To investigate CSC enrichment in formed spheroids as compared to their adherent counterparts, we applied three separate techniques: (1) secondary sphere formation capacity whereby self-renewal properties of CSCs are exploited for maintaining stem-like features of cancer cells; (2) assessment of key stemness genes expression including SOX2, C-MYC, OCT4, KLF4 and NANOG, as master regulators of pluripotency and self-renewal capacity in CSCs; and (3) quantifying the expression of potential CSCs surface markers by flow cytometry. Here, POU5F1 is linked to cancer.