Fig 1: ADM suppresses CD8+ T cell proliferation and induces exhaustion. (A) Representative bright-field images of CD8+ T cells cultured with PBS or adrenomedullin (200ng/ml) at 24, 48, and 72 hours. (B) Quantification of CD8+ T cell numbers at 24, 48, and 72 hours. ADM significantly reduced proliferation compared to PBS control. (C) Gating strategy for flow cytometric identification of live CD8+ T cells from culture. (D) Representative flow cytometry showing expression of cytotoxicity marker GZMB and exhaustion markers PD-1 and TIM3 in CD8+ T cells following adrenomedullin (200ng/ml) or PBS treatment at 72 hours. (E) Quantification of GZMB+, PD-1+, and TIM3+ CD8+ T cells. Statistic tests: two-sided t test. Significance levels are denoted as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 2: ADM promotes tumor cells proliferation, migration, invasion, and pro-angiogenesis in vitro and in vivo. (A) Barplot showing the mRNA level of ADM in LUAD cells (A549 and H1299) after si-ADM transfection. (B) Cell viability assay to evaluate the impact of si-ADM transfection on the proliferative ability of A549 and H1299 cells. (C) Colony formation assay to assess the impact of si-ADM transfection on the clonogenic capability of A549 and H1299 cells. (D, E) Barplot showing the mRNA level of CDH1, CDH2, MMP2, MMP9, TWIST1, TWIST2, VEGFA, VCAN in LUAD cells (A549 and H1299) after si-ADM transfection. (F, G) Transwell assay to evaluate the impact of si-ADM transfection on the migration and invasion ability of A549 and H1299 cells. (H, I) Endothelial tube-formation assay to evaluate the impact of si-ADM transfection on the promoting angiogenesis ability of A549 and H1299 cells. (J) In vivo LUAD tail vein injection model showing the effect of adrenomedullin on LUAD metastasis. (K, L) Fluorescence images and quantifications of metastatic lesions. (M) Ki-67 antibody was used to detect murine tumor cells. Statistic tests: two-sided t test. Significance levels are denoted as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 3: ADM as a key GPCR-associated gene linking metabolism and immune suppression. (A) Correlation heatmap showing the correlation between expression level of 13 genes in GPCRscore and metabolic enrichment score. (B) The expression of ADM in the different stage and pathological grade of LUAD patients. (C) Scatter plot showing the correlation between ADM and immune cells. (D) Survival analysis of ADM expression in immunotherapy cohorts. (E) Scatter plot showing the correlation between ADM and immunosuppressive checkpoint including PDCD1, IDO1, HAVCR2, and LAG3.
Fig 4: ADM expression in single-cell sequencing and pathological tissue. (A) UMAP plot showing cell types in GSE148071. (B) ADM expression in GSE148071. (C) Violin plot showing ADM expression in different cell types in GSE148071. (D) UMAP plot showing cell types in GSE162498. (E) ADM expression in GSE162498. (F) Violin plot showing ADM expression in different cell types in GSE162498. (G) UMAP plot showing cell types in GSE117570. (H) ADM expression in GSE117570. (I) Violin plot showing ADM expression in different cell types in GSE117570. (J) IHC staining of ADM in normal lung tissue and LUAD tissue from HPA database. (K) IHC staining of ADM in para-cancerous tissue (n=6) and LUAD tissue (n=6) from our center. The H-score showing the degree of positivity. Statistic tests: two-sided t test. Significance levels are denoted as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar: 100μm.
Fig 5: ADM expression in peripheral blood and spatial transcriptomics of LUAD patients. (A–D) Violin plot showing the level of adrenomedullin in different clinical stages (A), stage T (B), stage N (C), and stage M (D) of LUAD patients. (E–H) ROC curves showing diagnostic efficiency to evaluate the sensitivity, specificity, and the area under the ROC curves (AUC) for differentiating different clinical stages (E), stage T (F), stage N (G), and stage M (H) of LUAD patients. All ROC curve analyses were significant (p < 0.0001 from AUC of 0.5). (I) Spatial distribution of malignant cells inferred by SpaCET deconvolution. (J, K) Spatial enrichment of purine metabolism and pyrimidine metabolism pathway activity, demonstrating elevated metabolic activity in tumor-dense areas. (L–P) ADM, CD8A, PDCD1, IDO1, and HAVCR2 expression in spatial transcriptomics. Statistic tests: one-way ANOVA. Significance levels are denoted as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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