Cancer stemness contributes to cluster formation of colon cancer cells and high metastatic potentials.

The ability of cancer cells to form clusters is a characteristic feature in the development of metastatic tumours with drug resistance. Several studies demonstrated that clusters of circulating tumour cells (CTCs) have a greater metastatic potential to establish new tumours at secondary sites than single CTCs. However, the mechanism of cluster formation is not well understood. In this study, we investigated whether cancer stemness would contribute to cluster formation. We used a tumour sphere culture method to enrich cancer stem cells (CSCs) from colon cancer cells and found that during the second generation of sphere culture, clusters (between 3 and 5 cells) formed within the first 24 hours, whereas the rest remained as single cells. The clusters were analysed for stemness and metastatic potential, including gene expressions for cancer stemness (CD133 and Lgr5), epithelial-mesenchymal transition (E-cadherin and TGF-β 1-3) and hypoxia-induced factors (HIF-1α and HIF-2α). The results showed that the clusters expressed higher levels of these genes and colon CSC surface markers (including CD24, CD44 and CD133) than the single cells. Among these markers, CD24 seemed the major contributor linking the cells into the clusters. These clusters also showed a stronger ability to both form colonies and migrate. Our data collectively suggest that colon cancer stemness contributes to cluster formation and that clustered cells exhibit a great metastatic potential. Our study thus provides a method to study the CTC clusters and derive insight into oncogenesis and metastasis.