Isolation and characterization of spheroid cells from the HT29 colon cancer cell line.

Background: Colorectal cancer stem cells (Cr-CSCs) are involved in the growth of colon cancer, but their specific role in tumor biology, including metastasis, is still unclear. Currently, methods for sorting Cr-CSCs are based on the expression of surface markers (e.g., CD133(+), CD44(+), and aldehyde dehydrogenase 1 (ALDH1(+))); however, the specificity of these markers for Cr-CSCs is uncertain.

Purpose: This study aimed to develop more effective ways of isolating and purifying Cr-CSCs.

Methods: Suspension culture was used for isolation of Cr-CSCs. And spheroid cells were performed by side population technology, and the putative molecular marker analysis of colorectal cancer stem cell. Migration assay and chemoresistance experiment were conducted between the adherent cells and spheroid cells.

Results: HT29 colon cancer cells grew well in suspension culture. The percentage of CD44(+) cancer cell of spheroid cells was 68 times higher than that of adherent cells (89.5% vs. 1.3%), but there was no obvious difference in the percentage of CD133(+) cells (6.25% vs. 5.6%). Moreover, it is worth noting that the percent of CD133 (+)/CD44(+) cells remarkably rose (from 0.6% to 5.4%). The expression of ALDH1 was markedly increased (7.5% vs. 20.5%) for the spheroid cells than the adherent cells. The side population within the spheroid population dramatically increased from 0.2% to 6.3%. The resistance of spheroid cells to 5-FU was higher than that of adherent cells, as was their ability to migrate in the presence of SDF-1α.

Conclusion: Suspension culture is an effective approach for enriching Cr-CSCs and can provide an inexhaustible supply of genetically stable colon cancer stem cells for targeted Cr-CSC studies. Spheroid cell models also enable the study of colon cancer chemoresistance and metastasis and may help to elucidate the role of cancer stem cells in colon cancer.