Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model

Abstract

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

Keywords: EWS-FLI1; EWS-FLI1 driven transgenic mouse model; Ewing sarcoma.

Figure 1. Postnatal expression of EWS-FLI1 in cells with the activated Osterix promoter in E/F^+/- Osx-Cre⁺mice with or without p53 and pRb deletion in Model #2^OsxCre-EF

A. Representative presentation of deformed nasal bone of an E/F+/- Osx-Cre⁺ mouse compared to an E/F+/- littermate control. B. X-ray imaging of an E/F+/- Osx-Cre⁺ mouse showing structural abnormalities in facial bones as compared to the clear facial bone structure of an E/F+/- littermate control. C. Representative photographs of spleens and livers collected from E/F+/- p53fl/+ pRbfl/+ (control) and E/F+/- Osx-Cre+ p53fl/+ pRbfl/+ littermates. Hepatomegaly and splenomegaly were consistently observed in E/F+/- Osx-Cre+ p53fl/+ pRbfl/+ mice displaying leukemia-like symptoms. D. Kaplan-Meier survival plots for the indicated genotypes: E/F+/- p53fl/+ pRbfl/+ (n = 15); E/F+/- Osx-Cre+ p53fl/+ pRbfl/+ (n = 31); and Osx-Cre+ p53fl/fl pRbfl/fl, (n = 54) mice with doxycline diet until weaning age. In addition, survival plot for Osx-Cre+ p53fl/fl pRbfl/fl (n = 80) mice with no doxycycline diet is presented.

Figure 2. In vitro and in vivo expression of EWS-FLI1 and luciferase in Model #10^{COMET and COMETΔNeo}

A. Western blotting against EWS-FLI1 and beta-actin was performed in triple-transfected (COMET + Cre + rtTA) 293T cells treated with increasing amounts of doxycycline (DOX). The A673 ES cell line is shown as the positive control for endogenous EWS-FLI1 expression (right lane). B. The COMET construct was transiently co-transfected in vitro with (+) or without (-) Cre recombinase and rtTA and in the presence or absence of DOX. Varying amounts of DOX (0 to 1,000 ng/ml) were added to the media. C. Models #10^COMET (left) #10^COMETΔNeo (right) chimera were imaged for in vivo luminescence measurements. D. Quantitative RT-PCR for EWS-FLI1 and luciferase in #10^COMET (left panel) and #10^COMETΔNeo (right panel) expression in various organs extracted from the imaged mice in panel C. Relative expression (to testis expression level) of duplicates with respective SD is shown.

Figure 3. Somatic chromosomal translocation between endogenous Ewsr1 and Fli1 loci in Model #12^Cre-TL-EF

A. Targeting mouse embryonic stem cells to insert a lox-puromycinr-lox cassette between exons 8 and 9 of the Ewsr1 locus. Genomic DNAs from the embryonic stem cell clones were EcoRI/AgeI (left) or KpnI (right) digested and were analyzed for the 5’ and 3’ integrations using Southern blot. Green and purple horizontal bars represent the probes used in the Southern blots. B. Targeting mouse embryonic stem cells to insert a lox-hygromycinr-lox cassette between exons 5 and 6 of the Fli1 locus. Genomic DNAs from the embryonic stem cell clones were ApaI (left) or EcoRV/KpnI (right) digested and were analyzed for the 5’ and 3’ integrations using Southern blot. Green and purple bars represent the probes used in the Southern blots. C. Schematic illustration for adenoviral Cre infection of Ewslox/wt; Fli1lox/wt MEFs in vitro. Genomic PCR was used to detect the translocated and untranslocated Ews and Fli1 chromosomes. The locations of the primers used are presented in the schematic. D. qPCR for Ews-Fli1 on total RNA from adenoviral Cre-treated MEFs. Hprt was used as the control gene, and samples were normalized to uninfected MEFs.

Figure 4. Histopathological analysis of tumors from Model #13^RetroLTR-EF and Model #14^piggyBac-EF

A. Fibrosarcoma developed in Model #13^RetroLTR-EF. The storiform pattern of spindle-shaped, pleomorphic tumor cells is remarkable. Frequent mitotic figures (arrows) indicate aggressive tumor growth (left). α-SMA is a marker of smooth muscle and myofibroblastic cells. Human fibrosarcoma stains positive for α-SMA, whereas ES stains negative (middle). Expression of EWS-FLI1 in tumor tissue was confirmed using an anti-FLAG M2 antibody (right). B. Fibrosarcoma with a similar histology as (A) was also induced in Model #14^piggyBac-EF. Invasive growth of the tumor in lung tissue is noted (left). PDGF-RB is a mesenchymal marker that is frequently positive in human fibrosarcoma and negative in ES (middle). The expression of EWS-FLI1 was confirmed by western blotting (right).

Figure 5. Intramuscular and intraperitoneal delivery of adenovirus-Cre in 1-day-old E/F^+/+ mice in Model #16^Ad5Cre-EF

A. Penetrance of limping phenotype observed in Ad5-Cre-injected left leg vs. Ad5-eGFP-injected right leg (n = 11) at an age of 1 day. B. Comparison of quadriceps femoris muscle width between an Ad5-eGFP-injected right leg and Ad5-Cre-injected left leg. C. Representative image showing muscle atrophy observed in Ad5-Cre-injected leg. D. Penetrance of abdominal distention phenotype observed in 1-day-old mice IP-injected with Ad5-Cre (n = 9) vs. Ad5-eGFP (n = 7). E. Comparison of intestine length (from stomach to rectum) in 1-day-old mice IP injected with Ad5-Cre vs. Ad5-eGFP. F. Representative image showing shortened intestines observed in Ad5-Cre-injected mice compared to littermate mice of the same genotype injected with Ad5-eGFP.