Macropinocytosis of neutrophil extracellular trap by gastric cancer cells promotes immune escape and peritoneal metastasis

BACKGROUND: Patients with gastric cancer (GC) and peritoneal metastasis (PM) have poor prognoses due to drug resistance and metastatic relapse. The mechanism underlying PM recurrence remains unclear. OBJECTIVE: This study investigates the mechanisms of ascitic neutrophil extracellular traps (NETs) in promoting GC immune escape and PM recurrence. DESIGN: Ascites from a patient with GC were analysed using flow cytometry, immunofluorescence and electron microscopy. In vitro and in vivo experiments demonstrated NETs promoted GC immune evasion. RNA sequencing and functional models identified the immune checkpoint molecule CEACAM1. Internalisation assay combined with inhibitors determined tumour cells internalised NETs via macropinocytosis. Immunofluorescence and western blot revealed that NETs upregulated CEACAM1 and programmed death-ligand 1 (PD-L1) by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. The cotranscription factor interferon regulatory factor 1(IRF1) for CEACAM1 and PD-L1 were identified by dual-luciferase reporter assays and chromatin immunoprecipitation. Conditioned medium-culture systems identified CSF2 and CSF3 as NETs-inducing factors. Combination of DNase I and PD-L1 antibody was evaluated in humanised mice, organoid-T cell cocultures and ex-vivo ascites therapy models. RESULTS: We uncovered that macropinocytosis mediated uptake of NETs remodels an immunosuppressive microenvironment and promotes PM recurrence. Increased neutrophils and NETs in PM-relapsed ascites promote GC cell immune escape. Mechanistically, GC cells uptake NETs-DNA via macropinocytosis. Macropinocytic NETs-DNA activate the cGAS-STING-IRF1 pathway, upregulate immune-checkpoint molecules CEACAM1 and PD-L1, leading to immune escape. Moreover, clearing NETs-DNA combining with PD-L1 antibody achieves a synergistic inhibitory effect on PM. CONCLUSION: We highlight the importance of macropinocytosis mediated uptake of NETs in GC immune escape and PM recurrence, providing a potential combination therapeutic strategy for patients with PM.