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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

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

Fig. 4

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

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