Fig 2: Down‐regulating PPY ameliorated the immunosuppression microenvironment of PDAC and decelerated cancer progression. A) Following down‐regulating PPY expression in mouse cancer cells using recombinant lentivirus, an orthotopic allograft tumor model was established by conjecting these cancer cells and KPC mouse‐derived CAFs with a 4:1 ratio (n = 8). 20 days later, a vivo imaging system (IVIS) was applied to monitor the tumor growth. B) mIHC staining of PPY (pink), iCAFs (IL‐6, cyan; PDGFRa, magenta), M2 macrophages (CD206, red), MDSCs (Gr‐1, green), CD8+T cells (CD8, yellow), PD‐1+cells (orange) in TME of model mice. Scale bars, 50 µm. C) Summary and statistical analysis of PPY expression and the density of iCAFs and immune cells analyzed by mIHC in the TME of cancer tissues. D–H) Flow cytometry analysis of M2 macrophages (D), MDSCs (E), T cells (F), CD8+ T cells (G), exhausted CD8+T (H) in the TME of the group of cancer cells with down‐regulated PPY expression and respective control group. I–L) The human macrophages derived from THP‐1 cells (I and J) and mouse macrophages isolated from WT C57BL / 6J mice (K and L) were respectively co‐cultured with CAFs, that were respectively treated with the supernatants from cancer cells with reduced PPY expression. Flow cytometry (I and K) and qRT‐PCR (J and L) were applied to analyze the M2 macrophage polarization in these macrophages. M) Transwell migration assays showed the migration abilities in MDSCs stimulated by pancreatic cancer cells with reduced PPY expression. N) Transwell migration assays showed the alterations in the migratory abilities of MDSCs when a PPY antibody was added to their co‐culture system with pancreatic cancers. Scale bars, 50 µm. Statistics are shown in mean ± SD accessed by the paired t‐test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant.

Fig 3: The expression characteristics of PPY in PDAC and its correlation with the immune landscape and clinical prognosis of PDAC. A) The HPA database showed the expression of PPY in various organs. B) the different expression levels of PPY in cancer tissues (red) and non‐tumor tissues (black) were analyzed by the GEPIA database. C) The correlation between PPY expression and TME signature was analyzed using IOBR tool. D) The correlation between PPY expression and the overall survival (OS) was analyzed by the GEPIA database. E) qRT‐PCR analysis of PPY expression in human PDAC cell lines. F) qRT‐PCR analysis of PPY expression in wild type (WT) C57BL/6J mice and KPC mice. G) mIHC was applied to analyze the PPY expression in the paired tumor tissue and adjacent tissue of PDAC (n = 82). The statistical data is presented as mean ± SD and analyzed using the paired t‐test. *, p < 0.05. H) The multiplexed immunohistochemistry (mIHC) technique was employed to examine the correlation between PPY expression and iCAF density, as well as immune cell infiltration in PDAC tissues from PPY‐low and PPY‐high groups in a PDAC tissue microarray. The staining scheme was as follows: PPY (pink), iCAFs (IL‐6, cyan; PDGFRa, magenta), CD206+cells (red), CD33+cells (green), CD8+T cells (CD8, yellow), PD‐1+cells (orange). All scale bars, 100 µm. I) Kaplan‐Meier survival plot depicts overall survival of patients in PPY‐low group and PPY‐high group. J) Summary and statistical analysis of the density of iCAFs and infiltration of CD206+cells, CD33+cells, CD8+T cells, and PD‐1+CD8+T cells in cancer tissues between the PPY‐low group (n = 40) and the PPY‐high group (n = 42). Statistics are shown in mean ± SD and accessed by the unpaired t‐test. *, p < 0.05; ***, p < 0.001. mIHC, multiplexed immunohistochemistry.

Fig 4: Targeting iCAFs by interfering with PPY enhances the efficacy of anti‐PD‐1 monotherapy in PDAC. A) Schematic diagram illustrating the modeling and drug administration process in KPC mice (n = 6). B) Kaplan‐Meier survival plot depicts overall survival in KPC mice of different groups. *, p < 0.05; ns, not statistically significant. C) Summary and statistical analysis of tumor size before and after treatment using 18F‐FDG PET‐CT imaging scanning. D) Prior to the treatment, the 18F‐FDG PET‐CT imaging system was used to validate tumor formation and size in 15‐week‐old KPC mice. At the terminal stage of the experiments, the same imaging system was utilized again in living KPC mice to estimate tumor volume. E) mIHC staining of PPY (pink), iCAFs (IL‐6, cyan; PDGFRa, magenta), M2 macrophages (CD206, red), MDSCs (Gr‐1, green), CD8+T (CD8, yellow), PD‐1+cells(orange) in the TME of model mice. Scale bars, 50 µm. F) Summary and statistical analysis of PPY expression and the density of iCAFs and immune cells analyzed by mIHC in the TME of cancer tissues. G–L) Flow cytometric analysis was performed to evaluate the presence of iCAFs (G), M2 macrophages (H), MDSCs (I), T cells (J), CD8+ T cells (K), and exhausted CD8+T cells (L) within the tumor microenvironment of different treatment groups. Statistics are shown in mean ± SD accessed by the unpaired non‐parametric test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant.

Fig 5: PPY significantly induces the iCAF phenotype in PDAC CAFs both in vitro and in vivo. A–C) After treating CAFs derived from human PDAC tissues for 12 h, qRT‐PCR analysis was performed to assess their alterations in the expression of iCAF markers (CXCL12 (A), IL‐6 (B), CXCL12 (C)). D–F) qRT‐PCR analysis of iCAF markers (CXCL12 (D), IL‐6 (E), CXCL12 (F)) after treating the human CAFs for 24 h. G–I) qRT‐PCR analysis of iCAF markers (CXCL12 (G), IL‐6 (H), CXCL12 (I)) after treating the human CAFs for 36 h. J) qRT‐PCR analysis of the expression levels of myCAF markers (ACTA2 and CTGF) after treating the human CAFs with PPY proteins (40ng/ml) for 24 h. K) Flow cytometry analysis was performed to evaluate the populations of iCAFs (Ly6C+MHC‐II‐), myCAFs (Ly6C+MHC‐II‐), and apCAFs (Ly6C+MHC‐II‐), after treating CAFs derived from cancer tissues of KPC mice with PPY recombinant proteins. L,M) After co‐culturing the human CAFs together with BxPC‐3 cells overexpressing PPY, the expression levels of iCAF markers (IL‐6, CCL2, and CXCL12) and myCAF markers (ACTA2 and CTGF) were quantified using qRT‐PCR (L), and the secretion levels of IL‐6, CCL2, and CXCL12 were measured using ELISA (M). N) The CAFs derived from cancer tissues of KPC mice were cocultured with Panc02 overexpressed PPY, and flow cytometry was applied to analyze iCAF, myCAF, and apCAF populations. O,P) After co‐culturing the human CAFs with PANC‐1 cells that had down‐regulated PPY expression, the expression levels of iCAF markers (IL‐6, CCL2, and CXCL12) and myCAF markers (ACTA2 and CTGF) were analyzed by qRT‐PCR (L), and secretion levels of IL‐6, CCL2, and CXCL12 were assessed by ELISA (M). Q) The murine CAFs were cocultured with Panc02 that had down‐regulated PPY expression, and flow cytometry was applied to analyze iCAF, myCAF, and apCAF populations. R) Schematic diagram of co‐injection of mouse cancer cells and CAFs (4:1) derived from KPC mice to construct the orthotopic allograft tumor model in C57BL/6J mice (n = 8), and the tumor tissues were isolated and analyzed by flow cytometry. S,T) The tumor tissues of PPY upregulated and downregulated groups and their respective control groups were dissociated into single cells, and flow cytometry was utilized to analyze the iCAF, myCAF, and apCAF populations in the tumor tissue. Student's t‐test was used to analyze the data, and the results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant. OE, overexpression.