Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas

Abstract

Background: Several human cancers have been found to contain cancer stem-like cells (CSCs) having cancer-initiating ability. However, only a few reports have shown the existence of CSCs in bone and soft tissue sarcomas. In this study, we identified and characterised side population (SP) cells that showed drug-resistant features in human bone sarcoma cell lines.

Methods: In seven osteosarcoma cell lines (OS2000, KIKU, NY, Huo9, HOS, U2OS and Saos2) and in one bone malignant fibrous histiocytoma (MFH) cell line (MFH2003), the frequency of SP cells was analysed. Tumourigenicity of SP cells was assessed in vitro and in vivo. Gene profiles of SP cells and other populations (main population; MP) of cells were characterised using cDNA microarrays.

Results: SP cells were found in NY (0.31%) and MFH2003 (5.28%). SP cells of MFH2003 formed spherical colonies and re-populated into SP and MP cells. In an NOD/SCID mice xenograft model, 1 x 10(3) sorted SP cell-induced tumourigenesis. cDNA microarray analysis showed that 23 genes were upregulated in SP cells.

Conclusions: We showed that SP cells existed in bone sarcoma cell lines. SP cells of MFH2003 had cancer-initiating ability in vitro and in vivo. The gene profiles of SP cells could serve as candidate markers for CSCs in bone sarcomas.

Figure 1

Detection of side population cells in bone sarcoma cell lines. (A) The populations of SP cells of seven osteosarcoma cell lines (NY, OS2000, KIKU, Huo9, HOS, Saos2 and U2OS) and of one bone MFH cell line (MFH2003), in the presence or absence of verapamil, are shown. SP cells are marked by black dotted lines to show the proportion of SP cells among total living cells. (B) The mean proportions of SP cells in cell lines. These results were reproducible in at least two independent experiments.

Figure 1

Detection of side population cells in bone sarcoma cell lines. (A) The populations of SP cells of seven osteosarcoma cell lines (NY, OS2000, KIKU, Huo9, HOS, Saos2 and U2OS) and of one bone MFH cell line (MFH2003), in the presence or absence of verapamil, are shown. SP cells are marked by black dotted lines to show the proportion of SP cells among total living cells. (B) The mean proportions of SP cells in cell lines. These results were reproducible in at least two independent experiments.

Figure 2

The re-population of SP cells into both SP and MP cells. (A) (a) The populations of SP cells and MP cells before cell sorting are shown. SP cells were gated as G1, and MP cells were gated as G2. (b, c) The proportions of SP cells among the total living cells are indicated. Isolated SP cells (b) and MP cells (c) after cell sorting. The proportions of SP and MP cells among the total living cells are indicated. (d, e) The populations of SP cells (d) and MP cells (e) after 2-week in vitro culture with medium containing 10% FBS are also shown. Experiments were repeated in triplicate with similar results. (B) The relative expression of ABCG2 was evaluated in SP cells and MP cells by real-time PCR.

Figure 3

Tumourigenesis of SP and MP cells in vitro. (A and B) The features of spherical colonies derived from resultant SP cells (A) and MP cells (B) cultured without serum in an anchorage-independent manner for 2 weeks. (C) Spherical colony removed from the suspension culture and allowed to attach to a substratum. Adherent cells can be seen expanding from the sphere. (D) The numbers of resultant spherical colonies from SP cells and MP cells were counted. Data are representative of three independent experiments.

Figure 4

The features of xenotransplanted SP cells in vivo. (A) Macroscopic features of 1 × 10³ each of SP and MP cells in an NOD/SCID mouse at 12 weeks after xenotransplantation. Black arrow indicates the site of injection of MP cells. (B) Histological findings of the xenotransplanted tumour derived from SP cells (1 × 10⁴). Haematoxilin and eosin staining (original magnification: × 200) is shown.