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Zhang et al. Hepatoma Res 2019;5:27 I http://dx.doi.org/10.20517/2394-5079.2019.13 Page 9 of 18

tumorigenicity, decreased tumor differentiation potential, metastasis and invasion and poor prognosis in
HCC [160-163] , with profiling study shows that K19+ HCCs highly express invasion or metastasis-related genes
(TACSTD2,VASP, LAMC2, LAMB1, PDGFRA), biliary/HPC markers (NOTCH2, GSTP1, CD133, JAG1) and
members of the miRNA-200 family (miR-200c, miR-141) . A recent study showed that K19+ cells were not
[160]
involved in the early clonal expansion of rat hepatocarcinogenesis, and K19 expression arose in preneoplastic
hepatocyte lesions undergoing malignant transformation. In addition, they also indicated that K19 positivity
in HCCs did not necessarily reflect the cell of origin of the tumor, but rather the plasticity of preneoplastic
cells during the tumorigenic process .
[164]

LGR5
In 2007, LGR5, a G protein-coupled receptor with a seven transmembrane domains , was firstly identified
[165]
as a surface marker of intestinal stem cells . Later, it has been applied to identify homeostatic stem cells in
[166]
various organs such as ovaries, hair follicles, mammary gland, and stomach [166-169] . LGR5 has been reported to
involved in regeneration of damaged tissues in the small intestine and colon, liver, pancreas, and stomach [15,170-172]
and in CSCs that regulates tumor proliferation [173,174] . Carbon tetrachloride treatment enhances both fibrosis and
LGR5+ liver stem cell growth, whereas LGR5 downregulation aggravates fibrosis. HGF together with Rspo1
increases the number of LGR5+ liver stem cells and enhances hepatic function by inhibiting fibrosis . Both
[175]
Carbon tetrachloride-induced acute damage and oval cell response to damage can induce LGR5+ stem cells/
progenitors actively engaged in hepatic reconstitution via de novo generation of hepatocytes . Effendi et al.
[176]
[15]
addressed that LGR5 upregulated HCC cells showed more potent colony-forming capability and possessed
higher therapeutic resistant to a cytotoxic drug and weakened migration ability than the controls. Further,
LGR5 overexpressed HCC cells produces nodule-type metastases in the livers of immunodeficient mice,
whereas vector-transfected HCC cells generates more invasive tumors . Lei et al. unraveled that the LSD1/
[177]
[176]
Prickle1/APC/β-catenin signaling axis is engaged in regulating the stem characteristics and chemoresistance of
hepatic LGR5+ LCSCs.

TREATMENT TARGETING LCSCs
Using surface markers to identify and isolate LCSCs remains an initial and important step of CSC-targeting
therapy. Immunotherapy uses specific antibodies to target LCSC surface makers can be integrated with
conventional chemotherapy, radiotherapy and surgery to promote therapeutic effects. The most frequently-
used LCSC-associated surface markers along with clinical strategies that target them are demonstrated as
follows.

One of the current therapeutic approaches to target directly LCSCs is nanomedicine-based therapy, in
which medication delivery and intake are effectively controlled in nanoscale . Epirubicin-adsorbed
[178]
nanodiamonds displayed high efficacy in inducing the elimination of chemoresistant LCSCs . Poly lactic-
[179]
co-glycolic acid-encapsulated disulfiram strongly inhibits LCSCs and has a synergistic cytotoxicity with
5-FU or sorafenib . Gao et al. developed a GPC3-targeted CAR and found that it obviously suppressesed
[181]
[180]
HCC growth. Overexpression of ANXA3 increased the number of CD133+ cells and positively associated
with tumorigenicity of CD133+ cells. The underlying mechanism of ANXA3-mediated maintenance of
LCSCs stemness involved the HIF1A/Notch pathway. ANXA3 upregulated dendritic cells could induce
more active T cells ,which could preferentially kill CD133+ LCSCs . Xu et al. addressed that Hep-12
[183]
[182]
cells owing stemness properties, are susceptible to autologous-activated tumor-infiltrating lymphocytes-
mediated recognition and cytotoxicity. What’s more remarkable, the authors put forward that it may be the
first evidence to demonstrate the hypothesis that immunotherapy can be used to target recurrent HCC cells
with stem cell-like properties. Bone morphogenetic protein-9 is a potent growth inhibitor of hepatocellular
carcinoma and reduces the liver cancer stem cells population by suppressing the expression of five prominent
LCSC markers, including CD44, CD90, AFP, GPC3 and ANPEP . In current clinical practice (according to
[184]