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pad) implantation of patient’s tumor fragments [62,63] . These models preserve the histological and genetic
features of the patients they have been derived from; therefore, they represent powerful tools to discover
and validate new therapies for HB patients. In particular, as the majority of HB PDXs are developed from
surgical samples that received previous therapies or at relapse, they represent a valuable preclinical tool to
identify second-line treatments. The combination of duparlisib (a pan-PI3K inhibitor) and trametinib (a
[62]
MEK-1/2 inhibitor) was shown to inhibit tumor growth of a NRAS-mutated orthotopic PDX , and
evaluation of several potential second-line drugs such as irinotecan, temozolomide, temsirolimus, sirolimus,
sorafenib, crizotinib, paclitaxel, and volasertib on heterotopically implanted PDXs allowed the identification
of irinotecan alone or in combination with temozolomide or volasertib as promising anti-cancer treatment
for HB patients [63,64] . Moreover, the inhibition of CHKA using MN58b fully abrogated tumor growth by
lowering the proliferation rate and reverting the progenitor-like phenotype in a PDX model established
from a high-risk HB . The availability of a large HB PDX panel fully characterized at the molecular level is
[27]
also particularly relevant as it allows the investigation of the molecular markers associated to PDX response,
large-scale pre-clinical assays, and the development of companion biomarkers that are becoming an
important requirement to grant the authorization from the medicines regulatory authorities to run clinical
trials.

TUMOR CELL LINES DERIVED FOM PATIENT-DERIVED XENOGRAFTS
Recent advances have allowed the generation of cell lines derived from PDX models of pediatric liver
cancers by growing them under stem cell culturing conditions [62,63] . These second-generation models are
molecularly robust and reflect a variety of histological subtypes . As drugs identified in these models can
[64]
be easily validated in the corresponding PDX in mice [Table 1], this allows for a timely preclinical testing of
new drugs in a representative spectrum of pediatric liver tumors.

The first study using this kind of model applied trametinib, an inhibitor of the mitogen-activated protein
kinase kinase enzymes MEK1 and MEK2 to the B6 cell line, which has been derived from a xenograft from a
HB with HCC-like features with a mutation in the neuroblastoma ras viral oncogene homolog gene
(NRAS) . Strikingly, trametinib inhibited MEK1 activity and subsequently cell growth of B2 cells in a dose-
[62]
dependent manner in vitro. This inhibition was highly specific, as the subsequent testing in orthotopic PDX
models without NRAS mutation showed no sensitivity to trametinib treatment.

Expression profiling analyses have identified the polo-like kinase 1 (PLK1) gene to be overexpressed in a set
[65]
of 74 HB samples . Of note, high PLK1 expression was associated with a significantly poorer outcome.
Kats et al. tested the PLK1 inhibitor volasertib in six cell lines derived from PDX models and could show a
[64]
strong tumor-suppressive effect in half of the cell lines, as well as in HB-214 when transplanted into athymic
nude mice. Most interestingly, in vitro activity of volasertib was achieved at concentrations known to be safe
in phase I trials in children and adults .
[64]

To more accurately mimic the architecture and organization of solid tumors, the same PDX cell lines have
recently been further developed to be grown as three-dimensional spheroids . As a proof-of-concept,
[66]
[66]
Eloranta et al. assayed the well-documented anti-neoplastic activity of chloroquine in these models and
could show a reduced viability and induction of apoptosis. These organized cell culture models will bridge
the gap between cell lines grown on plastic and in vivo models and substantially further anticancer
therapeutic development in pediatric liver cancers.

Recently, a new study has been published reporting on a novel therapeutic strategy that reactivates p53 by
the inhibition of the p53 regulator murine double minute 4 (MDM4), which is highly expressed in HB