Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF synergistically induce genes involved in mesenchymal transition of mesothelial cells. (A) Synergistically induced genes of mesothelial cells treated as in Figure (n = 5 patients). Jitter plot of the calculated additive and the actually observed induction (FC) by TNF+IL‐17A treatment. The plot shows genes with a ≥2‐fold ratio (= 1 log2 unit) of observed/expected median fold change. (B) Molecular Signature (MSigDB) hallmark pathway analysis. ⁵⁴ The bubble graph presents the 10 most significantly enriched pathways of synergistic candidate genes defined in (A) and the relative overlap with all genes of the respective pathway.(C) RT‐qPCR of EMT marker gene expression after treatment of mesothelial cells for 48 h with rhIL‐17A, rhTNF or both cytokines. Expression values were normalized to the untreated control (cntr). (D) qPCR analysis of selected EMT‐transcription factor genes performed as in panel B. Bar plots show the mean±SD of biological replicates (n = 5). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 were determined by by one‐way ANOVA followed by Dunnett's multiple comparison test; ns: not significant.

Article Snippet: Th17 differentiation in RPMI 1640 was performed in the presence of recombinant cytokines rhIL‐6 (200‐06), rhTGFβ (100‐21), IL‐23 (200‐23) and rhIL‐1β (200‐01B; all from Peprotech, Cranbury, USA).

Techniques: Quantitative RT-PCR, Marker, Expressing, Comparison

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF perturb mesothelial monolayer integrity and promote adhesion of tumor cells. (A) Phalloidin staining of mesothelial cells treated with rhIL‐17A, rhTNF or both cytokines for 96 h. Medium and treatment were renewed after 48 h. Representative pictures are shown. (B, C) Quantification of ellipticity (B; Imaris software) and sphericity (C; ImageJ software) of experiments shown in panel A. Four random images were analyzed for each treatment condition and n = 4 patients. (D) ZO‐1 (green) and Hoechst (blue) staining of mesothelial cells treated as in panel A. The pictures are representative of three experiments with cells from different patients. (E) Quantification of ZO‐1 area at cell‐cell contacts with ImageJ software and the Labkit plugin. Three random images for each treatment condition were analyzed and n = 3 patients. (F) Schematic overview of the experimental design of the tumor cell adhesion assay. Mesothelial cells were seeded on 96‐well plates coated with collagen I, stimulated with rhIL‐17, rhTNF or both cytokines, and cultured for 4–5 days till formation of a dense monolayer. Subsequently, wells were washed and CellTracker Green‐stained tumor cells were added for 6 h. (G) Pictures of adherent OC‐tumor cells treated as described in panel F. Representative images are shown. (H) Quantification of adhesion assays as in panel G. Attached tumor cells were quantified by measuring the relative area covered by fluorescently labeled tumor cells using ImageJ software (n = 3 patients). The bar plots show the mean ±SD for biological replicates.*p<0.05, **p<0.01, ****p<0.0001 were determined in panels B, E and H by one‐way ANOVA followed by Dunnett's multiple comparison test, in panel C by Kruskal‐Wallis test followed by Dunn's multiple comparison test: ns: not significant.

Article Snippet: Th17 differentiation in RPMI 1640 was performed in the presence of recombinant cytokines rhIL‐6 (200‐06), rhTGFβ (100‐21), IL‐23 (200‐23) and rhIL‐1β (200‐01B; all from Peprotech, Cranbury, USA).

Techniques: Staining, Software, Cell Adhesion Assay, Cell Culture, Labeling, Comparison

Journal: Clinical and Translational Medicine

Article Title: Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

doi: 10.1002/ctm2.1604

Figure Lengend Snippet: IL‐17A and TNF synergistically direct the secretome of mesothelial cells towards a Th17‐promoting environment. (A) Affinity proteomics analysis of the mesothelial cell secretome after 48 h treatment with rhIL‐17A, rhTNF or both cytokines. (B) Heatmap showing the top 50 significantly upregulated proteins after combined treatment (p <0.05 by paired t test; synergism defined as FC of combined treatment > added individual FC values x 1.5). FC values were protein‐wise normalized. (C) Bubble graph of synergistically induced proteins. Relative expression normalized to combined IL‐17A/TNF treatment is shown. (D) Schematic overview of the experimental design. CM from mesothelial cells treated with rhIL‐17A and rhTNF and from untreated cells were collected after 24 h. CD4 + cells from PBMCs were treated with CM for 8 days. Induction of Th17 was measured by flow cytometry. (E) Representative staining of CD4 + cells after treatment with CM from untreated compared to IL‐17A/TNF‐treated mesothelial cells. (F) Increase (FC) of IL‐17A + /IFNγ − cell frequency after treatment as in panel E. Bars show the mean ±SD of n = 11 biological replicates. ***p<0.001 was determined by two‐tailed unpaired t‐test.

Article Snippet: Th17 differentiation in RPMI 1640 was performed in the presence of recombinant cytokines rhIL‐6 (200‐06), rhTGFβ (100‐21), IL‐23 (200‐23) and rhIL‐1β (200‐01B; all from Peprotech, Cranbury, USA).

Techniques: Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: Oncotarget

Article Title: INT-1B3, an LNP formulated miR-193a-3p mimic, promotes anti-tumor immunity by enhancing T cell mediated immune responses via modulation of the tumor microenvironment and induction of immunogenic cell death

doi: 10.18632/oncotarget.28608

Figure Lengend Snippet: ( A , B ) HCT116 cells were transfected with 1B3 or 3A1 at indicated concentrations or cells were mock (M) transfected and cultured for 72 hours. CD14+ cells were isolated from PBMC of healthy donors and cultured in presence of rhIL-4 and rhGM-CSF to induce differentiation into immature DC (iDC) for 6 days. After 6 days, immature DC were harvested and added to transfected tumor cells for 24 hours to induce DC maturation. Immature DC were also cultured in presence or absence of a cytokine cocktail (cyto mix) known to induce DC maturation as a control. Cells were analyzed for expression of DC maturation markers by flow cytometry before and after maturation ( n = 2–4). Histogram overlays and summary graphs showing MFI of CD80 and CD86 within CD45+ cells. ( C – F ) CTV-labelled PBMC were co-cultured for 5 days with DC matured by co-culture with HCT116 transfected with mock, 1B3 or 3A1 at a ratio of DC:PBMC of 1:10 ( n = 2–3). (C, D) Representative dot plots showing percentage IFNγ+ (C) or CTVlow (D) of CD4+ cells (top), CD8+ (middle), and CD4-CD8- (bottom) T cells. (E, F) Summary graphs showing percentage IFNγ+ and CTVlow of CD4+ cells (E) and CD8+ T cells (F). ( G ) CTV-labelled PBMC were co-cultured for 5 days with DC matured by co-culture with HCT116 transfected with mock, 1B3 or 3A1 at a ratio of DC:PBMC of 1:10, or with transfected HCT116 cells. Summary graphs showing percentage IFNγ+ and CTVlow of CD4-CD8- T cells. ( H ) NucLight Red labelled HCT116 cells were transfected overnight with 1B3 at indicated concentrations or mock transfected. After overnight transfection, cells were harvested, counted, reseeded into 96-well plates and allowed to adhere to the plate for 20–24 hours before adding PBMC. PBMC were added at ratio tumor cells:PBMC of 1:0, 1:8, 1:16 or 1:8 + aCD3CD28. Summary graph shows percentage tumor cell survival when normalized to tumor:PBMC of 1:0 (no PBMC) condition from one representative of 3 independent experiments. * p < 0.05. Bars in summary graphs represent mean.

Article Snippet: As a positive control for DC maturation, cells were cultured with a cytokine cocktail containing rhIL-1β (200 IU/ml), rhIL-6 (1000 IU/ml), rh-tumor necrosis factor alpha (TNFα) (1000 IU/ml) (premium grade, Miltenyi Biotech), prostaglandin E2 (PGE2) (1 μg/ml) (Stemcell Technologies) (cytokine mix).

Techniques: Transfection, Cell Culture, Isolation, Control, Expressing, Flow Cytometry, Co-Culture Assay