Isolation of colorectal cancer stem-like cells

Abstract

This study is aimed at isolating colorectal cancer stem-like cells in vitro using a neurosphere assay method employed in isolating gliobastoma multiforme tumor cells. This was followed with confirmation of the isolated cells by flow cytometry, pluripotent genes expression and in vivo tumorigenicity assay. Using this culture assay, stem-like and non-stem-like CRC cells were isolated and expanded in vitro from purchased Balb/c mice induced with CT26 colorectal cancer (CRC) cell line. The procedure includes an initial mechanical dissociation and chemical digestion of tumor tissue and subsequently plating the resulting single cell suspension in serum-free medium (SFM) or serum-containing medium (SCM). This selectively permits growth of cancer stem-like cells in SFM and eliminates non-stem-like cancer cells through the process of anoikis or apoptosis. CRC stem cells derived cultures proliferated as non-adherent spheres in vitro in different shapes and sizes. These cells expressed cell surface markers previously reported for tumor stem cells, including CD44, CD133, CD166 and CD26 and formed tumors when implanted in severe combined immunodeficient mice in a concentration dependent manner. Importantly, the stem-like cells had self-renewal properties with significantly higher expression of the pluripotent stem cell genes NANOG, OCT4, and SOX2 compared to the adherent non-stem cells. Collectively, the results of this study indicate that SFM is a defined culture medium that enriches for CRC stem-like cells and represents a suitable in vitro model for the study of CRC stem-like cells. This finding may be useful in developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation within colorectal cancer.

Establishment of a Novel Model for Anticancer Drug Resistance in Three‐Dimensional Primary Culture of Tumor Microenvironment

“…Expression of E-cadherin (Figure 3(a)), MUC2 (Figure 3(b)), vimentin (Figure 3(c)), and α -SMA (Figure 3(d)) was observed in these organoids. Since it was reported that expression of various stem cell markers was elevated in 3D culture compared with 2D culture [17], we next confirmed the existence of cancer stem cells in tumor ALI organoids (Figure 3(e)). Expression of a cancer stem cell marker, LGR5 in tumor organoids, was observed (Figure 3(e)).…”

“…A recent report showed that 3D culture exhibits higher stemness and tumorigenicity than 2D culture [17]. It was also known that higher stemness leads to the resistance to anticancer drugs [18].…”

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

“…Furthermore, analyses of short-term cultures (passages 2-7) showed stable expression of these different cell surface markers among the cultured CSC populations. The cell surface markers, CD26, CD29, CD44, CD54, CD49b, CD49f CD133, CD90 and CD326 (EpCam) have been reported to be present on human SC and CSC in various solid tumors [44-51]. CD24 and CD66 have been shown to identify mature/differentiated cells in normal and cancer tissues, including breast cancer [52, 53].…”

Self-renewal molecular mechanisms of colorectal cancer stem cells

“…The second is dependent on the characteristic of specific enzymes, such as aldehyde dehydrogenase 1 (ALDH1) (23) and ATP-binding cassette subfamily G member 2 (ABCG2) (24). The third is culturing the cells in serum-free, low-adhesion conditions in vitro and enriching suspending colospheres (25). The methods for identifying CCSC properties include evaluating the ability of continuous sphere formation in vitro , continuous tumorigenicity in NOD/SCID mice and the similarity of differentiation between xenograft tumors and primary tumors.…”