Phospholipase D2 downregulates interleukin-1β secretion from tumor-associated macrophages to suppress bladder cancer progression

The tumor microenvironment (TME) modulates therapeutic response and prognosis in patients with bladder cancer (BC). The roles of two phospholipase D (PLD) isoforms, PLD1 and PLD2 (hydrolysis of phosphatidylcholine to phosphatidic acid) in cancer cells have been well-studied in numerous cancer types, but their roles in the TME remain unclear. We used a mouse BC Pld2-knockout carcinogenesis model and global transcriptomic analysis to reveal that PLD2 was significantly involved in BC progression through immunosuppressive pathways in the TME. We therefore focused on PLD2 and tumor-associated macrophages (TAMs), which were increased in Pld2-knockout mice and further associated with poor prognoses in BC patients. In vitro, we found that Pld2- knockout mouse TAMs had significantly enhanced proliferation, correlating closely with increased IL-1β production. These results indicate that PLD2 suppresses BC progression by regulation of IL-1β secretion from TAMs in the TME, suggesting that PLD2 could serve as a potential therapeutic target for BC. To induce bladder cancer in mice, female 6-8-week-old WT, Pld1-/- and Pld2-/- mice were given drinking water with 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) (Tokyo Kasei Kogyo).Total RNA from extracted whole bladders was isolated using a NucleoSpin RNA kit (TaKaRa).Total RNA was extracted from sorted tumor-associated macrophages using TRIzol reagent (Invitrogen).For RNA sequencing, 500 ng total RNA were rRNA-depleted using the NEBNext rRNA Depletion Kit (New England Biolabs) according to the manufacturer’s instructions. Library preparation was performed using an NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). Libraries were validated via Bioanalyzer (Agilent Technologies) to determine size distribution and concentration. Validated libraries were then sequenced at Tsukuba i-Laboratory LLP (Tsukuba, Japan) on a NextSeq500 (Illumina) with the paired-end 36-base read option.Sequencing reads were mapped to the mm10 mouse reference genome, quantified, and then normalized with the quantile method using CLC Genomics Workbench version 10.1.1 (Qiagen).