A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis
- The School of Medicine, Nankai University, China
- Department of Lung Cancer Surgery, Tianjin Medical University General Hospital, China
- Research Institute of Obstetrics and Gynecology, Tianjin Central Hospital of Obstetrics and Gynecology, China
- Department of Gynecological Oncology, Tianjin Central Hospital of Obstetrics and Gynecology, China
Figures
Figure 1 with 1 supplement
High-throughput CRISPR screen identified IL20RA as a suppressor of the transcoelomic metastasis of ovarian cancer (OC).
(A) Schematics of experiment design to screen metastasis-related genes using CRISPR/Cas9 library in OC orthotopic murine model. (B) Venn diagram comparing the hits met the indicated enrichment …
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Figure 1—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
IL20RA suppresses the transcoelomic metastasis of ovarian cancer (OC) in syngeneic intraperitoneal OC mouse model.
(A) Western blot analysis of IL20RA in different OC cells. (B) qRT-PCR analysis of IL20RA in SK-OV-3 cells transfected with shCtrl or shIL20RA (means ± SEM, ***p<0.001, by unpaired two-sided …
Figure 2 with 2 supplements
Dramatically decreased IL20RA in human ovarian cancer (OC) peritoneal metastases and its correlation with the clinical outcome.
(A, B) Western blot analysis of IL20RA in primary OC tissues (Pri) and paired metastatic cancer cells in ascites (Asc) and metastatic nodules on the surfaces of the abdominal organs (Met) (A). …
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Figure 2—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
IL20RA expression is positively correlated with the clinical outcome of patients in many types of cancers.
Kaplan–Meier survival plots to show the correlations of different IL20RA expression with the overall survival in patients of bladder carcinoma, uterine corpus endometrial carcinoma, rectum …
Figure 2—figure supplement 2
IL20RB expression in human ovarian cancer (OC) tissues and its correlation with clinical outcome of OC patients.
(A, B) qRT-PCR analysis of IL20RB in human primary OC tissue and paired peritoneal metastases (A, data are plotted as means ± SEM from three independent measurements, *p<0.05, **p<0.01, ***p<0.001, …
Figure 3 with 1 supplement
Characterization of IL20RA-mediated modulation on the peritoneal immune microenvironment during the transcoelomic metastasis of ovarian cancer (OC).
(A, B) 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 60 days after …
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Figure 3—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
IL20RA has no effect on the proliferation and migration abilities of ovarian cancer (OC) cells.
(A) Growth curves of IL20RA-reconstituted or control (Vec) ID8 cells under the stimulation of IL-20. (B) Growth curves of shIL20RA- or shCtrl-transfected SK-OV-3 cells under the stimulation of …
Figure 4 with 1 supplement
IL20RA mediates a direct crosstalk between ovarian cancer (OC) cells and macrophages to regulate the polarization of macrophages.
(A) Schematic of the in vitro crosstalk experiment using conditioned medium (CM) from OC cells to educate macrophages. (B) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated …
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Figure 4—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
In vitro co-culture experiments to show the IL20RA-mediated crosstalk between ovarian cancer (OC) cells and macrophages for their polarization.
(A) Schematic of the in vitro co-culture experiments. (B, C) Transwell migration assay to show the migrated RAW 264.7 cells co-cultured with IL20RA-reconstituted or control (Vec) ID8 cells (B), …
Figure 5
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 …
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Figure 5—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
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 …
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Figure 6—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Mesothelial cells in peritoneum are the major source of IL-20 and IL-24 when challenged by ovarian cancer (OC).
(A, B) Immunofluorescent staining of IL-20/IL-24 (red), cytokeratin CK18 (green), and DAPI (blue) in peritoneum dissected from mice with intraperitoneal injection of ID8 cells (OC) or …
Figure 7 with 1 supplement
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 …
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Figure 7—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis shows that IL-20 activates the OAS1/RNase L-NLR/IL-18 signaling.
(A) KEGG gene set (based on organism systems) enrichment analysis on significantly changed genes in IL20RA-reconstituted ID8 cells. (B) KEGG pathway enrichment analysis on significantly changed, …
Figure 8 with 2 supplements
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 …
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Figure 8—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig8-data1-v1.xlsx
Figure 8—figure supplement 1
IL20RA signaling-inhibited ovarian cancer dissemination needs the functional IL20RA/IL20RB heterodimer receptor.
(A) qRT-PCR analysis of Il20rb in ID8Vec and ID8IL20RA cells transfected with shCtrl or shIL20RB (means ± SEM, ***p<0.001, by unpaired two-sided Student’s t-test). (B) Western blot analysis of …
Figure 8—figure supplement 2
The cross-reaction of cytokines involved in IL20RA-mediated signaling.
(A) qRT-PCR analysis of potential IL20RA ligands from the abdominal wall taken from C57BL/6 mice with intraperitoneal injection of SK-OV-3 cells or phosphate-buffered saline control (Ctrl) 9 days …
Figure 9
The therapeutic effect of recombinant IL-18 against the metastasis of ovarian cancer (OC).
(A) Schematic of the experiments. ID8 cells were orthotopic injected into the ovaries of C57BL/6 mice. The phosphate-buffered saline or IL-18 protein were intraperitoneal (i.p.) injected every 2 …
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Figure 9—source data 1
An Excel sheet with numerical quantification data.
- https://cdn.elifesciences.org/articles/66222/elife-66222-fig9-data1-v1.xlsx
Author response image 1
The mRNA levels of Tex14 in IL20RA-reconstituted ID8 cells and IL20RA-silenced SK-OV-3 cells.
The cells are treated with 2.5 ng/mL recombinant IL-20 protein before qRT-PCR analysis of Tex14 transcription.
Author response image 2
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 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Il20ra | GenBank | Gene ID: 237313 | |
| Strain, strain background (Escherichia coli) | DH5α | Shanghai Weidi Biotechnology | Cat.#: DL1002 | |
| Strain, strain background (Escherichia coli) | BL21(DE3) | CWBIO | Cat.#: CW0809S | |
| Cell line (Homo sapiens) | THP-1 | ATCC | RRID:CVCL_0006 | |
| Cell line (Homo sapiens) | HEK 293T | ATCC | RRID:CVCL_0063 | |
| Cell line (Homo sapiens) | SK-OV-3 | ATCC | RRID:CVCL_0532 | |
| Cell line (Mus musculus) | RAW 264.7 | ATCC | RRID:CVCL_0493 | |
| Cell line (Mus musculus) | ID8 | Merck | RRID:CVCL_IU14 | |
| Transfected construct (Homo sapiens) | IL20RA shRNA | Sangon Biotech | ||
| Transfected construct (Mus musculus) | Il18 shRNA | Sangon Biotech | ||
| Transfected construct (Mus musculus) | Il20rb shRNA | Sangon Biotech | ||
| Antibody | Anti-IL20RA (Rabbit polyclonal) | Abcam | RRID:AB_2123854 | WB (1:1000) IHC (1:200) |
| Antibody | Anti-IL20RB (Mouse monoclonal) | Proteintech | RRID:AB_11182930 | WB (1:1000) |
| Antibody | Anti-ACTB (Mouse monoclonal) | Immunoway | RRID:AB_2629465 | WB (1:5000) |
| Antibody | Anti-STAT3 (Mouse monoclonal) | Santa Cruz Biotechnology | RRID:AB_628293 | WB (1:1000) |
| Antibody | Anti-STAT3 (Mouse monoclonal) | Cell Signaling Technology | RRID:AB_331757 | ChIP (5 μL for 7 μg DNA) |
| Antibody | Anti-p-STAT3 (Mouse monoclonal) | Santa Cruz Biotechnology | RRID:AB_628292 | WB (1:1000) |
| Antibody | Anti-p-STAT3 (Rabbit polyclonal) | Signalway Antibody | RRID:AB_895980 | IHC (1:200) |
| Antibody | Anti-IL18 (Rabbit polyclonal) | Proteintech | RRID:AB_2123636 | WB (1:1000) IHC (1:200) |
| Antibody | Anti-OAS1A (Mouse monoclonal) | Santa Cruz Biotechnology | RRID:AB_10990911 | WB (1:1000) |
| Antibody | Anti-OAS1 (Rabbit polyclonal) | Proteintech | RRID:AB_2158292 | WB (1:1000) IHC (1:200) |
| Antibody | Anti-Caspase-1 (Rabbit polyclonal) | Proteintech | RRID:AB_2876874 | WB (1:1000) |
| Antibody | Anti-ARG1 (Rabbit polyclonal) | Proteintech | RRID:AB_2289842 | IHC (1:750) |
| Antibody | Anti-NOS2 (Rabbit polyclonal) | Proteintech | RRID:AB_2782960 | IHC (1:750) |
| Antibody | Anti-IL20 (Rabbit polyclonal) | Abclonal | RRID:AB_2750843 | IF (1:200) IHC (1:200) |
| Antibody | Anti-IL24 (Rabbit polyclonal) | Proteintech | RRID:AB_2880630 | IF (1:200) IHC (1:200) |
| Antibody | Anti-Cytokeratin 18 (Mouse monoclonal) | Abcam | RRID:AB_305647 | IF (1:200) |
| Antibody | Anti-CD16/CD32 (Mouse monoclonal) | eBioscience | RRID:AB_467133 | FACS (1:200) |
| Antibody | Anti-CD45-PE (Mouse monoclonal) | Biolegend | RRID:AB_312971 | FACS (1:200) |
| Antibody | Anti-CD11b-APC (Mouse monoclonal) | Biolegend | RRID:AB_312795 | FACS (1:200) |
| Antibody | Anti-F4/80-APC/CY7 (Mouse monoclonal) | Biolegend | RRID:AB_10803170 | FACS (1:200) |
| Antibody | Anti-I-A/I-E-PERCP/CY5.5 (rat monoclonal) | Biolegend | RRID:AB_2191071 | FACS (1:200) |
| Antibody | Anti-CD206-FITC (Mouse monoclonal) | Biolegend | RRID:AB_10900988 | FACS (1:200) |
| Antibody | Anti-CD11c-FITC (Mouse monoclonal) | BD Biosciences | RRID:AB_396683 | FACS (1:200) |
| Antibody | Anti-CD3-FITC (Mouse monoclonal) | Biolegend | RRID:AB_312661 | FACS (1:200) |
| Antibody | Anti-CD4-APC (Rat monoclonal) | BD Biosciences | RRID:AB_398528 | FACS (1:200) |
| Antibody | Anti-CD8-PE/CY7 (Mouse monoclonal) | eBioscience | RRID:AB_469584 | FACS (1:200) |
| Antibody | Anti-CD45R/B220-FITC (Rat monoclonal) | BD Biosciences | RRID:AB_394617 | FACS (1:200) |
| Recombinant DNA reagent | pLV-H1-EF1α-puro (plasmid) | Biosettia | Cat.#: SORT-B19 | |
| Recombinant DNA reagent | pLV-EF1α-MCS-IRES-Bsd (plasmid) | Biosettia | Cat.#: cDNA-pLV03 | |
| Recombinant DNA reagent | pET-20b(+) vector (plasmid) | Novagen | Cat.#: 69739–3 | |
| Peptide, recombinant protein | Murine recombinant IL-20 protein | Origene | Cat.#: TP723795 | |
| Peptide, recombinant protein | Murine recombinant IL-24 protein | R&D Systems | Cat.#: 7807 ML-010 | |
| Peptide, recombinant protein | Human recombinant IL-20 protein | Sino Biological | Cat.#: 13060-HNAE | |
| Peptide, recombinant protein | Murine recombinant M-CSF | Peprotech | Cat.#: 315-02-10 | |
| Chemical compound | PMA | Sigma-Aldrich | Cat.#: 16561-29-8 | |
| Chemical compound | Clodronate liposomes | LIPOSOMA | Cat.#: CP-030030 | |
| Commercial assay or kit | Murine IL18 ELISA Kit | Wuxin Donglin Sci & Tech Development | Cat.#: DL-IL18-Mu | |
| Commercial assay or kit | Human IL18 ELISA Kit | Wuxin Donglin Sci & Tech Development | Cat.#: DL-IL18-Hu | |
| Commercial assay or kit | Murine IL20 ELISA Kit | Wuxin Donglin Sci & Tech Development | Cat.#: DL-IL20-Mu | |
| Commercial assay or kit | Murine IL24 ELISA Kit | Wuxin Donglin Sci & Tech Development | Cat.#: DLIL24-Mu | |
| Commercial assay or kit | ChIP-IT Express Kits | Active Motif | Cat.#: 53035 |
Additional files
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Supplementary file 1
Primes for qPCR.
The primers used for qRT-PCR and ChIP- qPCR analyses.
- https://cdn.elifesciences.org/articles/66222/elife-66222-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66222/elife-66222-transrepform-v1.pdf
