Fig 1: GC with GPC3high CAFs is correlated with lower response of PD-1 blockage therapy. (A) The likelihood of the clinical response to anti-PD-1 therapy for high and low GPC3 expression GC patients from the TCGA cohorts. True represents immunotherapy responders, while false represents immunotherapy nonresponders. (B–D) The violin plots present of TIDE value, immune dysfunction and immune exclusion in high and low GPC3 expression GC patients. (E) The relationship between different GPC3 expression in CAFs and the response ratio of anti-PD-1 therapy in GC patients. (F–I) The proportion of interferon (IFN)-?-positive cells of CD4+ or CD8+ T cells in the tumours with different GPC3 expression in CAFs.
Fig 2: GC with GPC3high CAFs is insensitive to PD-1 blockage therapy in vivo. (A) The mice fibroblasts (L929 cells) after GPC3 overexpression lentivirus transfection. (B) The mice xenograft model construction and divide into four groups. (C) Tumour volume comparison after different treatment in the mice GC xenograft model. (D) Immunofluorescence were used to detect the GPC3 positive CAFs in xenograft tumour tissue. (E,F) The proportion of interferon (IFN)-?-positive cells of CD8+ T cells in the tumours of different groups (*p < .05; **p < .01, ***p < .001).
Fig 3: GPC3 is up-regulated in the CAFs of the advanced GC. (A) UMAP map showed single-cell trajectory of CAFs in normal gastric tissues, superficial and deep layers of tumours by pseudotime analysis. (B) The expression level of different expression genes, which included C7, CXCL14, DNAJB1, EGR1, FOSB, GPC3, JUNB, KCNN3, MGP, ZFP36 in the different clusters of CAFs in normal gastric tissues, superficial and deep layers of tumours by pseudotime analysis. (C) The scatter diagram showed the different expression genes of CAFs in superficial and deep layers of tumours. (D) The GPC3 expression level in the 15 clusters and the different clusters of CAFs.
Fig 4: Targeting GPC3 sensitizing the PD-1 blockage therapy in GC. (A) ELISA was used to detect the culture medium after GPC3 overexpression in mice fibroblasts. (B) The co-culture model to study the impact fibroblasts with GPC3 overexpression of to HGC-27 cells. (C) rt-qPCR was used to detect the immune checkpoints expression after co-culture with fibroblasts, the immune checkpoint genes included PD-L1, TIM3, CD24, CYCLIN D1, cMYC and PDK. (D) The immune checkpoint genes expression of HGC-27 cells under the anti-GPC3 treatment. (E) The mice xenograft model construction and divide into four groups. (F) Tumour volume comparison after different treatment in the mice GC xenograft model. (G,H) The proportion of interferon (IFN)-?-positive cells of CD8+ T cells in the tumours of different groups (*p < .05; **p < .01, ***p < .001).
Fig 5: GC with GPC3high CAFs is correlated with poor prognosis. (A,B) Kaplan–Meier survival curve shows the relationship between high- and low-GPC3 expression and GC prognosis with TCGA dataset. (C) Immunofluorescence were used to detect the GPC3 expression in CAFs of GC tissue and normal tissue (bar = 50 µm). (D) Comparison between the GPC3 expression level in CAFs of GC tissue and normal tissue. (E) Kaplan–Meier survival curve shows the relationship between high- and low-GPC3 expression in CAFs and OS of GC patients (***p < .001).
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