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Correnti et al. Hepatoma Res 2018;4:69 I http://dx.doi.org/10.20517/2394-5079.2018.96 Page 9 of 15

Since CSCs are characterized by metabolic changes, drugs that inhibit OXPHOS have been studied as
potential anticancer agents. Metformin, which interfere with OXPHOS by inhibiting NADH-coenzyme Q
oxidoreductase (complex I), is a key example and has been shown to be particularly cytotoxic for CSCs, as
well as for cells with mutations in OXPHOS complex I [148,149] . Despite metabolic studies in the field of liver
CSC are still at an early stage, the dual inhibition of glycolytic and mitochondrial energy pathways may
represent a promising superior therapeutic approach to effective eradicate heterogeneous liver CSCs and
to overcome therapeutic resistance.

Moreover, since EMT pathway and CSC features seem to be intimately linked, improving our
understanding of these cellular states may help to develop novel therapies. The plasticity of CSCs further
suggests that simultaneously targeting CSCs existing in both epithelial and mesenchymal states rather
[150]
than either state alone is needed to achieve complete tumor eradication . Hence, future investigations in
this direction are imperative.

It is important to underline that the development of CSC-specific therapeutic strategies imply the presence
of a common recognized method for isolation and subsequent characterization of liver CSCs. During
the last decade a large number of studies have aimed to identify liver CSCs and several attempts have
been made to enrich liver CSCs. Common strategies for PLC CSC enrichment, varied from the widely
used classical antigenic approach that relies on surface CSC markers detection (e.g., CD133 [46,56,151-154] ,
[161]
CD44 [71,153-155] , OV6 [156] , CD90 [129,130,157] , EpCAM [22,68,71,158] , CD13 [159] , CD24 [153,154,160] , CD47 ) to functional
techniques including side population (SP) analysis [65,162-165] , Aldefluor assay [166-169] and tumor-sphere
formation [65,67,70,170,171] . In all different published studies, enriched PLC CSC subsets have been then tested in
immune-deficient mice for the in vivo tumorigenic potential [22,56,65,67,68,129,130,151,152,155,156,159-162,166,170] .

One important challenge in developing new therapeutic strategies is the dynamic and plastic behavior of
tumor cells, especially of CSC. As it’s well known, a central role in the regulation of cancer cell plasticity
is played not only by genetic alterations, but also by epigenetic changes, including DNA methylation,
[58]
histone modifications and non-coding RNA (ncRNA) activity . By acting at transcriptional, post-
transcriptional and translational level, ncRNAs represent key regulators of CSCs by modulating several
biological processes including asymmetric division, unresponsiveness to treatments and EMT, thus
[58]
affecting tumor progression and recurrence . In addition, recent studies also suggest that similar to
normal stem cells, CSCs seem to reside in specialized microenvironment (“CSC-niche”) [46,50,70,172] , whose
signals can support self-renewal and drug-resistance features and, thereby, may influence the plasticity of
CSCs [173-177] . Therefore, targeting only CSCs may not be enough, and continued development of therapies
targeting CSCs and their microenvironment in combination with chemotherapy may be essential to
improve the outcomes of PLC patients.

DECLARATIONS
Authors’ contributions
Analysis of publications and drafting of the manuscript: Correnti M, Booijink R, Di Maira G
Critically revised the manuscript: Raggi C, Marra F
Read and approved the final manuscript: All authors

Availability of data and materials
Not applicable.

Financial support and sponsorship
Raggi C and Correnti M were supported by the Italian Foundation of Cancer Research (AIRC). Work on
liver cancer in Dr. Marra’s laboratory is supported by AIRC, Istituto Toscano Tumori (ITT), Fondazione
Umberto Veronesi, University of Florence.