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van der Ent et al. J Transl Genet Genom 2018;2:10. I https://doi.org/10.20517/jtgg.2018.09 Page 5 of 12

cells (MSCs) to the 3D culture, to investigate cross talk between stromal cues and those from biomechani-
cal forces. EWS and MSCs co-cultured in flow perfusion bioreactors stimulated each other’s growth and led
[91]
to altered responses to various inhibitors . Another model evaluates the effect of mechanical loading by
[89]
culturing cells in 3D scaffolds and subjecting these to cycles of compression . This led to an upregulation of
RUNX2, linked to drug resistance and poor prognosis when highly expressed in patients.

In vivo models
To accurately model tumours in a 3D fashion, and assess the effect of the tumour microenvironment on
drug response, good animal engraftment models are essential. Earliest xenotransplantation models were in-
jected either subcutaneously or intravenously in nude or NOD/scid mice [93-95] . Later murine models sought to
[96]
more closely resemble the native tumour environment by performing orthotopic xenografts in rib bones ,
[98]
[99]
[97]
femur , pretibial space , and gastrocnemius muscles . Not only new compounds are tested but also nov-
el drug delivery methods, such as silk gel to effectuate a sustained release of chemotherapeutics locally [100] .
Ultrasound visualization is being used for more accurate tissue implantation, as well as identifying early
response to treatment [101,102] .
In addition to murine models, serval groups have also used zebrafish embryonic xenograft models to test
novel therapeutics [95,103-105] . Here, cells are injected in the yolk of the embryo, show intra- and extravasation
and migration into muscles and fins within 4 days after implantation, making these models interesting for
rapid initial screening of a large number of possible therapeutic compounds.

A recent development is the establishment of patient-derived xenograft (PDX) models. A limitation of estab-
lished cell-lines is that they may have deviated from the original disease during years of culture. With PDX
models, pieces of resected tumour are directly implanted in mice, where they will remain until the tumour
has reached a size where they need to be respected again and implanted in new mice. This way, the artificial
environment of in vitro culture can be avoided. Being a rare tumour, the development of EWS and Ewing-
like PDX models is slow, but several models have been established already [106,107] .

THERAPEUTIC OPPORTUNITIES
A limited number of direct targets can be identified from genetics of EWS, with EWSR1-ETS being the most
prominent one. Targeting the fusion protein directly has proven to be challenging due to its flexibility, but
progress has been made and a phase I clinical trial (NCT02657005) is ongoing with a small molecule inhibi-
tor [108] . Aside from targeting the EWSR1-ETS fusion protein directly, inhibiting its dominant direct targets
has been investigated as therapeutic alternative, as EWSR1-ETS is a key driver in the epigenetics [23,24] , tran-
[27]
scription [109] , splicing [25,26] , and metabolic reprogramming of EWSR1-ETS.
[23]
Epigenetic remodelling inhibitors, including HDAC inhibitors and LSD1 , with success tested in the pre-
clinical phase, are being tested in clinical trials. EWSR1-ETS influences the transcription at such a level that
the cells become very sensitive to genotoxic agents, causing EWS to act like a BRCA1 deficient tumour with
impaired homologous recombination, as shown in a recent study [109] . The impaired homologous recombina-
tion opens therapeutic opportunities for PARP1 inhibitors, and these were effective in the induction of cell
death in in vitro and xenograft models. If this translates into the clinic is currently being investigated, as
clinical trials with combinations of genotoxic agents and PARP1 inhibitors are ongoing [110,111] . Recently a
study demonstrated the dysregulation of EWSR1-ETS at protein level and subsequent induction of a high un-
folded protein response (UPR). Inhibiting an important protein in this pathway, IRE1α-XBP1 led to reduced
cell viability in vitro and in vivo.

IMMUNOTHERAPY IN EWS
The low mutation rate of EWS suggest that corrective apoptosis pathways, such as the TNF-related apoptosis