CXCL12 upregulates PD-L1 expression and promotes M2 polarisation of tumour-associated macrophages to confer anti-PD-1 resistance in gastric cancer.

OBJECTIVE

Immunotherapy offers promise for gastric cancer (GC) patients, yet its efficacy is substantially limited by high rates of treatment resistance, the mechanisms of which remain incompletely characterised. We aimed to delineate the role and mechanistic basis of CXCL12 in conferring anti-PD-1 resistance in GC.

METHODS

An anti-PD-1-resistant GC mouse model was established through chronic anti-PD-1 induction and in vivo tumour tissue passaging. Human and murine GC cell lines stably overexpressing CXCL12 (OE-CXCL12) were generated via lentiviral transduction to construct corresponding OE-CXCL12 GC mouse models. Single-cell transcriptomics analysed the tumour microenvironment (TME) in anti-PD-1-resistant GC. Molecular docking and Drug Affinity Responsive Target Stability (DARTS) assays predicted and validated ligand-receptor binding, respectively. RNA or protein expression of specific molecules was quantified by RT-qPCR, ELISA, immunofluorescence, and western blotting.

RESULTS

Anti-PD-1-resistant GC models showed upregulated CXCL12, PD-L1, and CD206 expression with unchanged PD-1. OE-CXCL12 reduced anti-PD-1 sensitivity, elevated M2-TAM infiltration and TGF-β, IL-10, IL-4 levels in TME, reduced M1-TAM and CD8⁺ T cell infiltration and IL-12 level, with differentially expressed genes enriched in HIF-1, PD-L1/PD-1 checkpoint, and PI3K/AKT signalling pathways. M2-TAM-conditioned medium elevated SOX2, OCT-4, NANOG in GC cells. Mechanistically, CXCL12 binds CXCR4 to activate PI3K/AKT/HIF-1α, promoting PD-L1 expression and M2-TAM polarisation. CXCL12 inhibition recovered anti-PD-1 efficacy.

CONCLUSIONS

This identifies CXCL12 as a novel anti-PD-1 resistance gene in GC, with the CXCL12/CXCR4/PI3K/AKT/HIF-1α axis induces anti-PD-1 tolerance through a dual-pathway mechanism of promoting PD-L1 expression and M2-TAM polarisation, providing potential therapeutic strategies to prevent and reverse anti-PD-1 resistance in GC.