Figure 4

The EWSR1-FLI1^low→high transition increases seeding in lung and colonization in vivo. (a) DOX-pre-treated (EWSR1-FLI1 ^low→high group) or untreated (EWSR1-FLI1^high group) shA673-1c/GFP cells were injected in the mouse tail vein. During the experiments, no DOX was given to mice to enable EWSR1-FLI1 re-expression in the DOX-pre-treated group. (b) HES, GFP, CD99 and EWSR1-FLI1 staining on slides of lung from mice injected with shA673-1c/GFP-DOX-pre-treated clone and sacrificed after 4 weeks. Lung cryosections of 6 μm were prepared at different depth with a cryostat at −20 °C (Microm HM550). Ewing cells were revealed by positive GFP and CD99 immunostaining as described in Franzetti et al.²⁴ Section was scanned with Ultra-Fast Scanner (UFS, Philips). Scale bars represent 50 μm. (c) Percentage of mice positive for GFP/CD99 lung nodules in EWSR1-FLI1^high group or EWSR1-FLI1^low→high group, compared using a Fisher’s exact test (*P<0.05, ***P<0.001). (d) Number of GFP/CD99 lung nodules by group with lung nodules size distribution, measured with Image Management System (Philips Digital Pathology) and compared using a Mann–Whitney test.