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Abstract
BackgroundBecause few definitive markers are available for hepatic cancer stem cells (HCSCs), based on physical rather than immunochemical properties, we applied a novel method to enrich HCSCs.MethodologyAfter hepatic tumor cells (HTCs) were first isolated from diethylinitrosamine-induced F344 rat HCC model using percoll discontinuous gradient centrifugation (PDGC) and purified via differential trypsinization and differential attachment (DTDA), they were separated into four fractions using percoll continuous gradient centrifugation (PCGC) and sequentially designated as fractions I–IV (FI–IV). Morphological characteristics, mRNA and protein levels of stem cell markers, proliferative abilities, induced differentiation, in vitro migratory capacities, in vitro chemo-resistant capacities, and in vivo malignant capacities were determined for the cells of each fraction.FindingsAs the density of cells increased, 22.18%, 11.62%, 4.73% and 61.47% of primary cultured HTCs were segregated in FI–FIV, respectively. The cells from FIII (density between 1.041 and 1.062 g/ml) displayed a higher nuclear-cytoplasmic ratio and fewer organelles and expressed higher levels of stem cell markers (AFP, EpCAM and CD133) than cells from other fractions (P<0.01). Additionally, in vitro, the cells from FIII showed a greater capacity to self-renew, differentiate into mature HTCs, transit across membranes, close scratches, and carry resistance to chemotherapy than did cells from any other fraction; in vivo, injection of only 1×104 cells from FIII could generate tumors not only in subcutaneous tissue but also in the livers of nude mice.ConclusionsThrough our novel method, HCSC-like cells were successfully enriched in FIII. This study will greatly contribute to two important areas of biological interest: CSC isolation and HCC therapy.
Highlights
Over the last decade, the notion that tumors are maintained by their own stem cells, the so-called cancer stem cells (CSCs), has created great excitement in the research community [1]
Through our novel method, hepatic cancer stem cells (HCSCs)-like cells were successfully enriched in FIII
This study will greatly contribute to two important areas of biological interest: CSC isolation and Hepatocellular carcinoma (HCC) therapy
Summary
The notion that tumors are maintained by their own stem cells, the so-called cancer stem cells (CSCs), has created great excitement in the research community [1]. CSCs are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors [1]. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. The existence of CSCs was first proposed over 40 years ago [2]; they were only recently isolated from solid tumors, including tumors of the breast [3], prostate [4], brain [5] and colon [6]. CSCs have been isolated from some types of tumors, there is much debate surrounding this topic. Because few definitive markers are available for hepatic cancer stem cells (HCSCs), based on physical rather than immunochemical properties, we applied a novel method to enrich HCSCs
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