Cancer Stem Cells (CSCs) are well-known by not only their ability to undergo self-renewal and differentiate into more mature cancer cells but also by their tumor-initiating ability from relatively small number of cells. Little investigation into the exact role of isolated populations of CSCs has been undertaken and the prevalence of CSCs in malignancies is still a matter of some debate. We aim to identify specific CSC markers and isolate CSC sub-populations from colon cancer to allow for studies which will potentially make them more susceptible to chemotherapy.
Expression levels of colorectal CSCs markers including CD271, SSEA1, EPCAM, Cript-1, or ABCG2 were validated under both hypoxic and normoxic conditions in SW480 and CSC480, using Flow cytometry and immunofluorescence (IF). The relationship between hypoxia and cellular expression of Brn2, which is a transcription factor that could be a CSC marker, was explored via flow cytometry and IF. Furthermore, correlation between CSC marker expression in primary and metastasis tissues in human colorectal cancer was examined by IF and Immunohistochemistry.
ABCG2 and Cripto-1 were expressed in low levels on cell-subpopulations compared to CD271, EPCAM or SSEA1 consistent with the possibility that they are CSC markers. Interestingly, all the marker levels were increased in a subpopulation after 72 hours under hypoxia compared to normoxia conditions. However, comparing over the time course of hypoxia showed that EPCAM, Cripto-1, or ABCG2 expression were decrease at 48 hours and then increased again at 72 hours. The SW480 Brn2-EGFP showed a significant decrease in Brn2 positive cells between the normoxia and hypoxia samples at 24, 48, or 72 hours. We found that all markers were highly expressed in metastasis compared to primary in human caner tissues.
ABCG2 and Cripto-1 may be suitable markers for isolating and studying colon CSCs. Notably; colon CSCs increased under hypoxic conditions.