Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet:

Article Snippet: For overexpression of Cyp11a1 in TRAMP-C1 cells pcDNA3.1 plasmids (Addgene) were used.

Techniques: Virus, shRNA, Recombinant, Modification, Staining, RNA Library Preparation, TaqMan Assay, Competitive ELISA, RNA Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: IL30-dependent regulation of PC driver genes in murine and human PC cells. A Mouse Prostate Cancer PCR Array. Fold differences of the mRNAs of PC driver genes between rmIL30-treated (red bars) and untreated wild-type murine (m) PC-SLCs, and between IL30KO-mPC-SLCs (blue bars) and mPC-SLCs. Results obtained from control NTgRNA-treated and untreated mPC-SLCs were comparable to those from wild type cells. A significant threshold of twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2, in at least one condition, up- or downregulation, are shown. Experiments were performed in duplicate. Stripped boxes represent scale breaks. The dashed lines represent the twofold change cutoff. B Cytofluorimetric analyses of gp130 (CD130) and IL6Rα (CD126) expression in TRAMP-C1 cells. Red lines: isotype control. Experiments were performed in triplicate. C MTT assay of TRAMP-C1 cells after 48 h treatment with rmIL30 at concentrations of 10, 35, 50 and 100 ng/mL. ANOVA: p < 0.0001. * p < 0.05, Tukey HSD test compared with 0 ng/mL. ** p < 0.01, Tukey HSD test compared with 0, 10, 35 and 100 ng/mL. Results are expressed as mean ± SD. D , E The treatment with rmIL30 (6 h), significantly increased the number of TRAMP-C1 cells, which migrated ( D ) across the polycarbonate membrane insert, or which invaded ( E ) the basement membrane matrix layer. Results are expressed as mean ± SD. *Student’s t test: p = 0.0001 ( D ), p = 0.00001 ( E ), compared with untreated (CTRL) cells. Experiments were performed in triplicate. F Mouse Prostate Cancer PCR Array. Fold differences of the mRNAs of PC driver genes between rmIL30-treated (red bars) and untreated wild-type TRAMP-C1 cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2 are shown. Experiments were performed in duplicate. Stripped boxes represent scale breaks. The dashed lines represent the twofold change cutoff. G Venn diagram representing the “PC Driver Genes” which are up- and/or downregulated by IL30 (treatment with rmIL30 or human IL30 gene knockout) in TRAMP-C1 (purple circle), PIN-SC (red circle), DU145 (green circle) and PC3 cells (blue circle). Overlapping circles illustrate the sharing of IL30-regulated genes between different cell lines. H Human Prostate Cancer PCR Array. Fold differences of mRNAs of PC driver genes between IL30-overexpressing IL30-DU145 cells (red bars) and wild-type DU145 cells, and between IL30KO-DU145 cells (blue bars) and wild-type cells. Results obtained from control NTgRNA-treated and EV-transfected DU145 cells were comparable to those from wild-type cells. A significant threshold of twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2, in at least one condition, up- or downregulation, are shown. Experiments were performed in duplicate. The dashed lines represent the twofold change cutoff. I Fold differences of the mRNAs of PC driver genes between IL30-overexpressing IL30-PC3 cells (red bars) and wild-type PC3 cells, and between IL30KO-PC3 cells (blue bars) and wild-type cells. Results obtained from control NTgRNA-treated and EV-transfected PC3 cells were comparable to those from wild-type cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2, in at least one condition, up- or downregulation, are shown. Experiments were performed in duplicate. The stripped box represents a scale break. The dashed lines represent the twofold change cutoff. J Western blot analysis of BCL2 protein expression in WT, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-PC3 cells, and in WT, NTgRNA-treated and IL30KO-DU145 cells. K Western blot analysis of NFKB1 protein expression in EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO, PC3 and DU145 cells. Results obtained from control NTgRNA-treated and EV-transfected cells were comparable to those from wild-type cells. L Western blot analysis of DKK3 and SOCS3 protein expression in WT, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-PC3 cells, and in WT, NTgRNA-treated and IL30KO-DU145 cells

Article Snippet: Cell viability and proliferation were assessed, using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (#G3582; Promega, Madison, WI, USA), on TRAMP-C1 cells, after administration of recombinant murine IL30 (#7430-ML, R&D Systems, Minneapolis, MN, USA), and on PC3 and DU145 cells, after IL30 gene transfection or administration of recombinant human CXCL5 and IGF1 (#300-22 and #100-11, both from Peprotech, London, UK), or incubation with neutralizing anti-IL30, anti-CXCL5 (#AF1834, RRID:AB_355012 and #MAB254, RRID:AB_2261181, both from R&D Systems) and anti-IGF1 (#MAB2912, R&D Systems) antibodies (Abs).

Techniques: Control, Expressing, MTT Assay, Membrane, Gene Knockout, Transfection, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: Tumor growth and survival of mice-bearing IL30-deficient or IL30-overexpressing PC of murine or human origin. A Mean volume of tumors developed in NSG mice, after s.c. implantation of wild type, EV- or IL30-DU145 cells. ANOVA, p < 0.0001; Tukey HSD test, p < 0.01 versus wild type or EV-transfected DU145 cells. Results are expressed as mean ± SD. B Mean volume of tumors developed in NSG mice, after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. ANOVA, p < 0.0001; Tukey HSD test, p < 0.01 versus wild type or NTgRNA-treated DU145 cells. Results are expressed as mean ± SD. C Average number of lung metastasis spontaneously developed in NSG mice, which developed tumors after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test versus DU145 or NTgRNA-treated DU145 cells. Results are expressed as mean ± SD. D Kaplan–Meier survival curves of mice-bearing tumors developed after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. Log-rank test: p = 0.000009. E Immunopathological features of tumors developed in NSG mice, after s.c. implantation of IL30KO-DU145, wild type DU145 and IL30-DU145 cells. Expression of IGF1, proliferation (Ki67 Abs) and vascularization (CD31 Abs) were prominent in IL30-overexpressing tumors, and scanty in IL30KO tumors, compared with wild type tumors. Cytoplasmic and nuclear expression of NFKB1 was strong in IL30-overexpressing tumors and faint in IL30KO tumors. The immunopathological features of tumors developed after implantation of control, EV-transfected or NTgRNA-treated, cells were comparable to those of wild type tumors. Magnification: × 400; CD31, × 200. F Immunopathological features of tumors developed in NSG mice, after s.c. implantation of IL30KO-DU145 and wild type DU145 cells. Expression of tumor suppressor genes CDH1/E-Cadh, DKK3, PTEN, RARb and SOCS3 was stronger in IL30KO-DU145 tumors, when compared to wild type tumors, whereas the expression of PTGS2 was weaker. The immunopathological features of control tumors, developed in NSG after implantation of NTgRNA-treated cells, were comparable to those of wild type tumors. Magnification: × 400. G Automated immune cell count and microvessel density in tumors developed in NSG mice, after implantation of wild type, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-DU145 cells, assessed by immunohistochemistry, as described in Methods. ANOVA, p < 0.0001. * p < 0.01, Tukey HSD test compared with DU145, NTgRNA-treated DU145 and EV-transfected DU145. ** p < 0.01, Tukey HSD test compared with DU145, NTgRNA-treated DU145, IL30KO-DU145 and EV-transfected DU145. Results are expressed as mean ± SD. H The immune cell contexture of tumors developed in NSG mice, after s.c. implantation of IL30KO and IL30-DU145 cells, revealed a higher content of macrophages (anti-F4/80 Abs) and granulocytes (anti-Ly-6G Abs) in IL30-overexpressing tumors, compared to wild type tumors. By contrast, these immune cell populations were scarce to absent in IL30-deficient tumors. Magnification: × 400. Scale bars: 30 μm. I Western blot analysis of IL30 protein expression in wild type, EV- and IL30 gene-transfected TRAMP-C1 cells. J Mean volume of tumors developed in C57BL/6J mice, after s.c. implantation of wild type, EV- or IL30-TRAMP-C1 cells. ANOVA, p < 0.0001. Tukey HSD test, p < 0.01 versus wild type or EV-TRAMP-C1 cells. Results are expressed as mean ± SD. K Immunopathological features of tumors developed in C57BL/6J mice, after s.c. implantation of wild type or IL30-TRAMP-C1 cells revealed that proliferation (PCNA Abs), microvascular density (CD31 Abs) and granulocyte content (Ly-6G Abs) were higher in IL30-overexpressing tumors, than in control tumors. Cancer cells that formed IL30-TRAMP-C1 tumors showed reduced cytoplasmic expression of PTEN and an increased nuclear and cytoplasmic expression of NFKB1. Magnification: × 400; CD31, × 200. L Automated immune cell count in tumors developed in C57BL/6J mice, after s.c. implantation of wild type, EV or IL30 gene-transfected TRAMP-C1 cells, assessed by immunohistochemistry, as described in Methods. ANOVA, p < 0.001. * p < 0.01, Tukey HSD test compared with wild type or EV-TRAMP-C1 cells. Results are expressed as mean ± SD. M Percentage of lung metastasis spontaneously developed in C57BL/6J mice, which developed tumors after s.c. implantation of wild type or IL30 gene-transfected TRAMP-C1 cells. *Fisher’s exact test, p = 0.03 versus EV-TRAMP-C1 or TRAMP-C1. Results from mice implanted with EV-TRAMP-C1 cells were comparable to those obtained from mice implanted with wild type cells. N Kaplan–Meier survival curves of mice-bearing tumors developed after s.c. implantation of wild type, EV- or IL30 gene-transfected TRAMP-C1 cells. Log-rank test: p = 0.000124

Article Snippet: Cell viability and proliferation were assessed, using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (#G3582; Promega, Madison, WI, USA), on TRAMP-C1 cells, after administration of recombinant murine IL30 (#7430-ML, R&D Systems, Minneapolis, MN, USA), and on PC3 and DU145 cells, after IL30 gene transfection or administration of recombinant human CXCL5 and IGF1 (#300-22 and #100-11, both from Peprotech, London, UK), or incubation with neutralizing anti-IL30, anti-CXCL5 (#AF1834, RRID:AB_355012 and #MAB254, RRID:AB_2261181, both from R&D Systems) and anti-IGF1 (#MAB2912, R&D Systems) antibodies (Abs).

Techniques: Transfection, Expressing, Control, Cell Counting, Immunohistochemistry, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: IL30-dependent regulation of inflammation and immunity gene expression in murine and human PC cells. A Mouse Cancer Inflammation & Immunity Crosstalk PCR Array. Fold differences of mRNAs of inflammation and immunity-related genes between rmIL30-treated (red bars) and untreated wild-type TRAMP-C1 cells. A significant threshold of twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2 are shown. Experiments were performed in duplicate. The stripped box represents a scale break. The dashed line represents the twofold change cutoff. B Human Cancer Inflammation & Immunity Crosstalk PCR Array. Fold differences of mRNAs of inflammation and immunity-related genes between IL30KO-DU145 cells (blue bars) and wild-type cells. Results obtained from control NTgRNA-treated DU145 cells were comparable to those from wild type cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2 are shown. Experiments were performed in duplicate. The stripped box represents a scale break. The dashed line represents the twofold change cutoff. C Human Cancer Inflammation & Immunity Crosstalk PCR Array. Fold differences of mRNAs of inflammation and immunity-related genes between IL30KO-PC3 cells (blue bars) and wild-type cells. Results obtained from control NTgRNA-treated PC3 cells were comparable to those from wild-type cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2 are shown. Experiments were performed in duplicate. Stripped boxes represent scale breaks. The dashed lines represent the twofold change cutoff. D Venn diagram representing the genes that govern “Cancer Inflammation & Immunity Crosstalk” which are up- and/or downregulated by IL30 (treatment with rmIL30 or human IL30 gene knockout) in TRAMP-C1 (purple circle), DU145 (green circle) and PC3 cells (blue circle). Overlapping circles illustrate the sharing of IL30-regulated genes between different cell lines. E , F Western blot analysis of the expression of STAT3 α and β isoforms in EV-, IL30 gene-, NTgRNA-treated and IL30KO-DU145 ( E ) and PC3 ( F ) cells . Results obtained from control NTgRNA-treated and EV-transfected cells were comparable to those from wild-type cells. G Immunostaining of STAT3 in tumors developed in NSG mice, after s.c. implantation of IL30 gene-transfected, or deleted, DU145 cells, revealed that the cytoplasmic and nuclear expression of STAT3 was strong in IL30-overexpressing tumors, scanty in IL30-deficient tumors and moderate in wild-type tumors. Immunostaining of control tumors, developed after implantation of EV-transfected or NTgRNA-treated cells, was comparable to that of wild-type tumors. Magnification: × 400. H Graphic representation of the suppression of cytokine and chemokine expression in DU145 and PC3 cells following IL30 gene deletion by CRISPR/Cas9 technology. The most downregulated molecular mediators are represented with a larger font size. Both IL23A and IL12B, which form the heterodimeric cytokine IL23, were suppressed

Article Snippet: Cell viability and proliferation were assessed, using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (#G3582; Promega, Madison, WI, USA), on TRAMP-C1 cells, after administration of recombinant murine IL30 (#7430-ML, R&D Systems, Minneapolis, MN, USA), and on PC3 and DU145 cells, after IL30 gene transfection or administration of recombinant human CXCL5 and IGF1 (#300-22 and #100-11, both from Peprotech, London, UK), or incubation with neutralizing anti-IL30, anti-CXCL5 (#AF1834, RRID:AB_355012 and #MAB254, RRID:AB_2261181, both from R&D Systems) and anti-IGF1 (#MAB2912, R&D Systems) antibodies (Abs).

Techniques: Expressing, Control, Gene Knockout, Western Blot, Transfection, Immunostaining, CRISPR