Recent studies had indicated the importance of the stem cells
(SC) in systemic and solid cancers, including in primary liver
cancers (PLC). The SC can contribute in the tumor-stromal cross
talk in the cancer microenvironment. However their flexibility in
the progression and the spread of the PLC is still unclear. In this
study, we isolated and characterized a cells population with SC
characteristic from paired neoplastic and non-neoplastic tissues
from a total 15 patients of hepatocellular carcinoma (HCC,
n=10), cholangiocarcinoma (CC, n=3) and non-tumoral liver
(n=2). The cells were maintained in selective media and characterized
by flow cytometry, RT-PCR, anchorage-independent
assay and in vitro cells-directed trans-differentiation using
inducer media. In vivo xenograft assay was performed by subcutaneous
and orthotopic inoculation of primary cells from
HCC and CC in male adult athymic nude and NOD/SCID
mice, respectively. Isolated cells were negative for the expression
of CD34, CD45, CD117 and CD133, but positive for
CD90 and CD44. They expressed mesenchymal SC and
pluripotency factors and had the ability to clone after low density
plating in 3D matrix. After induction into insulin-secreting
cells, they showed gene up-regulations of pancreatic cells markers
somatostatin and gastric inhibitory protein, showing
potency to trans-differentiate to endodermal lineage. Under adipogenic
stimulation, they differentiate into adipocytes as confirmed
by lipid droplets accumulation in the cytoplasm and high
expression of adipocytes regulator PPARG, detected by nile red
lipid staining and RT-PCR, respectively. Under osteogenic condition,
the cells became positive of alkaline phosphatase staining
and showed up-regulation of bone specific genes such as
bone sialoprotein and osteopontin. In vivo assay in nude mice,
alpha fetoprotein and albumin in primary cells were noticed 4
months after injection, albeit no visible tumor nodules were
detected. Interestingly, by orthotopic injection in NOD/SCID
mice, tumor nodules in the liver and tumor metastasis on lung
were observed, indicating the capacity of the SC to enter into
circulation. Further analysis on the xenograft cells showed a
mixed population between human and mouse cells as confirmed
by RT-PCR and DNA sequencing. The expression of liverspecific
markers were also identified in metastatic site. Our
data provide clear evidence of the plasticity of the SC-like cells
isolated from HCC and CC in the process of hepatocarcinogenesis
and metastasis. Their trans-differentiation flexibility may
804A AASLD ABSTRACTS HEPATOLOGY, October, 2012
induce a switch from normal to cancerous microenvironment. It
supports the importance of the presence of the SC in human
PLC.
Disclosures: The following people have nothing to disclose: Caecilia H. Sukowati, Beatrice Anfuso, Lory S. Crocé, Claudio Tiribelli
