Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells

Abstract

Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1^high states, characterized by highly active cell proliferation, and EWSR1-FLI1^low states where cells have a strong propensity to migrate, invade and metastasize. This new model of phenotypic plasticity proposes that the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential.

Figure 1

EWSR1-FLI1^low cells demonstrate increased migration and invasion in three-dimensional matrix and in zebrafish. (a) Western blot, (b) phalloidin-stained actin cytoskeleton and (c) measure of cell area of shA673-1c and shSK-E17T clones treated for 10 days (+DOX) with 1 μg/ml DOX or untreated (-DOX) (n=15 per group). Scale bars represent 20 μm. Error bars represent s.e.m. DOX-treated/untreated cells were compared using Welch t-test (***P<0.001). See also Supplementary Figures S1 and S2, Supplementary Tables S1 and S2. (d) Three-dimensional collagen-I multicellular spheroid invasion assay with shA673-1c and shSK-E17T spheroids prepared by the hanging droplet method. 24-h videos were acquired and three time-points are shown. Red dotted lines represent the initial spheroid perimeter. Scale bars represent 100 μm. See also Supplementary Movie S1. (e) Representative pictures of zebrafish xenotransplantation model 4 days post-injection and cumulative results of migration distance of shA673-1c/mCherry cells, DOX-treated or -untreated, from implantation site (X) into the yolk sac for all zebrafish embryos, where each colour represents an individual embryo, and each dot a cluster of one or more tumour cells. Scale bars represent 500 μm. (f) Mean cumulative distance of migration and relative tumour burden of shA673-1c/mCherry cells (n=22 to 27 fish per group), error bars represent 1-way ANOVA with Bonferroni post-test (*P<0.05, ***P<0.001).

Figure 2

EWSR1-FLI1 expression is heterogeneous in Ewing cell lines. (a) Gene expression profiling at the single cell level by RT-ddPCR of EWSR1-FLI1 and RPLP0 mRNA. Representation of EWSR1-FLI1/RPLP0 ratio in A673, SK-N-MC and TC71 Ewing cell lines; and in shA673-1c, shSK-E17T, shA673-1c+DOX, and shSK-E17T+DOX clones (inducible systems), each dot represents one cell. Bars represent median with interquartile range. The EWSR1-FLI1^low threshold is defined by the upper limit of the interquartile range of DOX-treated cells. DOX-treated/untreated cells were compared using Wilcoxon test (***P<0.001). See also Supplementary Figure S2. (b) Protein quantification by immunofluorescence using three-dimensional deconvolution microscopy completed with ImageJ quantification at the single cell level of EWSR1-FLI1 (FLI1) normalized to DNA content (DAPI). Three-dimensional microscopy was performed on Eclipse 90i upright microscope on Nikon Imaging centre (Institut Curie, Paris, France) with a 0.2 μm step for 3D stack and a X100 NA 1.4 oil-immersion objective. The deconvolution of each image stacks was performed automatically using an iterative 7 and algorithm Meinel by PICT-IBiSA imaging facility of the Curie Institut, Paris. Scale bar represents 10 μm (c) Representation of EWSR1-FLI1/DAPI ratio in A673 and TC71 Ewing cell lines; and in shA673-1c and shA673-1c+DOX clones (inducible systems), each dot represents one cell. Quantification experiments of the shA673 clone in the −/+ DOX conditions were repeated three times with fully consistent results showing highly significant differences in the quantification of the specific EWSR1-FLI1 staining between both conditions. Bars represent median with interquartile range. The EWSR1-FLI1^low threshold is defined by the upper limit of the interquartile range of shA673-1c+DOX cells. DOX-treated/untreated cells were compared using Wilcoxon test (***P<0.001). (d) Characterization of ICAM1⁻ and ICAM1⁺ subpopulations by FACS analysis of A673 cell line stained with APC anti-ICAM1 (1:500, Biolegend, APC anti-CD54 HA58) and (e) mRNA expression analysis by RT-qPCR of direct EWSR1-FLI1-targets, data are represented as mean +/−s.e.m. (f) Characterization of ICAM1⁻ and ICAM1⁺ subpopulations at day 0, 20 and 38 days post-sorting.

Figure 3

Downstream markers of EWSR1-FLI1 activity confirm the presence of EWSR1-FLI1^low subpopulation. (a) Immunofluorescence of LOX (EWSR1-FLI1-downregulated target) and actin cytoskeleton in A673 and SK-N-MC cell lines. Images were acquired using upright widefield Apotome microscope (Zeiss, Marly-Le-Roi, France) equipped with a Coolsnap HQ2 camera through a x63 NA 1.4 oil-immersion objective lens and driven by Axiovision software (Zeiss). White arrows point the cells of interest, exhibiting LOX expression associated to an important cell-spreading and a well-organized actin stress fibre cytoskeleton. Scale bars represent 20 μm. (b) IHC of LOX in shA673-1c+/−DOX xenografts. Black arrows indicate the cells of interest. Right panels represent enlargement of left panels. Scale bars represent 50 μm. (c) IHC and quantification of LOX in eight representative samples from Ewing tumours TMA. Black arrows point cells expressing LOX. Scale bars represent 20 μm. Error bars mean s.d.

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. 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.

Figure 5

Model of Ewing cells dissemination based on cell-to-cell heterogeneity of EWSR1-FLI1 expression. In this model, undifferentiated EWSR1-FLI1^high cells proliferate with robust cell–cell adhesion, whereas mesenchymal-like EWSR1-FLI1^low cells migrate and invade environment through important cell–matrix interactions.