Results showed that the agar-based 3D cultured prostatospheres, derived from three prostate cancer cell lines [androgen receptor (AR)-positive: LNCaP and VCaP; AR-negative: DU145], expressed significantly higher levels of PCSC markers (including the commonly expressed CD44, CD133, and NANOG) as compared with their counterpart cells grown under the conventional adherent 2D culture conditions (Fig. 4a). Here, CD44 is linked to prostate cancer.