Comparison of enrichment model of cancer stem cells in colon cancer DLD-1 cells

Abstract

Objective To compare the effects of 3 enrichment models of cancer stem cells (CSCs) in colon cancer DLD-1 cells. Methods DLD-1 cells were cultured in 3 kinds of serum-free medium containing various growth factors to generate spheroid cells, including G1 group using 10 ng/mL EGF+ 10 ng/mL bFGF, G2 group using 20 ng/mL EGF+ 10 ng/mL bFGF and G3 group using 20 ng/mL EGF+ 20 ng/mL bFGF. The cell proportion of CD133+ CD44+ , CD133+ , CD44+ and ALDH+ were confirmed by flow cytometery. The mRNA expression of stem cells related genes (Oct4, Sox2, Nanog) and colon CSCs genes (CD133, CD44, CD166, ALDH, CD66c) were detected by real-time polymerase chain reaction (RT-PCR). The capacity of self-renewal was detected by sphere-forming assay. The tumorigenesis in vivo was measured by colony formation assay. Measurement data with normal distribution were presented as ±s, comparisons among groups were analyzed using the one-way ANOVA, and pairwise comparisons were done by the paired-samples t test. Results The results of flow cytometery: the proportion of CD133+ CD44+ cells in the G1, G2 and G3 groups were 0.030±0.010, 0.113±0.012 and 0.043±0.006, respectively, with a significant difference among the 3 groups (F=67.625, P 0.05). The proportion of CD133+ cells in the G1, G2 and G3 groups were respectively 0.037±0.015, 0.027±0.006 and 0.023±0.006, with no significant difference among the 3 groups (F=1.444, P>0.05). The proportions of CD44+ cells in the G1, G2 and G3 groups were 20.103±4.761, 44.600±2.138 and 44.387±2.117, respectively, with a significant difference among the 3 groups (F=56.269, P 0.05). The proportion of ALDH+ cells in the G1, G2 and G3 groups were 0.060±0.010, 0.627±0.068 and 0.043±0.021, respectively, with a significant difference among the 3 groups (F=192.097, P 0.05). The results of RT-PCR: mRNA expressions of G1, G2 and G3 groups were respectively 1.272±0.152, 11.636±3.164 and 1.420±0.201 in stem cell related genes Oct4, 4.717±0.409, 8.262±1.079 and 4.827±0.446 in stem cell related genes Sox2, 1.836±0.417, 12.883±4.030 and 1.636± 0.589 in stem cell related genes Nanog, 1.555±0.397, 4.367±1.109 and 1.208±0.165 in colon CSCs genes CD133, 25.853±4.544, 59.483±8.950 and 26.924±4.134 in colon CSCs genes CD44, 21.223±1.088, 57.847±5.880 and 21.710±4.010 in colon CSCs genes CD166, 19.378±2.479, 78.211±9.879 and 18.475±3.571 in colon CSCs genes ALDH, 4.737±2.105, 18.003±3.826 and 5.211±1.474 in colon CSCs genes CD66c, showing significant differences among the 3 groups (F=31.529, 23.865, 21.557, 19.102, 27.837, 76.608, 90.530, 23.997, P 0.05). The results of sphere-forming assay: the numbers of spheres in the G1, G2 and G3 groups were 182±34, 396±22 and 217±22, respectively, showing a significant difference among the 3 groups (F=56.128, P 0.05). The results of colony formation assay: the numbers of colonies in the G1, G2 and G3 groups were 230±33, 403±32 and 266±36, showing a significant difference (F=22.111, P 0.05). Conclusion The culturing cells using 20 ng/mL EGF+ 10 ng/mL bFGF could effectively promote the enrichment of CSCs in colon cancer DLD-1 cells. Key words: Colonic neoplasms; Neoplastic stem cells; Enrichment; Suspension culture