The efficacy of
hepatocellular carcinoma (HCC) treatment is very low because of the high percentage of recurrence and resistance to
anticancer agents.
Hepatic cancer stem cells (HCSCs) are considered the origin of such recurrence and resistance. Our aim was to evaluate the stemness of
doxorubicin and
5-fluorouracil resistant
hepatic cancer cells and establish the new method to isolate the HCSCs from primary cultured HCC
tumors. HCC biopsies were used to establish primary cultures. Then, primary cells were selected for HCSCs by culture in medium supplemented with
doxorubicin (0, 0.1, 0.25, 0.5 or 1 μg/mL),
5-fluorouracil (0, 0.1, 0.25, 0.5 or 1 μg/mL) or their combination. Selection was confirmed by detection of HCSC markers such as CD133, CD13, CD90, and the side population was identified by
rhodamine 123 efflux. The cell population with the strongest expression of these markers was used to evaluate the cell cycle, gene expression profile,
tumor sphere formation, marker
protein expression, and in vivo
tumorigenesis. Selective culture of primary cells in medium supplemented with 0.5 μg/mL
doxorubicin and 1 μg/mL
5-fluorouracil selected
cancer cells with the highest stemness properties. Selected cells strongly expressed CD13, CD133, CD90, and CD326, efflux
rhodamine 123 and formed
tumor spheres in
suspension. Moreover, selected cells were induced to differentiate into cells with high expression of CD19 and AFP (
alpha-fetoprotein), and importantly, could form
tumors in NOD/SCID mice upon injection of 1 × 10(5) cells/mouse. Selective culture with
doxorubicin and
5-fluorouracil will enrich HCSCs, is an easy method to obtain HCSCs that can be used to develop better therapeutic strategies for patients with HCC, and particularly HCSC-targeting
therapy.