Single-cell sequencing combined with spatial transcriptomics reveals that the IRF7 gene in M1 macrophages inhibits the occurrence of pancreatic cancer by regulating lipid metabolism-related mechanisms

Aim: The main focus of this study is to explore the molecular mechanism of IRF7 regulation on RPS18 transcription in M1-type macrophages in pancreatic adenocarcinoma (PAAD) tissue, as well as the transfer of RPS18 by IRF7 via exosomes to PAAD cells and the regulation of ILF3 expression.

Methods: By utilising single-cell RNA Sequencing (scRNA-seq) data and spatial transcriptomics (ST) data from the Gene Expression Omnibus database, we identified distinct cell types with significant expression differences in PAAD tissue. Among these cell types, we identified those closely associated with lipid metabolism. The differentially expressed genes within these cell types were analysed, and target genes relevant to prognosis were identified. Flow cytometry was employed to assess the expression levels of target genes in M1 and M2 macrophages. Cell lines with target gene knockout were constructed using CRISPR/Cas9 editing technology, and cell lines with target gene knockdown and overexpression were established using lentiviral vectors. Additionally, a co-culture model of exosomes derived from M1 macrophages with PAAD cells was developed. The impact of M1 macrophage-derived exosomes on the lipid metabolism of PAAD cells in the model was evaluated through metabolomics analysis. The effects of M1 macrophage-derived exosomes on the viability, proliferation, division, migration and Apoptosis of PAAD cells were assessed using MTT assay, flow cytometry, EdU assay, wound healing assay, Transwell assay and TUNEL staining. Furthermore, a mouse PAAD orthotopic implantation model was established, and bioluminescence imaging was utilised to assess the influence of M1 macrophage-derived exosomes on the intratumoural formation capacity of PAAD cells, as well as measuring tumour weight and volume. The expression of proliferation-associated proteins in tumour tissues was examined using immunohistochemistry.

Results: Through combined analysis of scRNA-seq and ST technologies, we discovered a close association between M1 macrophages in PAAD samples and lipid metabolism signals, as well as a negative correlation between M1 macrophages and Cancer cells. The construction of a prognostic risk score model identified RPS18 and IRF7 as two prognostically relevant genes in M1 macrophages, exhibiting negative and positive correlations, respectively. Mechanistically, it was found that IRF7 in M1 macrophages can inhibit the transcription of RPS18, reducing the transfer of RPS18 to PAAD cells via exosomes, consequently affecting the expression of ILF3 in PAAD cells. IRF7/RPS18 in M1 macrophages can also suppress lipid metabolism, cell viability, proliferation, migration, invasion and intratumoural formation capacity of PAAD cells, while promoting cell Apoptosis.

Conclusion: Overexpression of IRF7 in M1 macrophages may inhibit RPS18 transcription, reduce the transfer of RPS18 from M1 macrophage-derived exosomes to PAAD cells, thereby suppressing ILF3 expression in PAAD cells, inhibiting the lipid metabolism pathway, and curtailing the viability, proliferation, migration, invasion of PAAD cells, as well as enhancing cell Apoptosis, ultimately inhibiting tumour formation in PAAD cells in vivo. Targeting IRF7/RPS18 in M1 macrophages could represent a promising immunotherapeutic approach for PAAD in the future.

ILF3; IRF7; M1 macrophages; RPS18; exosomes; pancreatic adenocarcinoma; scRNA‐seq; spatial transcriptomics.