Dysregulated heparan sulfate proteoglycan metabolism promotes Ewing sarcoma tumor growth
- Cancer and Blood Disease Institute, Children's Hospital Los Angeles, United States
- Division of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, United States
- Department of Pathology, Keck School of Medicine, University of Southern California, United States
- Department of Pediatrics, Keck School of Medicine, University of Southern California, United States
- Department of Medicine, Keck School of Medicine, University of Southern California, United States
Figures
Figure 1 with 3 supplements
Cre-inducible expression of EWSR1-FLI1 drives small round blue cell tumor (SRBCT) development in zebrafish.
(A) Overview of experimental method. The Tol2 transposon system was used to integrate human EWSR1-FLI1 into the wild-type zebrafish genome by microinjection into single-cell-stage embryos. (enhanced …
Figure 1—figure supplement 1
The Tol2 transposon-based system was used to integrate EWSR1-FLI1 into the zebrafish genome by microinjection into single-cell-stage zebrafish embryos.
(A) Scheme of constructs for expression of EWSR1-FLI1 under cmv, fli1, mitfa, ubi promoters. (B) Scheme of constructs for Cre-inducible expression of eGFP-2A-EWSR1-FLI1 under cmv, fli1, b-actin, ubi …
Figure 1—figure supplement 2
The Tol2 transposon-based system was used to integrate b-actin:RSG-EWSR1-FLI1 into the wild-type zebrafish genome.
Coinjection of Cre mRNA or GFP mRNA was used to generate EWSR1-FLI1-expressing or control zebrafish, respectively. (A) TUNEL assay (red) made on zebrafish expressing eGFP (green) or eGFP2A-EWSR1-FLI1…
Figure 1—figure supplement 3
Mosaic model of Ewing sarcoma generated by the microinjection of ubi:RSG-EWSR1-FLI1 plus Cre mRNA into single-cell-stage embryos.
(A) Plot representing the incidence of one, two, three, or four tumors per zebrafish. (B) EWSR1-FLI1 triggers the formation of ectopic fins in zebrafish. (C) Examples of tumors observed in mosaic …
Figure 2 with 1 supplement
Zebrafish tumors phenocopy human Ewing sarcoma.
(A) Representative image of Ewing sarcoma tumor in WT zebrafish. H&E staining, immunohistochemistry (IHC) staining with anti-FLI1 and anti-CD99 antibodies. Scale bars, 100 μm. (B) H&E and Periodic …
Figure 2—figure supplement 1
Validation of zebrafish model of Ewing sarcoma.
(A) Immunohistochemistry (IHC) staining of sections of zebrafish tumor and control zebrafish with anti-FLI1 antibodies. (B) Gene set enrichment analysis (GSEA) showing the enrichment of proteins …
Figure 3
Validation of EWSR1-FLI expression in zebrafish embryos.
(A) Rate of embryos expressing EWSR1-FLI1 during the first 10 days of development (N = 524 Survival). Uninjected embryos (N = 528) as well as embryos injected with ubi:RSG-EWSR1-FLI1 plus GFP mRNA (N…
Figure 4 with 1 supplement
EWSR1-FLI expression activates the ERK1/2 signaling pathway in vivo.
(A) Immunofluorescent staining of eGFP (control) and eGFP2A-EWSR1-FLI1-expressing embryos at 48 hpf. Blue: phosphohistone H3. Green: eGFP or eGFP2A-EWSR1-FLI1. A small region of the dorsal surface …
Figure 4—figure supplement 1
Immunostaining of zebrafish tumors for pERK1/2.
(A) Immunofluorescence staining of zebrafish outgrowth at 72 hpf for pERK1/2 (red) eGFP2A-EWSR1-FLI1 (green). Arrows indicate cells positive for pERK1/2 and negative for EWSR1-FLI1. Scale bars, 20 …
Figure 5
Liquid chromatography–mass spectrometry (LC–MS/MS) analysis of proteins affected by EWSR1-FLI1 expression in developing zebrafish.
(A) Heat map representing the differentially expressed proteins dysregulated by EWSR1-FLI1 at 24 and 48 hpf. (B) Quantitative analysis of the differentially expressed proteins at 24 and 48 hpf. (C, D…
Figure 6
Surfen treatment impairs ERK1/2 signaling and growth of Ewing sarcoma cells.
(A) Immunoblot analysis of pERK1/2, ERK1/2, and tubulin levels in TC32 and EWS502 cells treated with surfen or DMSO. (B) Immunoblot quantification of pERK1/2 expression level relative to ERK1/2 in …
Figure 7 with 3 supplements
Surfen inhibits EWSR1-FLI1-mediated growth in the zebrafish model.
(A) Surfen inhibits the development of EWSR1-FLI1-driven outgrowths in zebrafish larvae. (B) Surfen inhibits progression of EWSR1-FLI1-driven tumors. Animals with eGFP-positive tumors were grouped …
Figure 7—figure supplement 1
Targeting ERK signaling pathway in embryonic model of Ewing sarcoma.
(A) Survival rate of embryos during the first 100 hr of treatment with surfen (0.2 and 1 μM), trametinib (1, 10, and 20 μM), or DMSO. (B) Immunoblot confirming the level of pERK1/2 in embryos …
Figure 7—figure supplement 2
Changes in the fin shape driven by EWSR1-FLI1 expression as a readout for drug screening.
(A) Changes in the fin shape driven by EWSR1-FLI1 expression. Embryos injected with ubi:RSG-EWSR1-FLI1 plus GFP mRNA were used as control. (B) Scheme of surfen treatment. Wild-type embryos were used …
Figure 7—figure supplement 3
Surfen inhibits progression of EWSR1-FLI1-driven tumors.
Representative image of animals with trunk (A) and head (B) tumors. Timeline of zebrafish tumor progression after 0, 3, 9, and 15 days of surfen or DMSO treatment.
Videos
Video 1
Immunofluorescence staining of a 48 hpf zebrafish embryo for pERK1/2 (red), EWSR1-FLI1 (green), and pH3 (blue).
Confocal Z-stack focusing on a region of the trunk and dorsal fin.
Video 2
Immunofluorescence staining of a tumor outgrowth in 48 hpf zebrafish embryo for pERK1/2 (red), EWSR1-FLI1 (green), and pH3 (blue).
Confocal Z-stack focusing on an outgrowth arising from the dorsal surface of the embryo.
Additional files
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Supplementary file 1
Detailed characterization of zebrafish tumors driven by EWSR1-FLI1 expression.
- https://cdn.elifesciences.org/articles/69734/elife-69734-supp1-v2.xlsx
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Supplementary file 2
List of downregulated and upregulated proteins identified by LC–MS/MS analysis in EWSR1-FLI1-expressing tumors, p < 0.05.
- https://cdn.elifesciences.org/articles/69734/elife-69734-supp2-v2.xlsx
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Supplementary file 3
List of downregulated and upregulated proteins identified by LC–MS/MS analysis in EWSR1-FLI1-expressing embryos, p < 0.05.
- https://cdn.elifesciences.org/articles/69734/elife-69734-supp3-v2.xlsx
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Supplementary file 4
List of primers for RT-PCR.
- https://cdn.elifesciences.org/articles/69734/elife-69734-supp4-v2.xlsx
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Source data 1
Western Blot - Raw Data.
- https://cdn.elifesciences.org/articles/69734/elife-69734-data1-v2.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69734/elife-69734-transrepform1-v2.docx
