mcp 1 ek287  (Multi Sciences (Lianke) Biotech Co Ltd)

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: Screening and initial validation of cancer cell‐secreted proteins capable of significantly inducing iCAF phenotype. A) The t‐distributed stochastic neighbor embedding (t‐SNE) plot of the 92,222 cells in the single‐cell sequencing profile revealed distinct cell types observed in PDAC. B) The t‐SNE plot exhibited diverse subtypes of fibroblasts observed in PDAC. C) The top 10 up‐regulated and down‐regulated expressed marker genes of each CAF subgroup. D) The expression levels of myCAF markers (ACTA2, COL1A1, COL11A1, MMP11), iCAF markers (CXCL12, IL‐6, CCL2, CXCL2), and apCAF markers (HLA‐DRA, HLA‐DRB1) in different fibroblast subsets. E) Pathway activities scored by GSVA between different fibroblast subsets. F) The volcano plot depicts the differential expression of genes encoding secreted proteins in cancer cells derived from patients with high versus low iCAF. G–I) qRT‐PCR analysis was conducted to assess alterations in the expression levels of iCAF markers (IL‐6, CXCL12, and CCL2) in CAFs isolated from KPC mice following treatment with conditioned medium (CM) containing potential candidates for 12 (G), 24 (H), and 36 h (I). J) qRT‐PCR analysis of changes in the expression levels of myCAF markers (ACTA2 and CTGF) in CAFs isolated from KPC mice after treating them with CM containing PPY for 12, 24, and 36 h. K) Flow cytometry analysis of iCAF (Ly6C+MHC‐II‐), myCAF (Ly6C+MHC‐II‐), and apCAF (Ly6C+MHC‐II‐) populations after treating CAFs with CM containing PPY for 24 h. L,M) The changes in expression of iCAF markers (IL‐6, CXCL12, and CCL2) (L) and myCAF markers (ACTA2 and CTGF) (M) in CAFs isolated from three patients with PDAC were quantified by qRT‐PCR; following a 24‐h treatment with CM containing PPY. N) After treating CAFs derived from three PDAC patients with CM containing PPY for 24 h, ELISA was performed to assess the secretion of IL‐6, CCL2, and CXCL12. Each experiment was performed three times independently, and Student's t ‐test was used to analyze the data. The results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Biomarker Discovery, Sequencing, Marker, Expressing, Quantitative Proteomics, Derivative Assay, Quantitative RT-PCR, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: 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.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: In Vitro, In Vivo, Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Construct, Isolation, Control, Over Expression

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: The inhibition of EGFR expression in CAFs impeded the induction of iCAFs by PPY. A) qRT‐PCR (A) and B) ELISA analyses of the expression levels of IL‐6, CCL2, and CXCL12 in human EGFR‐knockdown CAFs treated with PPY proteins. C) The efficiency of EGFR knockdown in KPC CAFs was examined by qRT‐PCR. D,E) qRT‐PCR (D) and ELISA (E) analyses of the expression levels of IL‐6, CCL2, and CXCL12 in murine EGFR knockdown CAFs treated with PPY proteins. F) Flow cytometry analysis was performed to evaluate the populations of iCAFs, myCAFs, and apCAFs in murine EGFR‐knockdown CAFs treated with PPY proteins. G,H) The IVIS image (G) and gross image (H) of tumors in model mice (n = 7), that was constructed by co‐injecting cancer cells with up‐regulated PPY expression and KPC CAFs with down‐regulated EGFR expression. I–N) Flow cytometric analysis was performed to evaluate the presence of iCAFs (I), M2 macrophages (J), MDSCs (K), T cells (L), CD8 + T cells (M), and exhausted CD8 + T cells (N) within the tumor microenvironment. O) Map of scientific hypotheses of this article. The statistical data is presented as mean ± SD and analyzed using the unpaired t‐ test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knockdown, Flow Cytometry, Construct