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. 2021 Oct 25;9(10):e002699. doi: 10.1136/jitc-2021-002699

Figure 7.

Figure 7

Blockade of PD-L2 glycosylation enhances the response to cetux in the orthotopic model of HNSCC. (A) Representative bioluminescence images of C3H mice with tumors derived from SCC7 cells transfected with PD-L2 WT or REY mutant and treated with cetux every week (n=7 mice per group). *p<0.05; **p<0.01; #, not significant. (B) The intracellular cetux content in SCC7 tumor mass from the experiment shown in (A) was detected using surface plasmon resonance (SPR). **p<0.01. (C) Effect of cetux, Stattic, or their combination on tumor growth in C3H mice bearing SCC7 tumors. The mice were treated with STAT3 inhibitor (Stattic; 4 mg/kg bodyweight; every 2 days) with or without cetux (40 mg/kg bodyweight; once a week) for 3 weeks. The tumor burden was examined using bioluminescence imaging (n=7 mice per group). *p<0.05; **p<0.01; #, not significant. (D) Survival of C3H mice bearing SCC7 tumors from the experiment show in (C). **p<0.01. (E) Intracellular cytokine staining of interferon gamma in CD8+ and CD3+ T cell populations of C3H mice bearing SCC7 tumors from the experiment shown in (C) (n=7 mice per group). **p<0.01; ***p<0.001; #, not significant. (F) Interaction between EGFR and PD-L2 in C3H mice bearing SCC7 tumors after treatment with cetux, Stattic, or the combination of Cetux and Stattic was assessed using Co-immunoprecipitation. (G) The intracellular cetux content in SCC7 tumor mass treated with cetux alone or in combination with Stattic was detected using SPR. Significance was determined using one-way analysis of variance. **p<0.01. (H) A proposed model illustrating glycosylation of PD-L2 is an important mechanism for cetux refractoriness and immune evasion in HNSCC. Glycosylated PD-L2 forms a complex with EGFR, activating EGFR/STAT3 signaling and reducing cetuximab binding affinity to EGFR. N-glycosyltransferase FUT8, a transcriptional target of STAT3, is required for EGF induced PD-L2 glycosylation. This supports the development of a therapeutic strategy for cancer involving the combination of PD-L2 glycosylation blockade and cetux therapy. EGFR, epidermal growth factor receptor; ESCRT, endosomal sorting complexes required for transport; FUTA8, fucosyltransferase; HNSCC, head and neck squamous cell carcinoma; PD-L2, programmed death ligand 2; STAT3, signal transducer and activator of transcription 3; REY, PD-L2 REY mutant, in which the amino acid of R168, E171 and Y174 were substituted by Alanine; WT, wild type.