Journal: Cancer Research

Article Title: Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization

doi: 10.1158/0008-5472.can-17-3117

Figure Lengend Snippet: Figure 1. Expression of IL30 and IL30R by PCSLCs, murine, and human prostate tissues. A, Cytofluorimetric analyses of gp130-(CD130) and IL6Ra- (CD126) expression in PIN-SCs. B, Relative expression SD of IL30 mRNA. CTRL, PIN-SCs. ANOVA, P < 0.0001. , P < 0.01, Tukey HSD test compared with CTRL, EV, or EV-IL30shRNA (clones D and B). C, Western blot analyses of IL30 protein expression. D, ELISA assay of IL30 release by CTRL (182.82 6.9 pg/mL), EV-PIN-SCs (168.21 10.82 pg/mL), IL30PIN-SCs (2424.46 83.9 pg/mL), EV-IL30shPIN-SCs (170.44 13.09 pg/mL), and IL30shPIN-SCs (clone D, 6.76 1.87 pg/mL; clone B, 7.53 1.38 pg/mL). ANOVA, P < 0.0001. , P < 0.01, Tukey HSD test compared with CTRL, EV, or EV-IL30shRNA (clones D and B). E, IL30 immunostaining in normal prostate, PIN (11 weeks), and in poorly differentiated tumor (28 weeks) of TRAMP mice. IL30 (brown) colocalizes with Sca-1 (red) in PIN; scale bars, 50 mm (left two); 30 mm (right two and inset). F, Expression of IL6Ra and gp130 in PIN and in poorly differentiated adenocarcinoma (AC) of TRAMP mice; scale bars, 50 mm (left two); 30 mm (right two). G, IL30 immunostaining in normal prostate, PIN (IL30/CD133 colocalization), and in poorly differentiated adenocarcinoma; scale bars, 20 mm (left two and inset); 30 mm (right two).

Article Snippet: The quantitation of Epstein-Barr virus–induced gene 3 (EBI3, also known as IL27 subunit beta), IL27, and IL27p28/IL30 proteins in the supernatant (sup) derived from (wild type and transfected) PIN-SCs, was assessed using the Mouse EBI3 ELISA Kit (LifeSpan BioSciences Inc.; detection sensitivity, 5.79 pg/mL), the LEGEND MAX Mouse IL27 Heterodimer (BioLegend; detection sensitivity, 16.9 pg/mL) and mIL27p28/IL30 Quantikine ELISA kit (R&DSystems; detection sensitivity, 4.27 pg/mL), respectively, according to the manufacturer's instructions.

Techniques: Expressing, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining

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 IGF1 production in human PC cells and IGF1-mediated autocrine growth loop. A , B Elisa assay of IGF1 release by wild-type, EV and IL30 gene-transfected DU145 ( A ) and PC3 ( B ) cells . ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with WT and EV-transfected cells. Results are expressed as mean ± SD. C Cytofluorimetric analyses of IGF1R expression in PC3 and DU145 cells. Red lines: isotype control. Experiments were performed in triplicate. D , E Elisa assay of IGF1 release by wild-type DU145 ( D ) and PC3 ( E ) cells, after the treatment with anti-IL30 Abs. (D) ANOVA: p < 0.05. * p < 0.05, Tukey HSD test compared with DU145 cells untreated or treated with 5 μg/mL. E ANOVA: p < 0.001. * p < 0.01, Tukey HSD test compared with untreated PC3 cells. Results are expressed as mean ± SD. F , G MTT assay of DU145 ( F ) and PC3 ( G ) cells, untreated (0.0 ng/mL) or treated with rhIGF1 (5.0, 10, 30, 50 ng/mL). ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0 ng/mL. ** p < 0.01, Tukey HSD test compared with 0 and 5 ng/mL. *** p < 0.01, Tukey HSD test compared with 0, 5 and 10 ng/mL. Results are expressed as mean ± SD. H , I MTT assay of DU145 (H) and PC3 (I) cells, untreated (0.0 μg/mL) or treated with anti-IGF1 Abs (0.1, 0.4, 0.8 μg/mL in DU145; 0.25, 0.50, 0.70 μg/mL in PC3). (H) ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0.0 μg/mL. ** p < 0.01, Tukey HSD test compared with 0.0 and 0.1 μg/mL. *** p < 0.05, Tukey HSD test compared with 0.0, 0.1 and 0.4 μg/mL. (I) ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0.00 µg/mL. ** p < 0.01, Tukey HSD test compared with 0.00 and 0.25 μg/mL. Results are expressed as mean ± SD. J , K MTT assay of wild-type and IL30 gene-transfected DU145 ( J ) and PC3 ( K ) cells, untreated (0.0 μg/mL), or treated with anti-IGF1 Abs (30 μg/mL). ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with wild-type cells. ** p < 0.01, Tukey HSD test compared with wild-type and IL30-transfected cells. Results are expressed as mean ± SD

Article Snippet: Quantitation of IL30, CXCL5 and IGF1, in the supernatant derived from murine or human PC cells, was carried out using the following ELISA kits, according to manufacturer’s protocols: Human CXCL5/ENA-78 Quantikine ELISA Kit (#DX000, R&D Systems, Minneapolis, MN, USA); human IGF1 ELISA Kit (#ab211651, Abcam, Cambridge, UK); and mouse Interleukin-27 subunit alpha ELISA Kit (#CSB-E08466m, Cusabio, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, MTT Assay

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: Quantitation of IL30, CXCL5 and IGF1, in the supernatant derived from murine or human PC cells, was carried out using the following ELISA kits, according to manufacturer’s protocols: Human CXCL5/ENA-78 Quantikine ELISA Kit (#DX000, R&D Systems, Minneapolis, MN, USA); human IGF1 ELISA Kit (#ab211651, Abcam, Cambridge, UK); and mouse Interleukin-27 subunit alpha ELISA Kit (#CSB-E08466m, Cusabio, Wuhan, China).

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