Ann Surg Treat Res.  2016 Apr;90(4):183-193. 10.4174/astr.2016.90.4.183.

Characterization of sphere-forming HCT116 clones by whole RNA sequencing

• Chung E
• Oh I
• Lee KY

• KMID: 2160947
• DOI: http://doi.org/10.4174/astr.2016.90.4.183

Abstract

PURPOSE
To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential.
METHODS
Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0.4 Gy γ-irradiation.
RESULTS
Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones.
CONCLUSION
Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions.

Keyword

Neoplastic stem cell; Colon neoploasms; RNA sequence analysis; HCT116 cells

MeSH Terms

Cell Separation
Clinical Coding
Clone Cells*
Colonic Neoplasms
HCT116 Cells
Humans
Neoplastic Stem Cells
Parents
RNA*
Sequence Analysis, RNA*
Transcriptome
RNA

Figure

• 

  Fig. 1 HCT116 cells show colonosphere forming capacity and radioresistance, regardless of CD133 expression. (A) Light microscopy photomicrographs of colonospheres over the course of 2 weeks. Upper panel: magnification ×4, scale bar denotes 500 µM; lower panel: magnification ×10, scale bar denotes 100 µM. (B) Radiosensitization of clonogenic cell survival curves were obtained from HCT116 parental, CD133+ and CD133- clones cells cells pretreated with different exposed to 0–4 Gy γ-irradiation.

• 

  Fig. 2 Comparison of each subclone of HCT116 in attached monolayer and colonosphere cultures by whole RNA sequencing.

• 

  Fig. 3 Heatmap analysis of the top 30 up-regulated and down-regulated genes in colonosphere culture conditions discriminated spheres and monolayers of each HCT116 subclone.

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