Fig 1: IL-18 is the essential factor downstream IL-20/IL20RA signaling to prevent the ovarian cancer dissemination.(A) qRT-PCR analysis of Il18 in ID8Vec and ID8IL20RA cells transfected with shCtrl or shIL-18 (means ± SEM, ***p<0.001, by unpaired two-sided Student’s t-test). (B) Western blot analysis of IL-18 in ID8Vec and ID8IL20RA cells transfected with shCtrl or shIL-18. (C) ELISA measurement of IL-18 in ascites from mice with intraperitoneal injection of ID8Vec, ID8IL20RA, and ID8IL20RA/shIL-18 cells 45 days post-injection. Data are shown as means ± SEM, n = 6, ***p<0.001, by unpaired two-sided Student’s t-test. (D–G) Representative images of ascites formation (D) and the metastatic nodules in peritoneal cavity (E) of C57BL/6 mice at day 45 after intraperitoneal injected with ID8Vec, ID8IL20RA, and ID8IL20RA/shIL-18 cells. The quantification of ascites and metastatic nodules is shown in (F) and (G), respectively. Data are shown as means ± SEM, n = 6, **p<0.01, ***p<0.001, by unpaired two-sided Student’s t-test. (H) Flow cytometry analysis of macrophages (CD45+ CD11b+ F4/80+) and M1-like (MHC II+ CD206-) and M2-like (MHC II- CD206+) subpopulations in ascites formed in C57BL/6 mice at day 45 after intraperitoneal injected with ID8Vec, ID8IL20RA, and ID8IL20RA/shIL-18 cells. Data are shown as means ± SEM, n = 3, *p<0.05, ns, not significant, by unpaired two-sided Student’s t-test. Figure 8—source data 1.An Excel sheet with numerical quantification data.
Fig 2: Macrophages play a prominent role in IL20RA-mediated suppression of ovarian cancer (OC) metastasis.(A) Schematic of the experiments. ID8 cells alone or mixed with CMIL20RA IL-20 (+)- or CMVec IL-20 (+)-stimulated bone marrow-derived macrophage were injected into the peritoneal cavity of C57BL/6 mice. (B–E) Representative images of the ascites formation (B) and metastatic nodules in peritoneal cavity (D) at day 45 post-inoculation. The quantification of ascites and metastatic nodules is shown in (C) and (E), respectively. Data are shown as means ± SEM, n = 6, *p<0.05; **p<0.01; ***p<0.001, by unpaired two-sided Student’s t-test. (F) Schematic of the experiments. IL20RA-reconsitituted or control (Vec) ID8 cells were injected into the peritoneal cavity of C57BL/6 mice. The phosphate-buffered saline liposomes (PBS) or clodronate liposomes (CLO) were intraperitoneal (i.p.) injected every 3 days. (G–J) Representative images of the ascites formation (G) and metastatic nodules in peritoneal cavity (I) at day 45 post-inoculation. The quantification of ascites and metastatic nodules is shown in (H) and (J), respectively. Data are shown as means ± SEM, n = 6 (ID8Vec/PBS and ID8IL20RA/PBS), n = 3 (ID8Vec/CLO and ID8IL20RA/CLO), ns, not significant; **p<0.01; ***p<0.001, by unpaired two-sided Student’s t-test. Figure 5—source data 1.An Excel sheet with numerical quantification data.
Fig 3: The expression of IL20RA ligands in the abdominal wall challenged by colon cancer cells and hepatocellular carcinoma cells.A-B. qRT-PCR analysis of IL20RA ligands from the abdominal wall in mice with the intraperitoneal injection of CT-26 cells (A) and Hepa 1-6 cells (B). C-D. ELISA measurement of IL-20 and IL-24 in peritoneal flushing fluid from mice with intraperitoneal injection of CT-26 cells (C) and Hepa 1-6 cells (D).
Fig 4: IL-20 activates the IL20RA-STAT3-OAS1/RNase L-NLR signaling to produce IL-18 to regulate the macrophages polarization.(A) qRT-PCR analysis of Oas1a and Il18 upon IL20RA reconstitution in ID8 cells. (B) The secreted IL-18 from IL20RA-reconstituted and control ID8 cells was measured by ELISA. (C) Western blot analysis of indicated proteins in IL20RA-reconstituted and control ID8 cells under the stimulation of IL-20. (D) Western blot analysis of indicated proteins in SK-OV-3 cells stimulated with IL-20 or phosphate-buffered saline (PBS) (Ctrl) for 24 hr. (E) ELISA measurement of IL-18 secreted from SK-OV-3 cells stimulated by IL-20 for 24 hr. (F) Schematic of the Oas1a promoter and Il18 promoter with predicted STAT3-binding sites and the primer sets. (G) IL20RA-reconstituted ID8 cells were stimulated with IL-20 or PBS (Ctrl) for 24 hr before the STAT3 binding on Oas1a promoter and Il18 promoter was analyzed by ChIP-qPCR. (H, I) Immunohistochemical analysis of IL20RA, p-STAT3, OAS1, and IL-18 in human primary ovarian cancer tissues (Pri) and paired peritoneal metastatic nodules (Met) (H) and quantification (I). **p<0.01; ***p<0.001, by paired two-sided Student’s t-test. Scale bar: 20 µm. (J) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated with IL-20 protein for 72 hr. (K) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells treated by CMIL20RA IL-20 (+) or CMVec IL-20 (+) together with IL-18 neutralizing antibody (nAb) or nonspecific lgG (IgG) for 72 hr. All the qRT-PCR and ELISA data are shown as means ± SEM from three independent experiments, *p<0.05, **p<0.01, ***p<0.001, by unpaired two-sided Student’s t-test. Figure 7—source data 1.An Excel sheet with numerical quantification data.
Fig 5: The mesothelial cells in peritoneum produce IL-20 and IL-24 when challenged by disseminated ovarian cancer (OC) cells in the peritoneal cavity.(A, B) qRT-PCR analysis of IL20RA ligands (Il19, Il20, and Il24) in peritoneal organs (intestinal, abdominal wall, ovary) (A) or peritoneal macrophages (CD11b+ F4/80+) (B) taken from C57BL/6 mice with intraperitoneal injection of ID8 cells (OC) or phosphate-buffered saline (PBS) control (Ctrl) 9 days before. Data are shown as means ± SEM, n = 18 for Ctrl group and n = 6 for OC group, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test. (C) qRT-PCR analysis of IL20RA ligands in IL20RA-reconstituted or control (Vec) ID8 cells (means ± SEM, ns, not significant). (D) ELISA measurement of IL-20 and IL-24 in peritoneal flushing fluid from mice with intraperitoneal injection of ID8 cells (OC) or PBS control (Ctrl) 9 days before. n = 6 for each group. Data are shown as means ± SEM, ***p<0.001, by unpaired two-sided Student’s t-test. (E) qRT-PCR analysis of the abdominal walls dissected from C57BL/6 mice and co-cultured with medium (Ctrl) or ID8 cells for 48 hr (means ± SEM from three independent experiments, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test). (F) Hematoxylin and eosin staining of the abdominal wall of C57BL/6 mice. Scale bar: 50 µm (upper panel); 20 µm (lower panel). (G) Immunohistochemical staining of IL-20 and IL-24 in abdominal walls dissected from mice with intraperitoneal injection of ID8 cells (OC) or PBS control (Ctrl) 9 days before. Scale bar: 20 µm. Figure 6—source data 1.An Excel sheet with numerical quantification data.
Supplier Page from Sino Biological, Inc. for Human IL20 / Interleukin-20 Protein