Clonospheres formed due to modified culture conditions are often studied for their stem cell like behaviour. The main objective of the current study is to compare the stem cell markers and link it to hTERT levels by monitoring their quantitative gene expression as they are potential targets for new generation combination therapeutics.
In the present study we created stable colonospheres of Human colon cancer cell line HCT-116 long term culture conditions of Serum deprivation. Clonospheres formed after 15 days were collected by gentle and enzymatic dissociation was performed. Single cell suspension was obtained by mechanically dissociating the cells through a 22G needle. Single cells were replanted at a density 1200 cells/ml in Serum Free Medium in the 6 well plates for further passage. Passaging of cells was done at an interval of 8 days. The spheres formed were cyto-spun in special slides for Immunocytochemistry (ICC) studies for β-catenin protein and hTERT. The colonospheres were also processed for real time PCR expression studies for the same genes to confirm.
In this present study, immunofluorescence studies revealed high β-catenin expression in the nucleus in colonospheres as compared to that of differentiated cancer cell line HCT-116 where the signal was localized mostly in the membranous and non-nuclear regions. Also increased TRF2 signal in colonospheres indicated higher activity of hTERT gene as TRF2 is the direct activator of hTERT to protect the telomere. Quantitative PCR studies showed that there was a significant over expression (p<0.05) at the mRNA level of the hTERT, TRF2, Rap1 genes along with the β-catenin over expression. Immunofluorescence analysis also revealed higher expression of CSC marker CD44 and ALDH1in colonospheres compared to the parental population.
Clonospheres sub-population is showing higher degree of hTERT gene expression along with β-catenin when compared to the parental HCT-116 cancer cells. We also checked the co expression of other telomere maintenance genes mainly TRF 2 and Rap1 which also showed similar results. Therefore, we conclude that not only hTERT but possibly other Sheltrin proteins are regulated by β-catenin which is co expressed.