Fig 2: Y126S reversed CTGF-mediated immunosuppressive TME. (A) Correlation analysis of CTGF with PD-L1 expression and exhausted CD8+ T cells in TCGA database (n=179). (B) Gene set enrichment analysis (GSEA) correlates CAFs-CTGF function with T cell differentiation, immune response regulation, and immune response regulating cell surface receptor signaling pathway in TCGA database (n=179). (C, D) Primary hCAFs isolated from patients with PDAC were treated with 20 ng/mL rhCTGF for 48 hours and subjected to RNA-seq analysis. Heatmap of differentially expressed genes (C) and top pathways in GO enrichment (D). (E, F) PD-L1 expression of hCAFs (E) and KPC-CAFs (F) after treatment with CM from cancer cells or CTGF (20 ng/mL) with α-CTGF or Y126S for 48 hours. (G–P) The effects of α-CTGF injection in orthotopic PDAC-bearing mice were evaluated. Schematic illustration for the design (G), bioluminescence images and average radiance (H), tumor weight (I), and representative images and correlation analysis between CTGF and PD-L1 expression in IHC staining (J). Quantification for the percentage of CD3+ T cells (K), CD8+ T cells (L), PD-1+ TIM-3+ T cells in CD8+ T cells (M), and expression of CD206+ by TAMs (N) of tumors treated with α-CTGF by flow cytometry. Representative images and quantification of anti-CD8 (O) and anti-Ki67 (P) IHC staining in tumors treated with α-CTGF or IgG group. 7 mice per group (n = 7) in G–P. Data were represented as mean ± SEM. Statistical analysis was performed with two-tailed Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. Scale bar: 20 µm. AOD, average optimal density; CM, conditioned media; CTGF, connective tissue growth factor; FACS, fluorescence-activated cell sorting; FDR, false discovery rate; GO, gene oncology; hCAFs, human cancer-associated fibroblasts; IHC, immunohistochemistry; IVIS, In Vivo Imaging System; KPC-CAFs, KPC’s cancer-associated fibroblasts; mCTGF, mouse CTGF protein; NES, nonparametric estimation statistics; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death ligand 1; rhCTGF, recombinant human CTGF protein; TAMs, tumor-associated macrophages; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment.

Fig 3: Y126S inhibited desmoplasia induced by CAF activation through targeting CTGF. (A–E) The desmoplasia of tumors derived from PDAC mice treated with α-CTGF or IgG in figure 2G. Representative images (A), quantification of anti-α-SMA (B), anti-collagen I (C), Masson (D) staining, and tissue stiffness (E). (F, G) Relative expression of specific genes of hCAFs after treatment with CM from PANC-1 cells or rhCTGF (20 ng/mL) plus α-CTGF or Y126S for 48 hours by qRT-PCR (F) and western blotting (G). (H) Representative images of hCAFs stimulated by rhCTGF (20 ng/mL) plus α-CTGF or Y126S for 48 hours for α-SMA (green), collagen I (red), and DAPI (blue). (I, J) Relative expression of specific genes of KPC-CAFs after treatment with CM from Panc-02 cells or mCTGF (20 ng/mL) plus α-CTGF or Y126S for 48 hours by qRT-PCR (I) and western blotting (J). (K–M) Representative fast force MAP AFM image (K), Young’s modulus of hCAFs (L), and KPC-CAFs (M) by AFM with a CSC38 cantilever (spring constant: 0.02–0.04 N/m). (N) Gel contraction of hCAFs treated with 20 ng/mL rhCTGF plus α-CTGF or Y126S. 7 replicates per group (n = 7) in B–E, 3 replicates per group (n = 3) in F, J, N, and 5 replicates per group (n = 5) in L, M. Data were represented as mean ± SEM. Statistical analysis in B–E was performed with two-tailed Student’s t-tests, and in F, J, L–N was performed with ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Scale bar: 20 µm in A, 50 µm in H, and 10 µm in K. AFM, atomic force microscopy; ANOVA, analysis of variance; α-SMA, alpha-smooth muscle actin; CM, conditioned media; CTGF, connective tissue growth factor; DAPI, 4',6-diamidino-2-phenylindole; FAP, fibroblast activation protein; hCAFs, human cancer-associated fibroblasts; KPC-CAFs, KPC’s cancer-associated fibroblasts; mCTGF, mouse CTGF protein; mRNA, messenger RNA; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative reverse transcription PCR; rhCTGF, recombinant human CTGF protein.

Fig 4: Y126S counteracts CTGF-caused immunosuppression by downregulating PD-L1 expression and reactivating T cell immune response. (A) KPC-CAFs were pretreated with the stimulation of 20 ng/mL mCTGF plus multiple antibodies for 48 hours and then co-cultured with CD8+ T cells isolated from C57BL/6J spleen for 18 hours (effector/target ratio = 2:1). Representative flow cytometric images and corresponding quantifications of PD-L1 expression by KPC-CAFs (top), ratio of PD-1+ TIM-3+ T cells in CD8+ T cells (center), and apoptosis of CD8+ T cells by flow cytometry (bottom). (B) Level of IL-2, IFN-γ, and TNF-α secreted by T cells in a co-culture system. 3 replicates per group (n = 3) in all experiments. Data were represented as mean ± SEM. Statistical analysis was performed with ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. ANOVA, analysis of variance; comb, combination of α-CTGF and α-PD-1; IFN-γ, interferon-gamma; KPC-CAFs, KPC’s cancer-associated fibroblasts; mCTGF, mouse connective tissue growth factor protein; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1; TNF-α, tumor necrosis factor-alpha.

Fig 5: Y126S exhibits tumor-specific accumulation through targeting CTGF. (A–C) The expression of CTGF in tumor tissues and adjacent normal tissue from patients with PDAC by RT-qPCR (A), western blotting (B), and IHC staining (C). For α-CTGF staining in C, representative histological plots were captured both in 100× and 200× (left panel), and quantification was conducted by measuring the positive area percentage of CTGF. n=5 (individual donors, illustrated as three technical replicates) (right panel). (D) Schematic illustration of the procedure of mice sacrifices and sample collection over time (0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, and 48 hours) after receiving a single administration of Y126S or α-PD-1. (E) Concentration curve of Y126S in serum. (F, G) Molecular weight-normalized-tumor concentration of Y126S and α-PD-1 in orthotopic tumor-bearing mice over time (F) and at 2 hours (G), expressed as molar concentration. (H–K) Further mass-normalized antibody levels of Y126S in tumor compared with liver (H) or lung (I), and of α-PD-1 in tumor compared with liver (J) or lung (K) were expressed as the percentage of molar concentration per gram of tissue. (L) Schematic illustration depicting the mechanism of Y126S treatment repressing desmoplastic and immunosuppressive TME in PDAC (created in BioRender.com). 3 replicates per group (n = 3) in A, 3 mice per group at each time point (n = 3) in D–K. Data were represented as mean ± SEM. Statistical analysis was performed with two-tailed Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. CAF, cancer-associated fibroblast; CTGF, connective tissue growth factor; ECM, extracellular matrix; IHC, immunohistochemistry; mRNA, messenger RNA; PD-1, programmed cell death 1; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death ligand 1; RT-qPCR, reverse transcription quantitative PCR; TAM, tumor-associated macrophage; TME, tumor microenvironment.