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.

Methods
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 bisulfite sequencing

WGBS profiling of GM-CSF-grown + IFNg+LPS 24 hours stimulated cells (Control), PGE2-treated EP2 WT and EP2 KO Bone marrow cells.
DNA was harvested using QIAamp DNA Kits for DNA Extraction kit (Qiagen). 100ng of total RNA was used for the construction of sequencing libraries. 
libraries for WGBS were prepared using Accel-NGS Methyl-Seq DNA Library Kit (Illumina) following manufacturer's protocols.