼠及经尾静脉注射路易斯肺癌细胞的小鼠体内，经流式细胞术分选（Fluorescence-Activated Cell Sorting, FACS）得到的肺巨噬细胞（CD45+Ly6G-Ly6C-MerTK+CD64+SiglecF-CD11b+）。
总RNA采用RNeasy Mini Plus试剂盒（Qiagen）提取，取10ng总RNA用于测序文库构建。RNA-seq文库采用SMARTer Ultra Low RNA试剂盒（Takara），严格按照制造商说明书进行制备。
对应样本文件：Naive-IM1-3.fastq.gz / LLC-IM1-3.fastq.gz
样本获取流程如下：
* mRNA测序分析对象为野生型C57BL/6雄性小鼠及经尾静脉注射路易斯肺癌细胞的小鼠体内，经流式细胞术分选的肺巨噬细胞（CD45+Ly6G-Ly6C-MerTK+CD64+SiglecF-CD11b+）。
* 总RNA采用RNeasy Mini Plus试剂盒（Qiagen）提取，取10ng总RNA用于测序文库构建。
* RNA-seq文库采用SMARTer Ultra Low RNA试剂盒（Takara），严格按照制造商说明书进行制备。

## 体外实验：PGE2处理的EP2野生型与EP2敲除骨髓细胞RNA测序
mRNA测序分析对象包括三组样本：经粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）培养并联合干扰素γ（Interferon-γ, IFN-γ）、脂多糖（Lipopolysaccharide, LPS）刺激24小时的骨髓细胞（对照组）、经PGE2处理的EP2野生型骨髓细胞、经PGE2处理的EP2敲除骨髓细胞。
总RNA采用RNeasy Mini Plus试剂盒（Qiagen）提取，取1μg总RNA用于测序文库构建。RNA-seq文库采用TruSeq Stranded mRNA Library Prep试剂盒（Illumina），严格按照制造商说明书进行制备。

### 其他补充说明
1. 所有原始测序数据均由Macrogen公司（https://dna.macrogen.com/）完成处理与制备。
2. 该研究相关DOI：https://doi.org/10.5061/dryad.jm63xsjjc