Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line - PubMed (original) (raw)

Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line

Toru Kondo et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Paradoxically, however, some cancers seem to contain stem-like cells (cancer stem cells). To help resolve this paradox, we investigated whether established malignant cell lines, which have been maintained for years in culture, contain a subpopulation of stem cells. In this article, we show that many cancer cell lines contain a small side population (SP), which, in many normal tissues, is thought to contain the stem cells of the tissue. We demonstrate that in the absence of serum the combination of basic fibroblast growth factor and platelet-derived growth factor maintains SP cells in the C6 glioma cell line. Moreover, we show that C6 SP cells, but not non-SP cells, can generate both SP and non-SP cells in culture and are largely responsible for the in vivo malignancy of this cell line. Finally, we provide evidence that C6 SP cells can produce both neurons and glial cells in vitro and in vivo. We propose that many cancer cell lines contain a minor subpopulation of stem cells that is enriched in an SP, can be maintained indefinitely in culture, and is crucial for their malignancy.

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Figures

Fig. 1.

Existence of SP cells in established cancer cell lines. Cells of the rat C6 glioma (A), human MCF7 breast carcinoma (B), rat B104 neuroblastoma (C), and human HeLa carcinoma (D) cell lines were labeled with the Hoechst 33342 and then analyzed by flow cytometry. (Lower) Results when the cells were treated with 50 μM verapamil during the labeling procedure. The SP, which disappears in the presence of verapamil, is outlined and shown as a percentage of the total cell population. These experiments were repeated at least three times with similar results.

Fig. 2.

The roles of PDGF and bFGF on C6 SP cells. C6 cells were cultured in FCS (A) or serum-free medium with bFGF (B), PDGF (C), or bFGF plus PDGF (D) for 3 wk and were then photographed while alive in an inverted phase-contrast microscope. C6 SP cells were cultured for 3 wk in FCS (E) or serum-free medium with bFGF (F), PDGF (G), or both (H) and were analyzed by flow cytometry as shown in Fig. 1. (I) C6 cells were cultured in FCS (○), bFGF (▴), PDGF (▪), or bFGF plus PDGF (•) for the indicated times, and the proportion of SP cells was analyzed by flow cytometry. (J) C6 cells were cultured as in A_–_D for 3 wk, and then the expression of bcrp1, mdr1, or g3pdh mRNAs was analyzed by RT-PCR. All experiments were repeated at least three times with similar results. (Scale bar in D, 100 μm.)

Fig. 3.

Different roles of bFGF and PDGF on C6 SP cells. C6 cells were cultured in PDGF (A), bFGF (B), or both (C) for 2 wk and then expanded in bFGF plus PDGF for an additional 2 wk. They were then analyzed by flow cytometry as described for Fig. 1. (D) C6 cells were cultured as shown in A_–_C, and the expression of bcrp1, mdr1, or g3pdh mRNAs was analyzed by RT-PCR. All experiments were repeated at least three times with similar results.

Fig. 4.

SP cells in the C6 glioma cell line can generate both SP and non-SP C6 cells. C6 cells were cultured in bFGF plus PDGF for 2 wk and then sorted by flow cytometry as described for Fig. 1. The SP (A) and non-SP (B) cells were then cultured separately in the same conditions for an additional 2 wk. The cells were then photographed while alive and analyzed by flow cytometry (C) as shown in Fig. 1. (Scale bar in B, 100 μm.)

Fig. 5.

C6 SP cells can differentiate into both neurons and glia. C6 SP cells were prepared as described for Fig. 4_A_ and then cultured in serum-free medium with bFGF plus PDGF on ornithine/fibronectin-coated chamber slides for 1 (A) or 10 (B) d. Cells were then immunolabeled for neuronal and glial markers. (Scale bar in B, 30 μm.)

Fig. 6.

Evidence of malignancy of C6 SP cells in vivo. SP or non-SP C6 cells (105) were injected i.p. into 4-wk-old female nude mice (A, right and left mice, respectively). The mice were killed 18 d later. (B_–_E) The hematocrit was measured (B), and tumor tissue in the mesentery (C), uterus (D), and lung (E) was photographed (right, SP-injected tissue; left, non-SP-injected tissue). Frozen sections of the tumors in the mesentery in SP-injected mice were immunolabeled for nestin (F), GFAP (G), or low molecular weight neurofilament (NF-L; H). The sections were labeled also with Hoechst 33342 (blue) to identify nuclei. (Scale bar in H, 50 μm.)

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Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line - PubMed (original) (raw)