Isolation and enrichment of PC-3 prostate cancer stem-like cells using MACS and serum-free medium

Abstract

Prostate cancer stem-like cells (PCSLCs) are considered to be the 'seed' of prostate cancer. The aim of this study was to confirm that the PC-3 cells, which we isolated and enriched from PC-3 cells through magnetic bead cell sorting (MACS) and serum-free medium (SFM) culture, were PCSLCs. Combinations of MACS, flow cytometry (FCM), SFM and immunocytochemistry (ICC) were used to ensure the positive expression of CD133 and CD44 on PC-3 and sphere-forming cell membranes. Self-renewal, multi-potential differentiation, unlimited proliferation and permanency assays were also applied to indentify whether the PC-3 cells exhibited the characteristics of cancer stem cells (CSCs). As a result, there was a low proportion of PCSLCs in the PC-3 cells. In the FCM assay, the proportion of cells expressing CD133 or CD44 in the PC-3 cells was 0.51 and 0.31%, respectively. In addition, we found that the proportion of PC-3 cells sorted by MACS that expressed CD133 was significantly increased compared with that of the sphere-forming cells cultured in SFM (99.09 vs. 84.80%, P<0.05), while no difference was observed in the proportion of cells expressing CD44 between them (99.88 vs. 99.82%, P>0.05). The expression of PAP and AR as detected by western blot analysis of induced PCSLCs was significantly increased compared with that of uninduced PCSLCs (P<0.05); the proliferation capacity of PCSLCs was significantly higher than that of both the PC-3 cells (P<0.05) and induced PCSLCs (P<0.05). Furthermore, the PCSLCs that were isolated from SFM and MACS both demonstrated certain characteristics of stem cells and should be considered as stem cell-like. These data may hold potential for further exploring the role of PCSLCs.

Keywords: CD133; CD44; PC-3 cell; magnetic bead cell sorting; prostate cancer; serum-free medium; stem cell.

Figure 1

Surface marker expression analysis by flow cytometry (FCM). Proportion of PC-3 cell membranes expressing CD133 (A); CD44 (B); CD133, following magnetic bead cell sorting (MACS) (C) and CD44, following MACS (D). Proportion of sphere-forming cell membranes expressing CD133 (E) and CD44 (F).

Figure 2

Formation process of prostate cancer stem-like spheres (PCSLSs) in serum-free medium (SFM). Three-day (A) and seven-day (B) phases of spheres in SFM (×400). Four-day phase of spheres (C), and one-day (D), two-day (E) and four-day (F) phases of TGF-β-induced spheres (×100). A comparison of sphere-forming cell immunofluorescence staining between CD133 (G), CD44 (H) and the nuclei phase (I) (×200). The merge phase of CD133 and CD44 is demonstrated in (J) and (K), respectively.

Figure 3

Western blot analysis of PAP (A) and AR (B). 1, spheres induced by TGF-β; 2, cells with the CD133⁺/CD44⁺ phenotype induced by TGF-β; 3, spheres and 4, cells with the CD133⁺/CD44⁺ phenotype.

Figure 4

Proliferation assay of the four cell phenotypes: G1, sphere cells following several subcultures; G2, PC-3 cells; G3, cells with the CD133⁺/CD44⁺ phenotype following MACS; G4, sphere cells following induction by TGF-β.