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.2010 Jul 6;5(7):e11456.
doi: 10.1371/journal.pone.0011456.

Characterization of side populations in HNSCC: highly invasive, chemoresistant and abnormal Wnt signaling

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Characterization of side populations in HNSCC: highly invasive, chemoresistant and abnormal Wnt signaling

Jun Song et al. PLoS One..

Abstract

Side Population (SP) cells, a subset of Hoechst-low cells, are enriched with stem cells. Originally, SP cells were isolated from bone marrow but recently have been found in various solid tumors and cancer cell lines that are clonogenic in vitro and tumorigenic in vivo. In this study, SP cells from lymph node metastatic head and neck squamous cell carcinoma (HNSCC) cell lines were examined using flow cytometry and Hoechst 3342 efflux assay. We found that highly metastatic HNSCC cell lines M3a2 and M4e contained more SP cells compared to the low metastatic parental HNSCC cell line 686LN. SP cells in HNSCC were highly invasive in vitro and tumorigenic in vivo compared to non-SP cells. Furthermore, SP cells highly expressed ABCG2 and were chemoresistant to Bortezomib and etoposide. Importantly, we found that SP cells in HNSCC had abnormal activation of Wnt/beta-catenin signaling as compared to non-SP cells. Together, these findings indicate that SP cells might be a major driving force of head and neck tumor formation and metastasis. The Wnt/beta-catenin signaling pathway may be an important target for eliminating cancer stem cells in HNSCC.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SP cells in high metastatic M3a2 and M4e cells.
(A): Flow cytometry analysis of 686LN cells. Cells were stained with Hoechst 33342. (B and C): M3a2, and M4e cells were stained with Hoechst 33342 in the absence or presence of either verapamil or reserpine. The SP cells were gated and shown as percentage as indicated. Flow cytometry analyses were performed in triplicate with similar results in each case.
Figure 2
Figure 2. Re-sorting of SP and non-SP cells.
(A): Cultured SP cells divide into both SP and non-SP cells. (B): non-SP cells only divide into non-SP cells. Flow cytometry analyses were performed in triplicate with similar results in each case.
Figure 3
Figure 3. Expression of stem cell markers in SP and Non-SP cells.
(A): The expression of CD29 and CD44 in SP and non-SP cells from M3a2 cells. Cells were stained with Hoechst 33342 in the presence of either CD29-APC or CD44-APC. Positive CD29 and CD44 were indicated as percentages in both SP and non-SP cells. (B): The expression of CD29 and CD44 in SP and non-SP cells from M4e cells. (C): Detection of BMI1 and ABCG2 in SP and Non-SP cells by RT-PCR. (D): Detection of BMI1 and ABCG2 in SP and non-SP cells by Real-time RT-PCR.
Figure 4
Figure 4. Anchorage-independent growth of SP and non-SP cells.
(A): Soft agar assay using SP and non-SP cells from M3a2 and M4e. (B): Quantification of soft agar assay of SP and non-SP cells. Relative colony numbers (fold) were indicated. (C): Soft agar assay using SP cells and their parental cells. Relative colony numbers (fold) were indicated. (D): Sphere assay of SP and non-SP cells. The sphere numbers were counted. (E): Sphere assay of SP cells and their parental cells. The sphere numbers were counted.
Figure 5
Figure 5. SP cells are highly invasive.
(A): Invasive growth of SP and non-SP cells. The invasion assay was performed using 24-well Matrigel invasion chambers. The results represent average value ± SD from three independent experiments. (B): Invasive growth of SP cells and their parental cells. The invasion assay was performed using 12-well Matrigel invasion chambers.
Figure 6
Figure 6. SP cells are resistant to chemotherapy.
(A): Treatment of SP cells, non-SP cells and the parental M4e cells with PS-341 and etoposide for 48 hr. Cells were counted using Trypan blue exclusion assay. (B): Overexpression of ABCG2 increases SP cell numbers. M4e cells were transduced with retroviruses expressing ABCG2 and empty vector. The expression of the HA-tagged ABCG2 was confirmed by Western blot analysis (left panel). M4e/V and M4e/ABCG2 cells were stained with Hoechst and sorting. (C): ABCG2 switched non-SP cells to SP cells. Non-SP cells from M4e were transduced with retroviruses expressing ABCG2 or the empty vector. M4e-NSP/V and M4e-NSP/ABCG2 cells were stained with Hoechst and sorting. (D): ABCG2 promoted chemoresistance. M4e-NSP/V and M4e-NSP/ABCG2 cells were treated with PS-341 for 24 and 48 hr.
Figure 7
Figure 7. Abnormal activation of Wnt/β-catenin signaling in SP cells.
(A): Increased Wnt/β-catenin activity in SP cells. SP and non-SP cells were transfected with TOPFLASH or FOPFLASH luciferase reporter assay. Relative luciferase activities were determined. (B): Endogenous Wnt target genes were highly expressed in SP cells. The expression of DKK1 and Axin2 was determined using Real-time RT-PCR. Relative mRNA expression levels were determined.
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