Being prepared by this single-cell 3D culture method, spheroids derived from different prostate cancer cell lines (DU145, LNCaP and VCaP) and CD133+-sorted primary-cultured prostate cancer cells displayed significant elevated levels of multiple CSC-associated transcription factors (SOX2, OCT4, KLF4, NANOG) and membrane antigens (CD44, CD133) (Fig. 3a–c, Additional file 1: Fig. S4a-c). This evidence concerns the gene KLF4 and prostate cancer.