CD44+ Cancer Stem-Like Cells In EBV-Associated ... - PLOS

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Figure 1.

Sphere-forming cells with stem cell-like properties in EBV-positive NPC.

(A) Free-floating tumor spheres were formed from EBV-positive C666-1 cells (left panel) and they demonstrated ability to differentiate comparably to monolayer cells upon administering complete medium (right panel). (B) By qRT-PCR, multiple stem cell-related genes (OCT4, NANOG, ALDH1, CKIT, CD44, CD133) were enriched in spheroids when compared to parental C666-1. Transcription of SOX2 was not increased in the spheroids. (C) SOX2 protein was frequently expressed in C666-1 sphere-forming cells. By flow cytometry, SOX2-positive (SOX2+) cells were found to be enriched in and constituted over 60% of sphere-forming cell population. (D) Over 80% of sphere-forming cells expressed cell surface marker CD44 while CD44+ cells in detected in parental C666-1 and other NPC xenografts were significantly lower (all P<0.001). Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (*P<0.05, **P<0.01, ***P<0.001).

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Figure 1 Expand

Table 1.

In vivo tumorigenic capacity of sphere-forming cells and unselected parental cells of C666-1 in nude mice.

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Figure 2.

CD44 and SOX2 as CSC markers.

By flow cytometry, SOX2 was found to be preferentially expressed on CD44+ cells and coincidentally, SOX2 expression was rarely detected in CD44− cells. Cells coexpressing both CD44 and SOX2 were found to be enriched in spheroids. Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (*P<0.05, **P<0.01, ***P<0.001).

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Figure 2 Expand

Figure 3.

CD44+ cells with stem cell-like properties in EBV-positive NPC.

CD44+ cell fraction exhibited a significantly higher (A) clone formation efficiency and (B) sphere-forming efficiency when compared to CD44− cell fraction. In addition, (C) CD44+ cells exhibited significantly higher proliferation rate than CD44− cells. (D) Developmental hierarchy feature of CD44+ cells. Percentage of CD44+ cells were continually reduced in the isolated CD44+ cell fraction over time. (E) CD44+ cells exhibited higher resistance to 5-FU treatment when compared to the CD44− and parental C666-1 cells. All graphs denoting mean ± SE (n≥3) with statistical significance calculated by t-test (*P<0.05, **P<0.01, ***P<0.001).

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Figure 3 Expand

Figure 4.

Overexpression of multiple EBV and cellular genes in sphere-forming NPC cells.

(A) By qRT-PCR, multiple EBV genes (EBER, BARF1, LMP1, LMP2A, EBNA1 and BZLF1) were found to be overexpressed in spheroids when compared to monolayer C666-1 cells. EBV copy number in these cells was determined by qPCR. (B) Selected genes aberrantly expressed in spheroids were confirmed by qRT-PCR. The significantly upregulated genes include chemokines and receptors (CCR7, CCL4, CX3CL1 and IL-8), cell adhesion molecule SELE, signaling molecules (GLI1, FOXN4) and ABC transporters (ABCC3, ABCC11). (C) Cell surface-expressed CCR7 was found to be frequently expressed in sphere-forming cells (>60%) by flow cytometry. The CCR7+ cell subpopulation was also detected in NPC lines and primary tumors (<5%). (D) CD44+CCR7+cells were also found to be enriched in spheroids. Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (*P<0.05, **P<0.01, *** P<0.001).

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Figure 4 Expand

Table 2.

Selection of aberrantly expressed genes in sphere-forming cells compared to monolayer C666-1 cells.

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Figure 5.

Immunohistochemical analysis of CCR7 and CD44 expression in primary NPC.

Representative primary NPC cases with high (A), medium (B), low (C) expression of CCR7. (D) Primary NPC with absence of CCR7 expression was shown. CCR7 staining were detected in few infiltrating lymphocytes, but not in the tumor cells. Primary tumors with high (E) and medium (F) CD44 expression were shown. In (G) and (H), weak CD44 expression was detected in the tumor cells while strong CD44 staining in infiltrating lymphocytes was commonly found.

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Table 3.

Correlation between CCR7 expression and clinicopathological features in primary tumors.

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Table 3 Expand

Figure 6.

Effect of CCR7 neutralization on NPC CSCs.

To evaluate the function of CCR7 in CSCs, C666-1 was treated with CCR7 blocking antibody and its proliferation, clone-forming and sphere-forming efficiency were investigated. (A) Proliferation of CD44+ cells was inhibited after treatment with CCR7 blocking antibody. (B) The clone formation efficiency of C666-1 cells was diminished after CCR7 blocking and (C) the spheroid-forming ability was significantly inihibited (P<0.001) when compared to untreated controls. Histograms denoting mean ± SE (n≥3) with statistical significance calculated by t-test (*P<0.05, ***P<0.001).

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