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ALDH1-Bright Epithelial Ovarian Cancer Cells Are Associated with CD44 Expression, Drug Resistance, and Poor Clinical Outcome

  • Yu-Chi Wang
    Affiliations
    Laboratory of Epigenetics and Cancer Stem Cells, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Graduate Institute of Medical Sciences, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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  • Yi-Te Yo
    Affiliations
    Laboratory of Epigenetics and Cancer Stem Cells, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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  • Hsin-Yi Lee
    Affiliations
    Laboratory of Epigenetics and Cancer Stem Cells, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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  • Yu-Ping Liao
    Affiliations
    Laboratory of Epigenetics and Cancer Stem Cells, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Graduate Institute of Life Sciences, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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  • Tai-Kuang Chao
    Affiliations
    Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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  • Po-Hsuan Su
    Affiliations
    Laboratory of Epigenetics and Cancer Stem Cells, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Graduate Institute of Medical Sciences, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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  • Hung-Cheng Lai
    Correspondence
    Address reprint requests to Hung-Cheng Lai, M.D., Ph.D., Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, 5F, 325, Sec 2, Cheng-Gong Rd., Neihu District, Taipei City 114, Taiwan, Republic of China
    Affiliations
    Laboratory of Epigenetics and Cancer Stem Cells, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Graduate Institute of Medical Sciences, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China

    Graduate Institute of Life Sciences, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, China
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Open ArchivePublished:January 04, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.11.015
      The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1br) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1br cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1br and ALDH1low cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1br cells. In the ES-2 and CP70 cells, ALDH1br cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1low cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.
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      Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance.
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      Ovarian cancer stem-like side-population cells are tumourigenic and chemoresistant.
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      Cancer stem cells and aneuploid populations within developing tumors are the major determinants of tumor dormancy.
      These studies have revealed that ovarian cancer is a heterogeneous disease. None of these markers are satisfactory for the identification of ovarian cancer stem cells.
      Aldehyde dehydrogenase (ALDH) is a detoxifying enzyme responsible for oxidation of intracellular aldehyde; it also protects organisms from harmful aldehydes and cytotoxic drugs.
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      Identification of a primitive brain-derived neural stem cell population based on aldehyde dehydrogenase activity.
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      Materials and Methods

      Cell Lines

      SKOV-3, OVCAR-3, A2780, and CP70 cells were maintained in RPMI-1640 medium (Gibco, Rockville, MD). TOV-21G cells were maintained in culture using MCDB 105 (Sigma-Aldrich, St. Louis, MO)/Medium 199 (Gibco) (1:1). ES-2 cells were maintained in McCoy's 5A medium (Invitrogen-Life Technologies, Carlsbad, CA). All media were supplemented with 10% fetal bovine serum (Invitrogen) and 100 IU/mL of penicillin-streptomycin at 37°C under a humidified atmosphere containing 5% CO2.

      Aldefluor Assay and FACSAria Sorting

      An Aldefluor kit (StemCell Technologies, Vancouver, BC, Canada; Tukwila, WA) was used to assess ALDH activity in the EOC cell lines. Briefly, cells were incubated in Aldefluor buffer containing ALDH substrate (1 μmol/L per 1 × 106 cells) and incubated for 30 minutes at 37°C. One sample was treated with 50 mmol of diethylaminobenzaldehyde (DEAB, an ALDH inhibitor), as a negative control. After incubation, cells were washed once with Hank's buffered saline solution (BioWhittaker, Walkersville, MD) and were resuspended in Hank's buffered saline solution supplemented with 5% fetal bovine serum. Cells were stained with 1 μg/mL of propidium iodide (Sigma-Aldrich), to assess viability and were then analyzed and sorted using flow cytometry on a FACSAria instrument (BD Biosciences, San Jose, CA). Accudrop beads (BD Biosciences) were used to confirm the purity of the FACS separation.

      Spheroid Culture

      Spheroid culturing was performed as described by Ponti et al.
      • Ponti D.
      • Costa A.
      • Zaffaroni N.
      • Pratesi G.
      • Petrangolini G.
      • Coradini D.
      • Pilotti S.
      • Pierotti M.A.
      • Daidone M.G.
      Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties.
      Cells from FACSAria sorting in the primary culture were plated immediately in ultralow attachment plates (Corning Life Sciences, Acton, MA) at a density of 2000 viable cells per milliliter in serum-free DMEM/F12 medium containing 5 mg insulin (Sigma-Aldrich), 0.4% bovine serum albumin (Sigma-Aldrich), 10 ng/mL basic fibroblast growth factor (Invitrogen), and 20 ng/mL human recombinant epidermal growth factor (Invitrogen) until the growth of spheres. Single cells were prepared using dissociation, both mechanically and with dissociation using Accutase (Millipore, Billerica, MA) in 0.5 μmol/L EDTA (Millipore), on day 4. At the next passage, cells were plated in six-well ultralow attachment plates (Corning) for 7 days and then were photographed. We assessed the size of spheroids based on images captured using a cooled, back-thinned CCD camera; images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD), which provides a computational algorithm for the analysis of cellular areas. We set the threshold for image capture at a size of 2000 pixels.

      Tumorigenic Assay

      NOD-SCID mice were purchased from the National Taiwan University. TOV-21G ALDH1br cells (suspended in 50 μL of Matrigel) and TOV-21G ALDH1low cells (suspended in 50 μL of PBS) were injected subcutaneously into the hind leg of NOD-SCID mice with different numbers of 105, 104, and 103 cells. Once xenografts were established, the size of the tumors in the mice was measured once a week; tumor volume was calculated as 0.5236 × L1 × (L2)2, where L1 is the length of the long axis and L2 is the length of the short axis of the tumor.
      • Liu C.Y.
      • Chao T.K.
      • Su P.H.
      • Lee H.Y.
      • Shih Y.L.
      • Su H.Y.
      • Chu T.Y.
      • Yu M.H.
      • Lin Y.W.
      • Lai H.C.
      Characterization of LMX-1A as a metastasis suppressor in cervical cancer.
      The mice were sacrificed after 6 to 9 weeks, and tumors were analyzed for the presence of outgrowth.
      All animal studies were approved by the Institutional Animal Care and Use Committee of the National Defense Medical Centre, Taipei, Taiwan.

      Cell Viability Assay

      Cells were plated at 1000 cells per 96-well plate for 1 day and then were treated with chemotherapeutic drugs for 72 hours. Subsequently, we evaluated cell viability using the CellTiter-Glo (Promega, Annandale, NSW, Australia) luminescent cell-viability assay (ATP assay). Briefly, the ATP reagent (0.01 μmol/L) was added to 100 μL of medium containing cells in each well of a 96-well plate, and the intensity of luminescence was measured 10 minutes after the addition of the reagent.

      Flow Cytometry Analysis

      The expression of cell markers was evaluated in ALDH1br and ALDH1low cells derived from ES-2 and CP70 cells using a FACSCalibur apparatus (BD Biosciences). The anti-CD133 antibody was purchased from Abcam (Cambridge, UK) and was indirectly conjugated with fluorescence compound. Nonconfluent cultures were trypsinized into a single-cell suspension and washed with PBS. Cells were incubated with the appropriate antibodies at room temperature for 30 minutes, washed, and incubated with fluorescein isothiocyanate-conjugated secondary antibodies for 20 minutes. Mouse anti-human CD44-PE antibody was purchased from BioLegend (San Diego, CA). Nonconfluent cultures were trypsinized into a single-cell suspension and washed with PBS. Cells were stained with 1 μg/mL of propidium iodide (Sigma-Aldrich), to assess viability, and were then analyzed using a FACSCalibur apparatus (BD Biosciences).

      Tissue Microarray

      Paraffin-embedded tumor tissues were retrieved from the Department of Pathology, National Defense Medical Center, Taipei, Taiwan, and tissue microarray slides were prepared. The tissue microarrays consisted of 84 ovarian surface epithelial carcinomas: 61 serous cystadenocarcinomas, 14 mucinous cystadenocarcinomas, 3 endometrioid adenocarcinomas, and 6 clear cell carcinomas. For the present study, two pathologists (T.-K. Chao and C.-K. Lin) screened the histological sections and selected areas of representative tumor cells. One tissue core (2 mm in diameter) was taken from each of the representative tumor samples and placed in a new recipient paraffin block. Pathological data corresponding to each specimen examined, which were collected using an Institutional Review Board-approved protocol, were analyzed.

      Immunohistochemistry

      Tissue microarray sections were dewaxed in xylene, rehydrated in alcohol, and immersed in 3% hydrogen peroxide for 10 minutes to suppress endogenous peroxidase activity. Antigen retrieval was performed by heating each section at 100°C for 30 minutes in 0.01 mol/L sodium citrate buffer (pH 6.0). After three 5-minute rinses in PBS, sections were incubated for 1 hour at room temperature with a mouse monoclonal anti-human ALDH1 antibody (clone 44/ALDH, 1:100; BD Biosciences) diluted in PBS. After three 5-minute washes in PBS, sections were incubated with horseradish-peroxidase-labeled rabbit anti-mouse immunoglobulin (Dako, Carpinteria, CA) for 1 hour at room temperature. After three additional washes, peroxidase activity was visualized using a solution of diaminobenzidine at room temperature.
      For evaluation of immunoreactivity and histological appearance, all tissue microarray slides were examined and scored by two pathologists (T.-K. Chao and C.-K. Lin) concurrently. The cytoplasmic immunostaining of ALDH1 in individual tumor cells was classified as 0% to 50% positive ALDH1 cells or >50% positive ALDH1 cells. As a negative control, the primary antibody was replaced with nonimmune serum.

      Statistical Analysis

      An SPSS software package (version 13 for Windows; SPSS, Chicago, IL) was used for statistical analysis. All values are expressed as means ± SEM. The Mann-Whitney U-test was used to compare cell proliferation, and the Student's t-test was used to compare the spheroid formation efficiency. The χ2 test or Fisher's exact test was used to identify correlations between ALDH1 expression and clinicopathological characteristics. Disease-free interval and overall survival time were assessed by Cox regression analysis. Kaplan-Meier survival curves were compared using the log rank test. The significance level was defined as P < 0.05.

      Results

      Subpopulations of ALDH1br Cells in EOC Cell Lines

      To examine ALDH1 enzymatic activity in ovarian cancers, we analyzed ALDH1br cells in human ovarian cancer cell lines using an Aldefluor assay (StemCell Technologies) and flow cytometry (FACSAria; BD Biosciences). Cells were re-sorted using Accudrop beads (BD Biosciences; one drop of beads in 1 mL of PBS) as a positive control. The purity detected in our experiments was 96.3% (see Supplemental Figure S1 at http://ajp.amjpathol.org). Activity of ALDH1 varied considerably among cell lines (Figure 1). ALDH1br cells were enriched in two clear cell carcinoma cell lines, TOV-21G (1%) and ES-2 (0.8%), and in a drug-resistant subclone (CP70; 2.8%) derived from A2780 (serous type), but not in parental A2780 or other serous-type cancers (SKOV-3 and OVCAR-3). TOV-21G and ES-2 cells were therefore chosen for further studies.
      Figure thumbnail gr1
      Figure 1ALDH1 enzymatic activity in six EOC cell lines. DEAB, a specific inhibitor of ALDH1, was used for confirmation of gating areas.

      Comparison of Repopulation Capability between ALDH1br and ALDH1low Cells

      The repopulation ability of ALDH1br cells in vitro was analyzed, to test their stem-like self-renewal properties. ALDH1br cells were assessed using an Aldefluor assay 1 week and 3 weeks later. The proportion of ALDH1br cells in TOV-21G and ES-2 cell populations decreased with passages, indicating that ALDH1br cells can rapidly generate ALDH1low populations (Figure 2). The ALDH1br cells of TOV-21G and ES-2 can complete their repopulation in 3 weeks, at which time the proportions of ALDH1br cells had returned to the original percentages. In addition, ALDH1low cells also seemed to be able to generate ALDH1br cells. Of note, in 3 weeks the ALDH1low cells of ES-2 cells repopulated their ALDH1br cells. TOV-21G ALDH1low cells generated 0.6% of ALDH1br cells in 3 weeks, which is about one half of its original proportion. Our results suggest that both ALDH1br and ALDH1low cells retain their repopulation potential. ALDH1br cells may give rise to ALDH1low cells more efficiently, whereas the opposite conversion was compromised in some cells.
      Figure thumbnail gr2
      Figure 2Repopulation of ALDH1br and ALDH1low cells. DEAB was used in each sorting round to confirm the gating area. SSC, side scatter.

      Comparison of Spheroid-Formation Capacity between ALDH1br and ALDH1low Cells

      To test the stem-like property further, we characterized the spheroid-formation ability of ALDH1br and ALDH1low cells. ALDH1br and ALDH1low cells derived from TOV-21G and ES-2 cells were cultured in serum-free suspension culture at a density of 104 cells per well (2000 cells/mL) in six-well plates. Spheroid formation was assessed at day 7. ALDH1br cells showed a high capacity for spheroid formation, regarding size and numbers (Figure 3, A and B), and for self-renewal, as assessed using serial passages (Figure 3A). In addition, ALDH1br cells were further enriched in spheroids: 10.5% of TOV-21G and 4.7% of ES-2 spheroids were ALDH1br cells (Figure 3C).
      Figure thumbnail gr3
      Figure 3Spheroid formation capacity of ALDH1br and ALDH1low cells. A: Typical morphology (top) and number (bottom) of spheroids in ALDH1br and ALDH1low cells in TOV-21G and ES-2 EOC cell lines. ALDH1br cells generated significantly larger and more spheroids, compared with ALDH1low subpopulations. Original magnification, ×200. The number of spheroids after serial passages reflects the self-renewal capability of these cells in vitro. *P < 0.05. B: The relative spheroid area derived from ALDH1br and ALDH1low cells was analyzed using Media Cybernetics Image-Pro Plus 6.0 software in TOV-21G and ES-2 cell lines. Images are at original magnification of ×50. *P < 0.05, **P < 0.01, and ***P < 0.001. C: Enrichment of ALDH1br cells in spheroids. SSC, side scatter.

      Tumorigenicity of ALDH1br and ALDH1low Cells

      To test the effects of ALDH1 on tumorigenicity, we established a xenograft tumor-formation assay using NOD-SCID mice. ALDH1br cells exhibited tumorigenic potential at a density as low as 103 cells. No mice injected with an equivalent amount of ALDH1low cells grew tumors (Figure 4A). After the injection of 104 cells, all ALDH1br and half of the ALDH1low cells grew tumors. The weekly growth curves of tumor volume at different weeks and tumor weight after implantation are shown in Figure 4B. Tumor weight was significantly greater in ALDH1br cells (103 cells injected), compared with ALDH1low cells (P = 0.0319). Histopathology confirmed that these were human ovarian tumors (Figure 4C). The expression of ALDH1 in these tumors was assessed using immunohistochemical staining. ALDH1 expression was identified in tumors derived from both ALDH1br and ALDH1low cells. We photographed high-power fields (×400) randomly in each section. There was a trend for increased expression of ALDH1 in cells of ALDH1br-derived tumors, compared with those of ALDHlow-derived tumors (Figure 4C). These results demonstrate that ALDH1br cells have higher tumorigenicity, compared with ALDH1low cells.
      Figure thumbnail gr4
      Figure 4Tumorigenicity of ALDH1br and ALDH1low cells. A: ALDH1br and ALDH1low cells were injected into each side of a mouse. B: Tumor growth curves and tumor weight were plotted for different numbers of TOV-21G cells injected [100,000 (left), 10,000 (middle), and 1000 (right) cells] for each ALDH1br and ALDH1low population. *P < 0.05. C: H&E staining of tumors derived from ALDH1br and ALDH1low cells (original magnification, ×200) and immunohistochemical staining of ALDH1 expression (original magnification, ×400).

      ALDH1br Cells Are Resistant to Chemotherapeutic Drugs

      The effects of ALDH1 expression on the sensitivity to chemotherapeutic drugs were investigated. ALDH1br and ALDH1low cells were incubated with increasing concentrations of various chemotherapeutic agents (taxol, cisplatin, epirubicin, topotecan, and etoposide). In general, ALDH1br cells showed mild but significant resistance to these agents, which are commonly used in clinical practice (Figure 5). Although minor, these differences were significant in general. Specifically, the difference in cell viability was >20% at a low dose of epirubicin in both cell lines tested. TOV-21G cells, which exhibited higher ALDH1 activity, were more resistant to cisplatin, whereas ES-2 cells were more resistant to topotecan. There was no significant difference in chemosensitivity to taxol, which stabilizes microtubules. This suggests that ALDH1br populations are more resistant to chemotherapeutic drugs.
      Figure thumbnail gr5
      Figure 5Comparison of drug sensitivity between ALDH1br and ALDH1low cells. All tests were performed in two biologically independent studies, with triplicate trials for every experiment. Data are expressed as means ± SE. *P < 0.05.

      ALDH1br Cells Are Associated with the Surface Marker CD44

      Although ALDH1br cells are superior to ALDH1low cells regarding repopulation ability, spheroid formation, chemoresistance, and tumorigenicity, ALDHlow cells also exhibit these properties, at lower efficiency. We hypothesized that ALDH1br cells are associated with other stem cell markers, an association that renders these cells more stem-like than ALDH1low cells. We investigated the expression of CD44 and CD133 in relation to ALDH1 activity in ovarian cancer. To exploit the association of ALDH1 with stem cell markers in clear cell and serous ovarian carcinoma, the ALDH1br and ALDH1low cells from ES-2 and CP70 cell lines were analyzed for the expression of CD44 and CD133 using flow cytometry. The results showed that, relative to ALDH1low cells, ALDH1br cells were more enriched in CD44 (by 1.74-fold and 5.18-fold for ES-2 and CP70, respectively) (Figure 6A) than in CD133 (by 1.39-fold and 1.17-fold for ES-2 and CP70, respectively) (Figure 6B). ES-2 and CP70 ALDH1br cells also expressed CD44, at 94% and 79%, respectively (Figure 7A). In contrast, only 1.7% of CD44+ ES-2 cells and 53.7% of CD44+ CP70 cells exhibited high ALDH1 activity (Figure 7B). CD44+ALDH1br cells were rare subpopulations (<1%) in both cell lines (Figure 7C). Our results support the hypothesis that ALDH1br is associated with CD44 in ovarian cancer.
      Figure thumbnail gr6
      Figure 6Relationship between ALDH1 and CD44/CD133. ALDH1br and ALDH1low cells were sorted and analyzed for expression of CD44 (A) and CD133 (B). CD44+ and CD133+ gates were defined by an isotypic control.
      Figure thumbnail gr7
      Figure 7ALDH1br/CD44+ cells in ovarian cancer cell lines. A: CD44+ in ALDH1br cells. B: ALDH1br cells in CD44+ cells. C: Cells costained with anti-CD44 PE monoclonal antibody (mAb) and Aldefluor. The gating area was defined by an isotypic control and DEAB.

      Correlations between ALDH1 Protein Expression and Clinicopathological Parameters

      To verify the role of ALDH1 in ovarian cancer, we performed an immunohistochemistry analysis using ovarian cancer tissue arrays. The expression of ALDH1 varied substantially, from no expression to strong expression. For comparison, patients were classified into two groups, according to the proportion of ALDH1 expression in tumors: ALDH1 high (> 50%) and ALDH1 low (< 50%) (Figure 8A). The expression of ALDH1 was significantly associated with histological type (P=0.01) (Figure 8B and Table 1). There were no significant associations between ALDH1 expression and age, stage, or nuclear grade. Patients with higher ALDH1 expression had poor overall survival, compared with those with lower ALDH1 (Figure 9A) (P=0.004). A subanalysis focusing on high-grade serous carcinoma yielded consistent results (see Supplemental Figure S2 at http://ajp.amjpathol.org). The multivariate analysis revealed that higher ALDH1 levels conferred an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) (Table 2). There was a trend toward higher recurrence in patients with higher ALDH1 expression; however, this result did not reach significance (Figure 9B).
      Figure thumbnail gr8
      Figure 8Expression and distribution of ALDH1 in four histological types of EOC: clear cell carcinoma (CCC), endometrioid carcinoma (EC), mucinous carcinoma (MC), and serous carcinoma (SC). A: Representative differential ALDH1 expression. B: Proportion of ALDH1 expression in cells of the different histological types.
      Table 1Clinicopathological Features
      ALDH1 positivity
      Characteristic0% to 50%>50%P value
      Patients (no.)5628
      Age (years)0.14
       Range16–7929–81
       Mean ± SEM52.0 ± 1.957.0 ± 2.8
      Stage [no. (%)]0.46
       I, II20 (74.1)7 (25.9)
       III, IV36 (63.2)21 (36.8)
      Nuclear grade [no. (%)]0.50
       G19 (69.2)4 (30.8)
       G222 (59.5)15 (40.5)
       G325 (73.5)9 (26.5)
      Histological type [no. (%)]0.01
      P < 0.05.
      Fisher's exact test.
       Serous type45 (73.8)16 (26.2)
       Mucinous type5 (35.7)9 (64.3)
       Endometrioid type1 (33.3)2 (66.7)
       Clear cell type5 (83.3)1 (16.7)
      Histological type [no. (%)]0.04
      P < 0.05.
       Serous type45 (73.8)16 (26.2)
       Other types11 (47.8)12 (52.2)
      low asterisk P < 0.05.
      Fisher's exact test.
      Figure thumbnail gr9
      Figure 9Kaplan-Meier analysis of overall survival (A) and recurrence (B) in ovarian cancer patients stratified according to ALDH1 expression (n = 84).
      Table 2Multivariate Analysis of Clinicopathological Factors in 84 Ovarian Cancer Patients
      Recurrence
      A Cox proportional hazards model was applied.
      Survival
      A Cox proportional hazards model was applied.
      VariableUnivariate analysis crude HR (95% CI)Multivariate adjusted HR (95% CI)
      The analysis was adjusted for stage, nuclear grade, and histological type.
      Univariate analysis crude HR (95% CI)Multivariate adjusted HR (95% CI)
      The analysis was adjusted for age, ALDH1 expression, stage, and nuclear grade.
      Age (years)1.02 (1.00–1.05)1.02 (0.99–1.05)1.05 (1.02–1.08)
      Significantly correlated with outcome (P < 0.05).
      1.04 (1.01–1.07)
      Significantly correlated with outcome (P < 0.05).
      ALDH1 expression
      Low expression of ALDH1 regarding survival is represented as 0% to 50%; high expression of ALDH1 regarding survival is represented as >50%.
       Low1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)
       High1.34 (0.66–2.75)1.70 (0.77–3.77)2.57 (1.31–5.05)
      Significantly correlated with outcome (P < 0.05).
      2.43 (1.12–5.28)
      Significantly correlated with outcome (P < 0.05).
      Stage
       I, II1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)
       III, IV9.62 (2.88–32.12)
      Significantly correlated with outcome (P < 0.05).
      7.88 (2.18–28.42)
      Significantly correlated with outcome (P < 0.05).
      23.70 (3.23–173.76)
      Significantly correlated with outcome (P < 0.05).
      23.25 (3.00–179.98)
      Significantly correlated with outcome (P < 0.05).
      Grade (nuclear)
       G11.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)
       G22.52 (0.57–11.11)0.90 (0.18–4.38)2.93 (0.67–12.76)1.43 (0.31–6.60)
       G35.69 (1.30–24.96)
      Significantly correlated with outcome (P < 0.05).
      2.51 (0.50–12.61)5.10 (1.16–22.38)
      Significantly correlated with outcome (P < 0.05).
      3.40 (0.71–16.36)
      Histological type
       Serous type3.37 (1.03–11.09)
      Significantly correlated with outcome (P < 0.05).
      1.29 (0.31–5.39)1.38 (0.57–3.34)0.49 (0.17–1.44)
       Other types1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)
      CI, confidence interval; HR, hazard ratio; Ref, reference value.
      low asterisk A Cox proportional hazards model was applied.
      The analysis was adjusted for stage, nuclear grade, and histological type.
      The analysis was adjusted for age, ALDH1 expression, stage, and nuclear grade.
      § Low expression of ALDH1 regarding survival is represented as 0% to 50%; high expression of ALDH1 regarding survival is represented as >50%.
      Significantly correlated with outcome (P < 0.05).

      Discussion

      ALDH1 activity, as assessed using an Aldefluor assay, provides a method for the isolation of normal and malignant progenitor cells.
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      which is consistent with our finding that ALDH1low can generate both ALDH1low and ALDH1br cells. The reason for this repopulation has not been clarified.
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      • Muñoz J.
      • Prat J.
      Gene expression analysis identifies two groups of ovarian high-grade serous carcinomas with different prognosis.
      A novel hypothesis regarding the possible origin of serous ovarian cancer in the uterus was also proposed recently.
      • Massuger L.
      • Roelofsen T.
      • Ham M.
      • Bulten J.
      The origin of serous ovarian cancer may be found in the uterus: a novel hypothesis.
      However, the origin of type I ovarian cancer remains elusive. As implied by the name, the mucinous type of ovarian cancer exhibits morphological similarities to the endocervical glands, whereas the endometrioid type of ovarian cancer is similar to the endometrial epithelium. Given that ALDH1 has been proposed as a stem cell marker of various origins, it is possible that ALDH1br ovarian cancers have a different origin, possibly in the uterus. Previous reports proposed that both ovarian clear cell and endometrioid carcinoma are associated with, and may arise from, endometriosis,
      • Ness R.B.
      Endometriosis and ovarian cancer: thoughts on shared pathophysiology.
      • Viganó P.
      • Somigliana E.
      • Chiodo I.
      • Abbiati A.
      • Vercellini P.
      Molecular mechanisms and biological plausibility underlying the malignant transformation of endometriosis: a critical analysis.
      • Wiegand K.C.
      • Shah S.P.
      • Al-Agha O.M.
      • Zhao Y.
      • Tse K.
      • Zeng T.
      • et al.
      ARID1A mutations in endometriosis-associated ovarian carcinomas.
      which was reported recently as expressing high levels of progenitor markers, including OCT4, c-KIT, and SOX2, indicating a stem cell origin for endometriosis.
      • Pacchiarotti A.
      • Caserta D.
      • Sbracia M.
      • Moscarini M.
      Expression of oct-4 and c-kit antigens in endometriosis.
      • Götte M.
      • Wolf M.
      • Staebler A.
      • Buchweitz O.
      • Kiesel L.
      • Schüring A.N.
      Aberrant expression of the pluripotency marker SOX-2 in endometriosis.
      However, the expression of ALDH1 in endometrial stem cells remains unexplored. The endometrial ALDH1br stem cell origin of certain types of ovarian cancers remains to be demonstrated.
      In conclusion, ALDH1br enriched the stem-cell properties of EOCs, and contributed to chemoresistance and poor prognosis, but ALDH1 alone was not sufficient as a cancer stem cell marker. The combination of ALDH1 with other stem cell markers, such as CD44 and CD133, may define ovarian cancer stem cells more stringently, or may represent different sets of progenitor populations. Novel therapies targeting ALDH1br ovarian cancer cells may help improve the survival of patients.

      Acknowledgments

      We thank Dr. Chi-Kuan Chen, Hui-Chen Wang, and Dr. Chih-Kung Lin (National Defense Medical Center, Taipei, Taiwan, China) for expert technical assistance and Dr. Mu-Hsien Yu (National Defense Medical Center, Taipei, Taiwan, China) for administrative support.

      Supplementary data

      • Supplemental Figure S1

        The purity of sorting was assessed by using Accudrop beads. Distinct subpopulations were gated as P2 and P3 (left panel). The P2 population (arrow) was sorted and reanalyzed with 96.36% purity (right panel).

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        The American Journal of PathologyVol. 181Issue 1
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          In the article entitled, “Toward Routine Use of 3D Histopathology as a Research Tool” (Volume 180, pages 1835–1842 of the May 2012 issue of The American Journal of Pathology), the support footnote should have included the following: “This work was partially funded through WELMEC, a Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC (grant WT088908/Z/09/Z ).”
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