Fig 1: Blocking Aebp1 in fibroblasts enhances T cell antitumor immunity.MC38 (A) and EO771 (B) tumor growth from WT versus Aebp1 cKO mice (p < 0.0001). Percentage of IFN-γ+ and TNF-α+ CD8+ T cells in MC38 (C) and EO771 (D) tumors from WT versus Aebp1 cKO mice. Percentage of CD8+ T cells in MC38 (E) and EO771 (F) tumors from WT versus Aebp1 cKO mice, p < 0.0001 (F). Representative images (G) and correlation between AEBP1+α-SMA+ cells and CD8+ or GZMB+CD8+ T cells expression in human TNBC tissues (two-sided Spearman correlation analysis) (H). I MC38 tumor growth with CD8+ T cells depleted by anti-CD8 antibodies. J tSNE plot of tumor infiltrating lymphocytes (TILs) overlaid with the expression of indicated markers from WT or Aebp1 cKO group. K Frequency of clusters of indicated immune cell subsets in MC38 tumors from WT and Aebp1 cKO group, p < 0.0001 for WT vs. cKO in cluster 1. n = 5 mice/group (A–F, I, K). Data are presented as the mean ± SEM (A–F, I, K). Data were analyzed by two-sided unpaired Student’s t-test (C–F, K), and two-way ANOVA with Sidak’s (A, B) or Tukey’s (I) multiple comparisons test. Source data are provided as a Source Data file.
Fig 2: Chem-0199 suppresses the interaction between AEBP1 and CKAP4.A Workflow of inhibitor screening. Created in BioRender. Xiaoyu, W. (2025) https://BioRender.com/s97i4nk. B Overall structures of AEBP1 (shown in silver) and CKAP4 (shown in pink). The figure demonstrates the formation of a hydrogen bond (represented by yellow dashed lines) between the amino acid Tyr874 in the AEBP1 protein and the amino acid Gln527 in the CKAP4 protein. C Close-up views of AEBP1-Chem-0199 complexes. The figure illustrates the competitive binding of Chem-0199 to AEBP1, forming hydrogen bonds and π-π interactions specifically with the amino acid Tyr874 in the AEBP1 protein. The green arrows indicate Chem-0199. Two perpendicular views are shown. D Western blot analysis of total AKT and p-AKT expression in vehicle or Chem-0199 treated WT and Aebp1-/- CAFs. GAPDH was used as a loading control. Blot is representative of n = 2 biological replicates. E Flow cytometry analysis of PD-L1 expression on vehicle or Chem-0199 treated WT and Aebp1-/- mCAFs. F Percentage of IFN-γ+ CD8+ T cells cocultured with WT and Aebp1-/- mCAFs treated with vehicle or Chem-0199. G MC38 tumor growth of vehicle or Chem-0199-treated WT and Aebp1 cKO mice. H Tumor growth of MC38 tumor-bearing WT mice treated with Chem-0199 alone, anti-CTLA-4 alone, or Chem-0199 + anti-CTLA-4. Percentage of IFN-γ+ (I) or TNF-α+ (J) CD8+ T cells in MC38 tumors from mice treated with indicated regimens, p < 0.0001. n = 3 biological replicates (E, F), n = 5 mice/group (G–J). Data are presented as the mean ± SEM (E–J), and were analyzed by one-way (E, F, I, J) or two-way ANOVA (G, H) with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.
Fig 3: CAF-derived AEBP1 is associated with T cell dysfunction and closely related to poor survival.A Illustration of the workflow, including the cohorts of tumor samples and an overview of analytical approaches used. Created in BioRender. Xiaoyu, W. (2025) https://BioRender.com/5uvgprp. B T cell dysfunction scores of indicated genes in pan-cancer based on TIDE system. C Expression levels of AEBP1 in the cell clusters in TME based on scRNA-seq data from COAD (CRC_GSE146771_Smartseq2). D Expression levels of AEBP1 in the cell clusters in TME based on scRNA-seq data from TNBC (BRCA_GSE114727_inDrop). IF staining showed representative images of AEBP1 (green) and α-SMA (red) in human COAD (E) or TNBC (F) tissues. Scale bar, 50 μm. n = 3 biological replicates. Correlation between GZMB and AEBP1 expression in human COAD (G) or TNBC (H) samples was detected by IHC (two-sided Spearman correlation analysis). I, J Overall survival of COAD or TNBC patients with AEBP1high or AEBP1low expression levels in an in-house cohort. Source data are provided as a Source Data file.
Fig 4: Blocking AEBP1 down-regulates PD-L1 expression on CAFs.A Gene ontology analysis by RNA sequencing of shNC or shAebp1 mouse CAFs (mCAFs) (n = 3 mice/group). Heatmap shows the differentially expressed genes (DEGs) and associated signatures. B scRNA-seq analysis showing the expression of DEGs including Cd274 in fibroblasts in EO771 tumors from WT (n = 184) or Aebp1 cKO (n = 445) group. C The expression of PD-L1 in AEBP1high or AEBP1low human COAD samples was detected by IHC (two-sided Spearman correlation analysis). Flow cytometry analysis of PD-L1 expression on CAFs in WT or Aebp1 cKO tumors of MC38 (D) and EO771 (E) models (n = 5 mice/group). Flow cytometry analysis of PD-L1 expression on shNC and shAEBP1 hCAFs (F), or WT and Aebp1-/- mCAFs (G). Flow cytometry analysis of PD-L1 expression on hCAFs or mCAFs treated with IgG or rhAEBP1 (2 μg/ml) (H)/rmAEBP1 (1 μg/ml) (I). J Percentages of IFN-γ+ or TNF-α+ CD8+ T cells co-cultured with shNC or shAEBP1 hCAFs pre-treated with IgG, or anti-PD-L1 antibodies (10 μg/mL). n = 3 biological replicates (F–J). Data are presented as the mean ± SEM (D–J). Data were analyzed by two-sided unpaired Student’s t-test (B, D, E, G–I), and one-way ANOVA with Dunnett’s (F) or Tukey’s (J) multiple comparisons test. p < 0.0001 (E, F). Source data are provided as a Source Data file.
Fig 5: Schematic illustration of the mechanism by which CAF-derived AEBP1 induces T cell dysfunction in tumors.CAF-derived AEBP1 activates the CKAP4/AKT/PD-L1 pathway, then leads to T cell dysfunction. Chem-0199, an AEBP1 inhibitor, disrupts the AEBP1-CKAP4 complex, suppresses the activation of Akt pathway and PD-L1 expression in CAFs, and finally enhances the cytotoxic activity of CD8+ T cells. Furthermore, genetic or pharmaceutical inhibition of AEBP1 synergizes with ICT.
Supplier Page from Abcam for Recombinant Mouse AE binding protein 1 (His tag)