| Abstract |
Several studies have indicated that bipotential liver stem cells, also
called oval cells (OC), may give rise to liver cancers. We have endeavored
to establish and characterize a cancer cell line from our model of
combined hepatocholangiocarcinoma (CHCC) in rats. This model is suggestive
of a stem cell origin of the primary liver tumor, since the induction of
OC proliferation followed by aflatoxin B1 (AFB1) exposure results in both
hepatocarcinoma and cholangiocarcinoma. F344 rats were subjected to
2-acetylaminofluorene (2AAF) followed by partial hepatectomy (PH) to induce
OC activation. At the point of maximal OC proliferation, animals were
injected with AFB1, and sacrificed at 8 months following carcinogen
exposure. Cancer cells were isolated from the tumor nodules by collagenase
digestion, and cultured in DMEM serum-free media for 72 hours. Cells were
then transferred into DMEM/F12 media supplemented with 10% FBS. The
established cell line was characterized for growth kinetics, cell
morphology, immunophenotype and karyotype. The primary tumors were well-
differentiated CHCC, intermingled with OC. The cancer cell line, LCSC-1,
was sub-cloned 3 weeks after establishment of the primary culture and has
been maintained through multiple passages. In culture these cells adopted
a cobble stone appearance, being compact, polygonal-shaped with an
epithelioid morphology. These cells demonstrated a complete loss of
contact inhibition and were able to grow in multiple layers after reaching
confluence. Doubling time at passage 4 was 18 hours. Immunostaining
demonstrated that LCSC-1 expressed the OC marker OV-6, which was also
expressed by the primary tumor cells. Additionally, RT-PCR showed that
LCSC-1 expressed mRNA for both albumin and alpha-fetoprotein. Cytogenetic
analysis at passage 4 revealed an aneuploid karyotype with a modal
chromosome number of 41 and evidence of chromosomal abnormalities. We
believe that LCSC-1 represents a cancer stem cell line. Transplantation
studies to assess the tumorigenicity of these cells are currently underway.
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