Journal: Theranostics

Article Title: Cancer-associated Fibroblast-derived IL-6 Promotes Head and Neck Cancer Progression via the Osteopontin-NF-kappa B Signaling Pathway

doi: 10.7150/thno.22182

Figure Lengend Snippet: Fibroblasts contribute to the up-regulation of OPN in HNC cells. (A) Immunohistochemical analysis of OPN protein expression in HNC tissues and matched adjacent normal tissues (Scale bar: 25 μm). Higher staining of OPN was observed in tumor cells of the edge of bulk tumors. (B) The mRNA and protein levels of OPN were determined in NFs, CAFs and cancer cells (derived from 4 HNC patients) using real-time PCR and western blotting. (C) OPN at the protein and mRNA level and in the supernatant was detected in 8 representative HNC cell lines and normal oral epithelial cells (titled normal) using western blotting, real-time PCR and ELISA. (D) The protein level, supernatant concentration and mRNA level of OPN were determined in CAL-27 and SCC-25 cells after co-culture with NFs. (E) The protein level, supernatant concentration and mRNA level of OPN were determined in NFs after co-culture with CAL-27 and SCC-25 cells. (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001)

Article Snippet: The supernatant OPN and IL-6 concentrations and the plasma OPN and IL-6 levels in patients with HNC were assessed using the Human OPN ELISA kit (Boster, China) and human IL-6 ELISA kit (Boster, China).

Techniques: Immunohistochemical staining, Expressing, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay

Journal: Theranostics

Article Title: Cancer-associated Fibroblast-derived IL-6 Promotes Head and Neck Cancer Progression via the Osteopontin-NF-kappa B Signaling Pathway

doi: 10.7150/thno.22182

Figure Lengend Snippet: Effects of stromal IL-6-induced OPN on promoting tumor growth and metastasis in vivo . (A) OPN overexpression in CAL-27 cells facilitated the xenograft tumor growth in nude mice. (B) Plasma OPN levels of the tumor-bearing mice from the normal group, CAL-27 group and CAL-27-OPN group. (C) Subcutaneous injection of OPN antibody (10 μg per tumor nodule) or IL-6 antibody (10 μg per tumor nodule) around the tumor partly inhibited NF-mediated tumor growth, and the combination of OPN antibody (5 μg per tumor nodule) and IL-6 antibody (5 μg per tumor nodule) exhibited a more powerful antitumor activity. (D) Plasma OPN levels of the tumor-bearing mice from the normal group, IgG treatment group, OPN antibody treatment group, IL-6 antibody treatment group and the combination group. (E) Overexpression of OPN in Rca-T cells promoted the formation and growth of metastatic nodules in nude mice (Scale bar, left: 5 mm; right: 100 μm). (F) Western blot analysis confirmed the exogenous expression of OPN in Rca-T cells, and plasma OPN levels of the mice involved in experimental metastasis were measured using ELISA. (G) Kaplan-Meier analyses of overall survival. (H, I and J) ROC curve analysis of the mRNA panel of OPN and IL-6 stratified by different groups in the validation set. ROC plots for the mRNA panel of OPN and IL-6 discriminated the five-year survival group from the death group (H), the TNM stage I group from the healthy controls (I), and the metastasis group from the non-metastasis group (J). AUC, area under the curve. (K) A proposed model illustrating the modulatory role of stromal IL-6-induced neoplastic OPN in controlling tumor growth and metastasis. (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001)

Article Snippet: The supernatant OPN and IL-6 concentrations and the plasma OPN and IL-6 levels in patients with HNC were assessed using the Human OPN ELISA kit (Boster, China) and human IL-6 ELISA kit (Boster, China).

Techniques: In Vivo, Over Expression, Clinical Proteomics, Injection, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

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