Targeting plasticity in the pyrimidine synthesis pathway potentiates macrophage-mediated phagocytosis in pancreatic cancer models
Jie Zhao, Xinghao Li, Xinyu Li, Pengfei Ren, Yilan Wu, Hao Gong, Lijian Wu, Junran Huang, Saisai Wang, Ziwei Guo, Mo Chen, Zexian Zeng, Deng Pan
Jie Zhao, Xinghao Li, Xinyu Li, Pengfei Ren, Yilan Wu, Hao Gong, Lijian Wu, Junran Huang, Saisai Wang, Ziwei Guo, Mo Chen, Zexian Zeng, Deng Pan
View: Text | PDF
Research Article Immunology Metabolism Oncology

Targeting plasticity in the pyrimidine synthesis pathway potentiates macrophage-mediated phagocytosis in pancreatic cancer models

Abstract

Macrophage-mediated phagocytosis plays a critical role in the elimination of cancer cells and shaping antitumor immunity. However, the tumor-intrinsic pathways that regulate cancer cell sensitivity to macrophage-mediated phagocytosis remain poorly defined. In this study, we performed a genome-wide CRISPR screen in murine pancreatic cancer cells cocultured with primary macrophages and identified that disruption of the tumor-intrinsic pyrimidine synthesis pathway enhances phagocytosis. Mechanistically, we discovered that macrophages inhibit the pyrimidine salvage pathway in tumor cells by upregulating Upp1-mediated uridine degradation through cytokines TNF-α and IL-1. This shift increased tumor cells’ reliance on de novo pyrimidine synthesis. As a result, tumor cells with impaired de novo pyrimidine synthesis showed depleted UMP and displayed enhanced exposure of phosphatidylserine (PtdSer), a major “eat-me” signal, thereby promoting macrophage-mediated phagocytosis. In multiple pancreatic cancer models, Cad-deficient tumors exhibited markedly reduced tumor burden with increased levels of phagocytosis by macrophages. Importantly, the Cad-mediated suppression of pancreatic cancer was dependent on TAMs and cytokines IL-1 and TNF-α. Pharmacological inhibition of DHODH, which blocks de novo pyrimidine synthesis, similarly decreased tumor burden with enhanced phagocytosis in pancreatic cancer models. These findings highlight the critical role of the tumor-intrinsic pyrimidine synthesis pathway in modulating macrophage-mediated antitumor immunity, with potential therapeutic implications.

Authors

Jie Zhao, Xinghao Li, Xinyu Li, Pengfei Ren, Yilan Wu, Hao Gong, Lijian Wu, Junran Huang, Saisai Wang, Ziwei Guo, Mo Chen, Zexian Zeng, Deng Pan

×

Figure 1

Loss of function of de novo pyrimidine synthesis pathway sensitized pancreatic tumor cells to macrophage-mediated phagocytosis.

Options: View larger image (or click on image) Download as PowerPoint
Loss of function of de novo pyrimidine synthesis pathway sensitized panc...
(A) Workflow of CRISPR screen for tumor-intrinsic regulators of macrophage-mediated phagocytosis. (B) Scatter plot showing the top depleted sgRNAs based on mean log2 fold change of sgRNA counts in BMDM coculture condition versus control condition. Annotated genes represent the CD47 pathway (red), the de novo pyrimidine synthesis pathway (blue), and endocytosis regulators (black). (C) Metascape analysis of pathways among top depleted hits (FDR < 0.1). (D and E) In vitro competition assay based on coculture of BMDMs and Panc02 tumor cells expressing Fc fragment (D) or parental Panc02 cells (E). Control Panc02-Fc cells were mixed with cells with indicated genes knocked out (labeled with CFSE) and then cocultured with BMDMs for 24 hours. Log2 fold change of the percentage of KO cells upon coculture with BMDMs was shown. (F and G) In vitro phagocytosis assay. BMDMs were cocultured with CFSE-labeled control or Cad-KO Panc02 (F) or KC-806 (G) cells for 24 hours. Percentages of phagocytosis (F4/80+ CSFE+) were quantified by FACS. (H) In vitro phagocytosis assay by using tumor-educated macrophages (TEMs). TEMs were cocultured with CFSE-labeled control or Cad-KO Panc02 cells for 24 hours. Percentages of phagocytosis (F4/80+CSFE+) were quantified by FACS. (I) In vitro phagocytosis assay by using TAMs isolated from an autochthonous pancreatic cancer model driven by KrasG12D expression and P53 inactivation. Coculture assays were performed using TAMs and CFSE-labeled Panc02 cells. Phagocytosis percentages (F4/80+CFSE+) were quantified by FACS. A schematic representation of TAM isolation from the autochthonous pancreatic tumor (created using BioRender.com) and statistical analysis are shown. Data are presented as mean ± SD and analyzed by 1-way ANOVA (D–G) or unpaired t test (H and I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are representative of at least 2 independent experiments (D–I).