Ovarian cancer stem cells are enriched in side population and aldehyde dehydrogenase bright overlapping population

Abstract

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH(Br)) cells from ovarian cancer cells. Both SP cells and ALDH(Br) cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH(Low) cells, respectively. We analyzed an SP and ALDH(Br) overlapping population (SP/ALDH(Br)), and the SP/ALDH(Br) population exhibited higher tumor-initiating ability than that of SP cells or ALDH(Br) cells, enabling initiation of tumor with as few as 10(2) cells. Furthermore, SP/ADLH(Br) population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH(Low), MP/ALDH(Br) and MP/ALDH(Low) cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH(Br) population was detected in several gynecological cancer cells with ratios of 0.1% for HEC-1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH(Br) overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.

Figure 1. MCAS CSCs/CICs are enriched in SP cells.

A. Detection of SP cells from MCAS cells. MCAS ovarian mucinous adenocarcinoma cells were stained with Hoechst 33342 dye and analyzed. The percentage represents the ratio of SP cells. B. Tumor initiation of SP cells derived from MCAS cells. 10³ and 10⁴ SP and MP cells derived from MCAS cells were inoculated subcutaneously into the backs of NOD/SCID mice, and tumor growth was measured weekly. Data represent means ± SD. Differences between SP and MP cells were examined for statistical significance using Student's t-test. *P values. C. qPCR of CSC/CIC markers in MCAS SP and MP cells. Data represent means ± SD. Asterisks indicate significant difference. *P<0.05. t-test.

Figure 2. MCAS CSCs/CICs are enriched in ALDH^Br cells.

A. Detection of ALDH^Br cells from MCAS cells. MCAS ovarian mucinous adenocarcinoma cells were stained with BAAA and analyzed. The percentage represents the ratio of ALDH^Br cells. Inhibitor indicate ALDH1 inhibitor (diethylamino- benzaldehyde (DEAB)). B. Tumor initiation of ALDH^Br cells derived from MCAS cells. 10⁴ ALDH^Br and ALDH^Low cells derived from MCAS cells were inoculated subcutaneously into the backs of NOD/SCID mice, and tumor growth was measured weekly. Data represent means ± SD. Differences between ALDH^Br and ALDH^Low cells were examined for statistical significance using Student's t-test. *P values. C. qPCR of CSC/CIC markers in MCAS SP and MP cells. Data represent means ± SD. Asterisks indicate significant difference. *P<0.05. t-test.

Figure 3. SP and ALDEFLUOR dual assay.

A. Summary of SP and ALDEFLUOR dual assay. MCAS cells were stained by Hoechst 33342 dye and then stained by BAAA and analyzed. The cells were divided into three groups according to ALDH intensity (ALDH^Br (ALDH bright), ALDH^Mid (ALDH middle), ALDH^Low (ALDH low)), then analyzed by SP assay. The ratio of ALDH^Br cells was 14.4%, and the ratio of SP cells was 5.3%. The ratios of SP cells in ALDH^Br cells, ALDH^Mid cells and ALDH^Low cells were 7.1%, 6.9% and 3.7%, respectively. The ratio of SP/ALDH^Br cells in total cells was 1.0%. B. Tumor initiation of SP/ALDH^Br, SP and ALDH^Br cells. 10² and 10³ SP/ALDH^Br, SP and ALDH^Br cells derived from MCAS cells were inoculated subcutaneously into the backs of NOD/SCID mice, and tumor growth was measured weekly. Data represent means ± SD. Differences between SP/ALDH^Br and SP cells or ALDH^Br cells were examined for statistical significance using Student's t-test. *P values. Daggers indicate mice death due to tumor. C. Immunohistochemical staining of ABCG2 and ALDH1. Ovarian carcinoma tissue was stained by anti-ABCG2 antibody and anti-ALDH1 antibody. Brown membrane staining indicates ABCG2 and cytoplasm pink staining indicates ALDH1. Asterisk indicates vessel, and arrows indicate ABCG2 and ALDH1 double-positive ovarian carcinoma cells. Magnification, ×400.

Figure 4. Characterization of SP/ADLH^Br cells.

A. Sphere formation assay. The numbers of colonies from four fractions (SP/ALDH^Br, SP/ALDH^Low, MP/ALDH^Br and MP/ALDH^Low) were evaluated at day 7. Data represent means ± SD. The differences were examined for statistical significance using Student's t-test. *P values. Representative images of spheres are shown (×100). B. Adipocyte differentiation assay. The cells were cultured under existence of trans-retinoic acid followed by adipocyte differentiation medium. Oli Red O-positive adipocytes were counted. Data represent means ± SD. The differences were examined for statistical significance using Student's t-test. *P values. Representative images of Oil Red O-staining are shown (×200). Red-staining indicate adipocyte differentiation.

Figure 5. Charasterization of SP/ALDHBr cells.

A. Cell viability assay. The cells were cultured under existence of serially diluted cisplatin. The viable cells were analyzed by WST-1 kit. Y-axis indicates the viability of cells. Data represent means ± SD. The differences were examined for statistical significance using Student's t-test. *P values. B. qPCR analysis. The expression of stem cell markers was examined using SP/ALDH^Br, SP/ALDH^Low, MP/ALDH^Br and MP/ALDH^Low cells. Data represent means ± SD. Asterisks indicate significant difference. *P<0.05. t-test. C. SOX2 knockdown suppress the expressions of ALDH1A1 and ABCG2. SOX2 mRNA was knocked down by siRNA. Two days after transfection of SOX2 siRNA, the expressions of ALDH1A1 and ABCG2 were investigated by RT-PCR. GAPDH was used as an internal control. Control siRNA (si-Cont) transfected cells were used as negative control. D. SOX2 knock down suppress the tumor-initiation. SOX2 mRNA was knocked down by siRNA. Ten thousand si-SOX2 and control siRNA (si-Cont) transfected cells were inoculated subcutaneously into the backs of NOD/SCID mice, and tumor growth was measured weekly. Data represent means ± SD. Differences were examined for statistical significance using Student's t-test. *P values.

Figure 6. SP, ALDEFLUOR and CD44 triple staining.

MCAS cells were stained by Hoechst 33342 dye, BAAA and anti-CD44 antibody, and analyzed. The ratios of SP, ALDH^Br, CD44⁺, SP/ALDH^Br, SP/CD44⁺, ALDH^Br/CD44⁺ and SP/ALDH^Br/CD44⁺ cells were 5.3%, 14.4%, 8.0%, 1.0%, 0.9%, 3.3% and 0.4%, respectively.