Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis

  • Authors:
    • Di Wu
    • Yi-Ping Mou
    • Ke Chen
    • Jia-Qin Cai
    • Yu-Cheng Zhou
    • Yu Pan
    • Xiao-Wu Xu
    • Wei Zhou
    • Jia-Qi Gao
    • Ding-Wei Chen
    • Ren-Chao Zhang
  • View Affiliations

  • Published online on: June 1, 2016     https://doi.org/10.3892/ijo.2016.3551
  • Pages: 611-622
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Enhanced aldehyde dehydrogenase (ALDH) activity has been shown to serve as a hallmark for cancer stem cells (CSCs). Recent evidence suggests that its role as a stem cell-related marker has come down to the specific isoform. However, little is known about the specific ALDH isoform contributing to aldefluor activity in gastric cancer. In this study, we isolated ALDHbright cells from 2 human gastric cancer cell lines MKN-45 and SGC‑7901 by using an Aldefluor assay and found elevated self-renewal, differentiation and tumorigenicity, as demonstration of stemness characteristics. We also found that ALDHbright cells expressed decreased levels of E-cadherin but increased levels of Snail and Vimentin, indication of an epithelial-mesenchymal transition (EMT) phenotype which may be responsible for the enhanced metastatic potential. Since further research and prognostic application based on ALDH prevalence require the quantification of the specific ALDH isoform, we characterized the expression of all 19 ALDH isoforms in the sorted gastric cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Compared with the non-stem counterparts, robust upregulation of ALDH-3A1 was observed in these gastric cancer stem-like cells. Furthermore, we performed immunohistological analysis on 93 fixed patient gastric tumor samples and found that ALDH-3A1 expression correlated well with gastric cancer dysplasia and grades, differentiation, lymph node metastasis and cancer stage. Our data, therefore, provide strong evidence that ALDH-3A1 is a novel gastric cancer stem cell related marker with potential prognostic values and demonstrate a clear association between ALDH-3A1 prevalence and gastric cancer progression.

Introduction

Gastric carcinoma is a highly aggressive tumor with approximately 50% of cases occurring in Eastern Asia, mainly China (1,2). Although the current operation techniques, chemo- and radio therapies are continuously improving, the 5-year survival rate remains low due to the highly invasive and metastatic properties of gastric carcinoma. Therefore, understanding the crucial events underlying gastric tumorigenesis and progression is urgently needed, which could pave the way for the future anti-anticancer drug discovery and efficacious therapeutic strategies.

It is well known that tumors are composed of heterogeneous cell populations (25), which are inconsistent in tumor-propagating ability. Cancer stem cells, also referred to as tumor initiating cells (TICs), which exhibit the self-renewal capacity and highly tumorigenic ability are responsible for tumor invasion, relapse and therapy resistance (69). Increasing evidence has suggested that high aldehyde dehydrogenase activity can be used to characterize cells with CSC properties (1022). The human ALDH enzymes are a family comprised of 19 isoforms (23,24), mainly functioning in oxidizing aldehydes to their corresponding carboxylic acid and converting retinol to retinoic acid (RA) which would activate the RA signaling pathway. Among the various ALDH isoforms, ALDH-1A1 is the most extensively investigated isoform and has always been considered to be the cause of aldefluor activity (18). However, recent studies have shown that not all tumors exhibit the correlation between ALDH-1A1 prevalence and tumor progression, therapeutic resistance or prognosis estimation, that is, ALDH activity is not necessarily ALDH-1A1 specific, but could be attributed to other ALDH isoforms (17,2530). Therefore, identifying the individual responsible ALDH isoforms in each cancer is of vital importance and can be the foundation of the following research.

ALDHbright gastric cancer cells have recently been demonstrated to possess certain CSC properties (3133). However, the existing research all regarded ALDH-1 as being responsible for aldefluor activity in gastric cancer, although ALDH-1 families contain 6 members (23,24,34). Few reports identified the specific isoforms responsible for ALDH activity in gastric carcinoma. We therefore investigated the expression of 19 human ALDH isoforms in ALDHbright gastric cancer cells which have been proved possessing certain stemness properties. Our study shows that ALDH-3A1, but not ALDH-1A1 is robustly upregulated in gastric cancer stem-like cells. Moreover, high levels of ALDH-3A1 expression associates with poorly differentiation degree in gastric cancer tissue which suggests that certain level of dedifferentiation may have occurred. Collectively, our research reveals that the aldefluor activity in gastric cancer stem-like cells is primarily due to ALDH-3A1 and its expression correlates well with gastric cancer progression.

Materials and methods

Cell lines and culture conditions

The human gastric cancer cell lines MKN-45 and AGS were obtained from American Type Culture Collection. The other four human gastric cancer cell lines SGC-7901, BGC-823, MGC-803 and HGC-27 were obtained from Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences. MKN-45, SGC-7901, BGC-823, MGC-803 were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovin serum (FBS, Invitrogen). HGC-27 and AGC were cultured in Dulbecco's modified Eagle's medium (Gibco) and F-12K (Boster), respectively supplemented with 10% FBS. All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2. Images of cell lines were taken by using a Nikon Eclipse TS100 inverted microscope.

Patients and clinical samples

Gastric cancer specimens used in this study were obtained from the 93 cases of gastric cancer patients, with written informed consent, who underwent a surgical resection from 2013 to 2014 at Sir Run Run Shaw Hospital. The samples of patients who underwent preoperative radiotherapy or chemotherapy were excluded. The clinical characteristics of the patients are presented in Table III. The study was approved by the Clinical Research Ethics Committee of Sir Run Run Shaw Hospital of Zhejiang University (no. 20150227–13).

Table III

Relationship between ALDH-3A1 and clinicopathological charateristics of gastric cancer.

Table III

Relationship between ALDH-3A1 and clinicopathological charateristics of gastric cancer.

Expression of ALDH-3A1

Clinical factorsNos.HighLowP-value
Age (years)
 <604729180.527
 >60462521
Gender
 Male6338250.237
 Female301515
T stage
 Tis, T11100.452
 T21688
 T3693831
 T4761
Lymph node metastasis
 Presence312380.015
 Absence623032
Tumor stage
 I12750.011
 II512229
 III20173
 IV1073
Differentiation
 Well1257<0.001
 Moderate401525
 Poor41338
Aldefluor assay and FACS isolation of cells

The ALDH activity of tumor cells was evaluated by Aldefluor kit (Stem Cell Technologies) according to the manufacturer's instructions. Briefly, 106 harvested gastric cancer cells were resuspended in 500 μl aldefluor buffer containing 2.5 μl ALDH substrate and incubated for 45 min at 37°C. The specific ALDH inhibitor diethylaminobenzaldehye (DEAB) served as a negative control. For FACS sorting, cells were labeled using Aldefluor kit and the desired populations were sorted by a FACS Aria cell sorter (BD Bioscience).

Tumorsphere formation assay

For sphere formation assay, cells were cultured in 100-mm ultralow attachment plates (Corning) at a density of 2,000 cells/ml in DMEM with nutrient mixture F-12 (Invitrogen), supplemented with 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 100 ng/ml epidermal growth factor (Pepro Tech), 20 ng/ml basic fibroblast growth factor (Pepro Tech) at 37°C in humidified air 5% CO2. To passage tumorspheres, spheres cultured for 7 days were collected, disaggregated with 0.05% trypsin/EDTA (Solarbio), sieved through a 40-μm filter and replated as described above.

Colony formation assay

For colony formation analysis, two hundred viable sorted cells were plated in each well of 6-well plates and cultured in RPMI-1640 containing 10% FBS. After incubation for 2 weeks at 37°C, colonies containing >50 cells were counted with Giemsa staining.

Immunofluorescence confocal microscopy

To analyze cell differentiation, the sorted ALDHbright cells were seeded on glass coverslips and cultured in DMEM containing 10% FBS for 7–10 days. The cells were then fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 1 h, blocked in 10% goat serum in PBS for 1 h and labeled overnight at 4°C with primary mouse anti-human CK-18 antibodies (Zhongshan Gold Bridge Biotechnology) and mouse anti-human CD44 antibodies (Thermo) respectively. After washing the cells with PBS, the cells were incubated for an additional 1 h at room temperature with goat anti-mouse IgG conjugated Alexa 594 (Invitrogen) and goat anti-mouse IgG conjugated FITC (Invitrogen) respectively. Cell nuclei were counterstained with DAPI. Samples were then observed and photographed under immunofluorenscence confocal microscope (Zeiss).

In vivo tumorigenicity assay

The sorted cells were resuspended in 150 μl of 1:1 PBS/Matrigel (BD Bioscience) and injected subcutaneously into the inguen of 6-week-old NOD/SCID mice with 1×103 or 5×103 cells per mouse. Xenograft tumors were removed at the end of 6th week and measured according to the formula: Tumor volume = (length × width2)/2. A portion of each tumor tissue was fixed in 4% formaldehyde for immunohistochemical (IHC) analysis.

Lung metastasis assay

For generation of lung metastasis, 1×106 sorted ALDHbright and ALDHlow were resuspended into 300 μl PBS and injected through the lateral tail vein of 6-week-old NOD/SCID mice. All mice were sacrificed at the 16th week, and the lungs were harvested and examined for tumor nodules.

RNA extraction and qRT-PCR analysis

The total RNA of cells was extracted with RNAiso reagent (Takara). cDNA was synthesized using equivalent amount of total RNA (1 μg) with primers in a 20-μl reverse transcriptase reaction mixture (Takara). The primers were designed and purchased from Invitrogen, the sequences of each primer pair were listed in Tables IV and V. qRT-PCR was performed using LightCycler® 480 Real-Time PCR system. The expression of each gene was calculated using 2−ΔΔCT method. Results were normalized against the level of GAPDH. All assays were performed in triplicates and results were plotted as the mean ± SD.

Table IV

Real-time PCR primers used for all 19 human ALDH isoforms and GAPDH.

Table IV

Real-time PCR primers used for all 19 human ALDH isoforms and GAPDH.

GenesSequences
ALDH1A1F: 5′-ACTGCTCTCCACGTGGCATCTTTA-3′
R: 5′-TGCCAACCTCTGTTGATCCTGTGA-3′
ALDH1A2F: 5′-AGGGCAGTTCTTGCAACCATGGAA-3′
R: 5′-CACACACTCCAATGGGTTCATGTC-3′
ALDH1A3F: 5′-ACCTGGAGGTCAAGTTCACCAAGA-3′
R: 5′-ACGTCGGGCTTATCTCCTTCTTCC-3′
ALDH1B1F: 5′-TGCTGCAGAGTGTCAGCAT-3′
R: 5′-GGTGGTAGGGTTGACCGTCG-3′
ALDH1L1F: 5′-ATCTTTGCTGACTGTGACCT-3′
R: 5′-GCACCTCTTCTACCACTCTC-3′
ALDH1L2F: 5′-GCCTGGTCTCGTTACCAAAA-3′
R: 5′-GCCACTTTCACCTCTTCAGC-3′
ALDH2F: 5′-CCAACCAGCAGCCCGAGGTC-3′
R: 5′-AAGGCCTTGTCCCCTTCAGCTACC-3′
ALDH3A1F: 5′-TGTGTCAAAGGCGCCATGAGCAAG-3′
R: 5′-GGCGTTCCATTCATTCTTGTGCAG-3′
ALDH3A2F: 5′-TGCACTTCACGCTCAACTCT-3′
R: 5′-GACTGGCTGTTGGGAGGATA-3′
ALDH3B1F: 5′-ACAAGTCAGCCTTCGAGTCGG-3′
R: 5′-AGCACCACACAGTTCCCTGC-3′
ALDH3B2F: 5′-ACAGAGAAGGTCCTGGCTGA-3′
R: 5′-CATGACAATCTTGCCCACAC-3′
ALDH4A1F: 5′-TGCAGTACCAAGTGTCGCCTTT-3′
R: 5′-AATCTCCGCTTGGATCACGGTCTT-3′
ALDH5A1F: 5′-ACCAATTCTTGGTGCAAAGG-3′
R: 5′-GTTGGTGTCGTTTTCCACCT-3′
ALDH6A1F: 5′-GGCTCTTTCAACAGCAGTCC-3′
R: 5′-ATGGAAGCTCCCTCCTTTGT-3′
ALDH7A1F: 5′-CGAGCCAATAGCAAGAGTCC-3′
R: 5′-CTTCACCCACACCTTCCACT-3′
ALDH8A1F: 5′-TGGTGAGCATAGGTGCTCTG-3′
R: 5′-GTTATCACCGTGGGAAGCAT-3′
ALDH9A1F: 5′-CACTCATCAACCGACCACAC-3′
R: 5′-GGACATAACAGGCCCAAAGA-3′
ALDH16A1F: 5′-GCTCCTCATCCAGGAGTCTG-3′
R: 5′-AAGGTTGGGGGATAGAATGG-3′
ALDH18A1F: 5′-CTGAGTATGGGGACCTGGAA-3′
R: 5′-GCGGTAACCATCAGAAAAGC-3′

Table V

Real-time PCR primers used for EMT associated transcription factors and stem cell related markers.

Table V

Real-time PCR primers used for EMT associated transcription factors and stem cell related markers.

GenesSequences
SnailF: 5′-GACCACTATGCCGCGCTCTT-3′
R: 5′-TCGCTGTAGTTAGGCTTCCGATT-3′
SlugF: 5′-AGCGAACTGGACACACATAC-3′
R: 5′-TCTAGACTGGGCATCGCAG-3′
Twist-1F: 5′-CACTGAAAGGAAAGGCATCA-3′
R: 5′-GGCCAGTTTGATCCCAGTAT-3′
Zeb-1F: 5′-CGAGTCAGATGCAGAAAATGAGCAA-3′
R: 5′-ACCCAGACTGCGTCACATGTCTT-3′
Zeb-2F: 5′-GAGTTGATGCCTCGGCTATTGC-3′
R: 5′-CTGGACATTGAGCTGCTTCGATC-3′
E-cadherinF: 5′-TACACTGCCCAGGAGCCAGA-3′
R: 5′-TGGCACCAGTGTCCGGATTA-3′
VimentinF: 5′-TGAGTACCGGAGACAGGTGCAG-3′
R: 5′-TAGCAGCTTCAACGGCAAAGTTC-3′
OCT-4F: 5′-GCAGCGACTATGCACAACGA-3′
R: 5′-CCAGAGTGGTGACGGAGACA-3′
NANOGF: 5′-GACTTGACCACCGAACC- CAT-3′
R: 5′-CTGGATGTTCTGGGTCTGGT-3′
BMI-1F: 5′-TCGTTCTTGTTATTACGCTGTTTT-3′
R: 5′-CGGTAGTACCCGCTTTTAGGC-3′
GAPDHF: 5′-GGTGTGAACCATGAGAAGTATG-3′
R: 5′-GATGGCATGGACTGTGGTCAT-3′
SnailF: 5′-GACCACTATGCCGCGCTCTT-3′
R: 5′-TCGCTGTAGTTAGGCTTCCGATT-3′
SlugF: 5′-AGCGAACTGGACACACATAC-3′
R: 5′-TCTAGACTGGGCATCGCAG-3′
Twist-1F: 5′-CACTGAAAGGAAAGGCATCA-3′
R: 5′-GGCCAGTTTGATCCCAGTAT-3′
Zeb-1F: 5′-CGAGTCAGATGCAGAAAATGAGCAA-3′
R: 5′-ACCCAGACTGCGTCACATGTCTT-3′
Zeb-2F: 5′-GAGTTGATGCCTCGGCTATTGC-3′
R: 5′-CTGGACATTGAGCTGCTTCGATC-3′
E-cadherinF: 5′-TACACTGCCCAGGAGCCAGA-3′
R: 5′-TGGCACCAGTGTCCGGATTA-3′
VimentinF: 5′-TGAGTACCGGAGACAGGTGCAG-3′
R: 5′-TAGCAGCTTCAACGGCAAAGTTC-3′
OCT-4F: 5′-GCAGCGACTATGCACAACGA-3′
R: 5′-CCAGAGTGGTGACGGAGACA-3′
Western blot analysis

Cultured cells were lysed with 200 μl of ice-cold RIPA buffer (Beyotime) containing 1 mM PMSF. Proteins were resolved by SDS-polyarylamide gel and transferred to PVDF membranes (Millipore). The membranes were then blocked with 5% non-fat milk and were then incubated with primary antibodies overnight at 4°C. The membranes were washed and incubated with appropriate HPR-conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected by using Pierce ECL Western blotting substrate (Thermo). GAPDH or TUBLIN were used as a loading control. The primary antibodies used for western blot analyses were as follows: NANOG (Abcam), OCT-4 (Abcam), BMI-1 (Abcam), ALDH-1A1 (BD Bioscience), ALDH-3A1 (Santa Cruz Biotechnology), GAPDH (Abcam), TUBLIN (Cwbiotech).

TMA and image analysis

TMAs were either constructed by author (Key Laboratory of Biotherapy of Zhejing University) or purchased from Alenabio Biotech Co.. Staining of tissue microarray slides was carried out according to the manufacturer's protocol. Briefly, IHC staining was performed on 10% phosphate-buffered formalin fixed and paraffin-embedded gastric cancer sections (4 μm) using xylene. The slides were hydrated by a graded series of ethanol washes and incubated in 0.3% H2O2. After incubation with blocking solution for 30 min at room temperature, the slides were incubated with primary antibodies at 4°C overnight. The secondary antibodies were added for incubation at 37°C. TMA slides were scanned by using Aperio Slide Scanner and analyzed by Image Scope software (Aperio). The IHC staining was scored independently by two pathologists blinded to the clinical data as follows: Score = percentage of immunoreactive cells (0, <10%; 1, 11–25%; 2, 26–50%; 3, 51–75%; 4, >75%) × mean stain intensity (0–3).

Statistical analysis

Statistical analysis was performed using SPASS statistical analysis software Version 17.0 (SPSS). Student's t-test was used to examine the statistical significance when two groups were compared. To determine differences among 3 groups, an ANOVA analysis was performed. P-value <0.05 was regarded as statistically significant.

Results

ALDHbright gastric cancer cells exibit cancer stem-like cell properties

We first examined the proportion of ALDHbright cells in the six human gastric cancer cell lines using Aldefluor kit and found that each of the 6 gastric cancer cell lines (MKN-45, SGC-7901, HGC-27, AGS, BGC-823, MGC-803) contained ALDH-positive cells (Fig. 1A). Compared with the DEAB-treated control groups, high ALDH activity was detected in 22.1% of the MKN-45 cells, 18.3% of the SGC-7901 cells, 1.40% of the HGC-27 cells, 1.25% of the MGC-803 cells, 1.24% of the AGS cells, 0.36% of the BGC-823 cells (Fig. 1D). We therefore chose MKN-45 and SGC-7901 for the follow-up research taking advantage that both cell lines contained prominent ALDHbright subpopulations (indicated by the bright green fluorescence), making the ALDH activity based separation more efficient and convincing (Fig. 1B and C). We present here that ALDHbright cells isolated from the 2 gastric cancer cell lines showed higher capabilities of tumorsphere formation compared with ALDHlow cells. These spheres could be passaged for at least 3 consecutive generations with an increasing forming efficiency, while the ALDHlow cells did not form typical tumorspheres, only a few loose cell aggregates (Fig. 2A). The number of colonies formed by ALDHbright MKN-45 cells and ALDHbright SGC-7901 cells were higher than those formed by ALDHlow cells (138 versus 36, 150 versus 49, respectively) (Fig. 2B). The ALDHbright cells from MKN-45 and SGC-7901 also showed multi-potent capacity of differentiation by expressing decreased level of CD44 but increased level of CK-18 after induction of differentiation (Fig. 2C). ALDHbright MKN-45 and SGC-7901 were also found to express higher levels of stem cell-related markers including OCT-4, BMI-1, NANOG than ALDHlow cells (Fig. 2D). The tumorigenicity assay with isolated ALDHbright and ALDHlow gastric cancer cells in NOD/SCID mice showed that ALDHbright MKN-45 and SGC-7901 cells have enhanced tumor initiation capacity as compared to ALDHlow cells at each cell dose (103, 5/6 versus 0/6, 6/6 versus 0/6; 5×103, 6/6 versus 1/6, 6/6 versus 2/6, respectively) (Fig. 2E and Table I). Also, the volume of ALDHbright cell derived xenografts were larger than those of ALDHlow cell-derived (Fig. 2E).

Table I

Tumorigenicity of ALDHbright and ALDHlow cells from MKN-45 and SGC-7901.

Table I

Tumorigenicity of ALDHbright and ALDHlow cells from MKN-45 and SGC-7901.

Cell dose

Cell lineSubpopulation 1×103 5×103
MKN-45 ALDHbright5/66/6
ALDHlow0/61/6
SGC-7901 ALDHbright6/66/6
ALDHlow0/62/6
ALDHbright cells display enhanced metastatic ability correlating with a mesenchymal transition phenotype

Accumulating evidence suggests that CSCs are responsible for tumor relapse, we therefore examined the metastatic capabilities and the expression of related molecules in the sorted ALDHbright cells. The ALDHbright cells derived from MKN-45 and SGC-7901 formed lung metastasis in four of six mice, and five of six mice, respectively, whereas few metastasis was formed by ALDHlow cells (Fig. 3A and Table II). Gene expression analysis further showed that MKN-45 ALDHbright cells express higher levels of Vimentin and Snail but lower level of E-cadherin, similar results were also obtained in SGC-7901 ALDHbright cells (Fig. 3B), suggesting EMT might be responsible for their enhanced metastatic capabilities.

Table II

Generation of lung metastasis of ALDHbright and ALDHlow cells from MKN-45 and SGC-7901.

Table II

Generation of lung metastasis of ALDHbright and ALDHlow cells from MKN-45 and SGC-7901.

Cell lineSubpopulationLung metastasis (yes/no)
MKN-45 ALDHbright4/2
ALDHlow0/6
SGC-7901 ALDHbright5/1
ALDHlow1/5
Specific increase in ALDH-3A1 expression in ALDHbright subpopulations

Since the ALDH isoform responsible for Aldefluor activity may vary depending on cancer type and tissue origin, we characterized the gene expression of all 19 ALDH isoforms between ALDHbright and ALDHlow cell populations sorted from MKN-45 and SGC-7901. Our results showed that among all the various human ALDH isoforms, ALDH-3A1 was the most elevated isoform in the sorted MKN-45 and SGC-7901 cells (15.9- and 11.2-fold, respectively) (Fig. 4A and B). In addition, several other isoforms were also found upregulated including ALDH-16A1, ALDH-2, ALDH-3B1, ALDH-4A1, ALDH-3B2 in MKN-45 derived ALDHbright cells, ALDH-18A1, ALDH-2, ALDH-3B2 in SGC-7901 derived ALDHbright cells, the elevated folds of which ranges from 4.4 to 5.8, 4.0 to 4.6, respectively. Notably, no fold change was detected in ALDH-1A1 in ALDHbright MKN-45 and SGC-7901 cells. Moreover, the mRNA levels of ALDH-1A1 were undetectable until cycle 35 for two cell lines. The expression of ALDH-3A1 was subsequently confirmed by western blot analysis (Fig. 4C), immunocytochemical (ICC) staining (Fig. 4D) and IHC staining (Fig. 4E).

ALDH-3A1 expression associates with gastric cancer evolution

To understand the possible role of ALDH-3A1 in the development and progression of gastric cancer, we examined the expression of ALDH-3A1 in a set of primary gastric tumors of different severity and metastatic gastric tumors. We were able to show here that ALDH-3A1 could not be detected or was only slightly expressed in the basal layer in normal gastric epithelia, but was detectable at low levels in the gastric dysplastic lesions, then the staining intensity of ALDH-3A1 progressed from weak to strong with increasing grades of dysplasia and carcinoma (Fig. 5B and C). The expression of ALDH-3A1 was associated with histological degrees (P<0.001), N stage (P=0.015) and cancer stage (P=0.011) of gastric cancer (Table III). Considering that CSCs play critical roles in tumor relapse, we subsequently examined the level of ALDH-3A1 in several pairs of local and lymph node metastatic gastric cancer and found higher levels of ALDH-3A1 in metastatic lesions compared with the local carcinomas (Fig. 5D). Collectively, these results indicate that the expression level of ALDH-3A1 correlates with the increasing severity of gastric cancer.

Discussion

In this study, we provide strong evidence that ALDHbright gastric cancer cells possess cancer stem-like properties and revealed for the first time that ALDH-3A1 is the highly expressed isoform in gastric cancer stem-like cells. Together with IHC analysis in tissue level, we show that ALDH-3A1-expressing cells play a vital role in the initiation and progression of gastric cancer. We convincingly demonstrate that ALDH-3A1 functions as a pivotal enzyme in regulating gastric cancer stem-like population, ALDH-3A1 prevalence therefore might be used as a novel indicator to predict gastric cancer patient outcome.

The human ALDH enzymes are a family comprised of 19 isoforms, mainly functioning in oxidizing various aldehydes to their corresponding carboxylic acids (27,34). High ALDH activity was originally identified as a marker for hematopoietic stem cells (25,35,36) and is thought to play a vital role in the self-renewal and differentiation of hematopoietic progenitor cells via converting retinal to retinoic acid (37). Recent studies suggest that high levels of ALDH activity characterize a subpopulation of cells with CSC properties in several malignancies (14,1921,38,39). Moreover, enhanced ALDH activity predicts poor patients outcome in breast cancer (11,22,4042), lung cancer (15,40), and esophageal squamous cell carcinoma (43), which is consistent with CSCs being responsible for cancer evolution and metastasis. Katsuno et al (32) have reported that ALDHbright cells derived from HSC-39 and OCUM-2ML gastric cancer cell lines exhibited certain stem cell traits but little is known about traits of ALDHbright cells in other gastric cancer cell lines. In this study, we isolated ALDHbright and ALDHlow subpopulations from 2 different gastric cancer cell lines MKN-45 and SGC-7901. Our data show that these sorted cells have capabilities of self-renewal, multilineage differential potential, high tumorigenicity and express high levels of stemness associated hallmarks, thereby representing a more reliable cancer stem-like cell marker in gastric cancer. We also found that the ALDHbright cells are more invasive and exhibit a phenotype of EMT by expressing increased level of Vimentin and Snail but decreased level of E-cadherin, in agreement with EMT being able to endow tumor cells with stem cell-like properties. We have previously reported that Snail is required for the maintenance of stem cell-like phenotype in pancreatic carcinoma (44), as such, it would be interesting to examine its contributions to gastric cancer stem-like compartment, which might provide novel therapeutic strategies.

With our discoveries that ALDHbright gastric cancer cells exhibit stemness charateristics and may contribute to tumor invasion, we hypothesized that the activity of ALDH in tumor might associate with gastric tumorigenesis and provide clinical prognostic values in gastric cancer patients. Generally, CSCs quantification in clinic requires immunohistological methods for detection of the specific protein expression in fixed tumor tissue. Therefore, the prognostic application by evaluating ALDH prevalence requires detection and quantification of ALDH expression at protein level in gastric tissue, but not activity. Although the Aldefluor assay is able to identify the celluar ALDH activity, it is not practical for clinical evaluation of ALDH at protein level. More importantly, the Aldefluor assay has been proved to be not merely ALDH-1A1-specific. Increasing evidence has demonstrated that the ALDH isoforms responsible for Aldefluor assay may vary depending on the cell origin and tumor types, the reason of which could be attributed to the cross-reactivity between the similar amino acid sequence sharing among 19 ALDH isoforms. Van den Hoogen et al (45) reported high expression of ALDH-7A1 in both prostate cancer cell lines and malignant tissues. Yan et al (46) demonstrated the association of ALDH-3A1 with prostate cancer progression. These findings suggest that more than one of these isoforms could be responsible for the Aldefluor activity. Chen et al (25) compared the expression levels of ALDH-1A1 and ALDH-3A1 in colon adenocarcinoma and reported that 98% of the tumor samples were ALDH-1B1 positive. Marcato et al (26) characterized the Aldefluor positive cells isolated from breast cancer patient tumor samples and found a better correlation with ALDH-1A3. Similar studies have successively been published and attracted broad attention in defining the responsible ALDH isoforms in different tumors. So far, the specific ALDH isoform responsible for Aldefluor activity in gastric cancer is still unclear. Furthermore, staining with ALDH-1A1 has shown no prognostic impact in gastric cancer patients (33), suggesting that ALDH-1A1 might not be attributed to ALDH activity, thus unable to provide clinical prognostic value. We show here the obvious upregulation of ALDH-3A1 in both MKN-45 and SGC-7901 derived ALDHbright cells compared with ALDHlow cells. In the tissue level, the staining intensity of ALDH-3A1 elevates along with increasing severity of gastric cancer, suggesting the expression of ALDH-3A1 may have resulted in certain level of dedifferentiation in which process gastric cancer stem-like cells may have been produced and contributed to gastric tumorigensis. Particularly, enhanced expression of ALDH-3A1 was observed in gastric cancer metastatic lesions compared to the matched primary carcinoma. This fits well with ALDHbright cells possessing increased metastatic capabilities with EMT phenotype. In addition, ALDH-1A1 expression could not be detected in sorted gastric cancer stem-like cells, suggesting ALDH-1A1 might not function in gastric cancer stem cell biology, thereby could not be deemed as a cancer stem cell related marker in gastric cancer. We speculate this possibility could account for the negative follow-up results of applying ALDH-1A1 prevalence to predict gastric cancer patients outcome.

ALDH-3A1 is an important isoform of ALDH family located in the cytoplasm (27) and shows multiple catalysis including serving as a corneal crystalline to protect eyes from UV radiation, contributing to the maintainance of hematopoietic stem cell population, regulating cell proliferation and apoptosis and conducing to the chemotherapeutic drug resistance (4754). All these functions in particular have been linked to tumor evolution. We demonstrate here that ALDH-3A1 is involved in the process of malignant transformation, tumor relapse in gastric cancer. These findings are consistent with those of Yan et al and Calderaro et al, who have shown upregulation of ALDH-3A1 correlated with tumorigenesis in hepatocellular carcinoma and protate cancer, respectively (46,55). It is necessary to clarify the mechanisms underlying ALDH-3A1 prevalence and gastric cancer development. With the recent discovery that EMT transcription factors, such as TWIST could promote cancer stem-like cells by directly binding to E-box sequences in promoter region of CD24 (56), it would be intriguingly to clarify whether the elevated EMT transcription factors would directly act on the ALDH-3A1 promoter region thereby contributing to the maintainance of ALDHbright gastric cancer stem-like subpopulation.

In conclusion, our discoveries identify ALDH-3A1 as a critical CSC marker with potential clinical prognostic application, and demonstrate a clear association between ALDH-3A1 prevalence and gastric cancer evolution.

Acknowledgements

This study was supported by grants by Natural Science Foundation of Zhejiang Province (CN) (grant no. LY15H160030 and no. LY15H160031) and Chinese Medicine Research Program of Zhejiang Province (CN) (grant no. 2012ZA087).

References

1 

Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar

2 

Jones RJ, Matsui WH and Smith BD: Cancer stem cells: Are we missing the target? J Natl Cancer Inst. 96:583–585. 2004. View Article : Google Scholar : PubMed/NCBI

3 

Fidler IJ and Hart IR: Biological diversity in metastatic neoplasms: Origins and implications. Science. 217:998–1003. 1982. View Article : Google Scholar : PubMed/NCBI

4 

Heppner GH and Miller BE: Tumor heterogeneity: Biological implications and therapeutic consequences. Cancer Metastasis Rev. 2:5–23. 1983. View Article : Google Scholar

5 

Nowell PC: Mechanisms of tumor progression. Cancer Res. 46:2203–2207. 1986.PubMed/NCBI

6 

Reya T, Morrison SJ, Clarke MF and Weissman IL: Stem cells, cancer, and cancer stem cells. Nature. 414:105–111. 2001. View Article : Google Scholar : PubMed/NCBI

7 

Visvader JE and Lindeman GJ: Cancer stem cells in solid tumours: Accumulating evidence and unresolved questions. Nat Rev Cancer. 8:755–768. 2008. View Article : Google Scholar : PubMed/NCBI

8 

Greaves M: Cancer stem cells as ‘units of selection’. Evol Appl. 6:102–108. 2013. View Article : Google Scholar : PubMed/NCBI

9 

Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL and Wahl GM: Cancer stem cells - perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 66:9339–9344. 2006. View Article : Google Scholar : PubMed/NCBI

10 

Luo Y, Dallaglio K, Chen Y, Robinson WA, Robinson SE, McCarter MD, Wang J, Gonzalez R, Thompson DC, Norris DA, et al: ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets. Stem Cells. 30:2100–2113. 2012. View Article : Google Scholar :

11 

Landen CN Jr, Goodman B, Katre AA, Steg AD, Nick AM, Stone RL, Miller LD, Mejia PV, Jennings NB, Gershenson DM, et al: Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer. Mol Cancer Ther. 9:3186–3199. 2010. View Article : Google Scholar : PubMed/NCBI

12 

Rahadiani N, Ikeda J, Mamat S, Matsuzaki S, Ueda Y, Umehara R, Tian T, Wang Y, Enomoto T, Kimura T, et al: Expression of aldehyde dehydrogenase 1 (ALDH1) in endometrioid adenocarcinoma and its clinical implications. Cancer Sci. 102:903–908. 2011. View Article : Google Scholar

13 

Clay MR, Tabor M, Owen JH, Carey TE, Bradford CR, Wolf GT, Wicha MS and Prince ME: Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase. Head Neck. 32:1195–1201. 2010. View Article : Google Scholar : PubMed/NCBI

14 

Kim MP, Fleming JB, Wang H, Abbruzzese JL, Choi W, Kopetz S, McConkey DJ, Evans DB and Gallick GE: ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma. PLoS One. 6:e206362011. View Article : Google Scholar : PubMed/NCBI

15 

Sullivan JP, Spinola M, Dodge M, Raso MG, Behrens C, Gao B, Schuster K, Shao C, Larsen JE, Sullivan LA, et al: Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. Cancer Res. 70:9937–9948. 2010. View Article : Google Scholar : PubMed/NCBI

16 

Qin J, Liu X, Laffin B, Chen X, Choy G, Jeter CR, Calhoun-Davis T, Li H, Palapattu GS, Pang S, et al: The PSA(−/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration. Cell Stem Cell. 10:556–569. 2012. View Article : Google Scholar : PubMed/NCBI

17 

van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Lippitt JM, Guzmán-Ramírez N, Hamdy FC, Eaton CL, Thalmann GN, Cecchini MG, et al: High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer. Cancer Res. 70:5163–5173. 2010. View Article : Google Scholar : PubMed/NCBI

18 

Su Y, Qiu Q, Zhang X, Jiang Z, Leng Q, Liu Z, Stass SA and Jiang F: Aldehyde dehydrogenase 1 A1-positive cell population is enriched in tumor-initiating cells and associated with progression of bladder cancer. Cancer Epidemiol Biomarkers Prev. 19:327–337. 2010. View Article : Google Scholar

19 

Chu P, Clanton DJ, Snipas TS, Lee J, Mitchell E, Nguyen ML, Hare E and Peach RJ: Characterization of a subpopulation of colon cancer cells with stem cell-like properties. Int J Cancer. 124:1312–1321. 2009. View Article : Google Scholar

20 

Carpentino JE, Hynes MJ, Appelman HD, Zheng T, Steindler DA, Scott EW and Huang EH: Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer. Cancer Res. 69:8208–8215. 2009. View Article : Google Scholar : PubMed/NCBI

21 

Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G, Appelman H, Fields JZ, Wicha MS and Boman BM: Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res. 69:3382–3389. 2009. View Article : Google Scholar : PubMed/NCBI

22 

Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, et al: ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 1:555–567. 2007. View Article : Google Scholar

23 

Vasiliou V and Nebert DW: Analysis and update of the human aldehyde dehydrogenase (ALDH) gene family. Hum Genomics. 2:138–143. 2005.PubMed/NCBI

24 

Marchitti SA, Brocker C, Stagos D and Vasiliou V: Non-P450 aldehyde oxidizing enzymes: The aldehyde dehydrogenase superfamily. Expert Opin Drug Metab Toxicol. 4:697–720. 2008. View Article : Google Scholar : PubMed/NCBI

25 

Chen Y, Orlicky DJ, Matsumoto A, Singh S, Thompson DC and Vasiliou V: Aldehyde dehydrogenase 1B1 (ALDH1B1) is a potential biomarker for human colon cancer. Biochem Biophys Res Commun. 405:173–179. 2011. View Article : Google Scholar : PubMed/NCBI

26 

Marcato P, Dean CA, Pan D, Araslanova R, Gillis M, Joshi M, Helyer L, Pan L, Leidal A, Gujar S, et al: Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis. Stem Cells. 29:32–45. 2011. View Article : Google Scholar

27 

Vasiliou V, Thompson DC, Smith C, Fujita M and Chen Y: Aldehyde dehydrogenases: From eye crystallins to metabolic disease and cancer stem cells. Chem Biol Interact. 202:2–10. 2013. View Article : Google Scholar

28 

Levi BP, Yilmaz OH, Duester G and Morrison SJ: Aldehyde dehydrogenase 1a1 is dispensable for stem cell function in the mouse hematopoietic and nervous systems. Blood. 113:1670–1680. 2009. View Article : Google Scholar :

29 

Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP and Leach SD: Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci USA. 107:75–80. 2010. View Article : Google Scholar :

30 

Marchitti SA, Orlicky DJ, Brocker C and Vasiliou V: Aldehyde dehydrogenase 3B1 (ALDH3B1): Immunohistochemical tissue distribution and cellular-specific localization in normal and cancerous human tissues. J Histochem Cytochem. 58:765–783. 2010. View Article : Google Scholar : PubMed/NCBI

31 

Nishikawa S, Konno M, Hamabe A, Hasegawa S, Kano Y, Ohta K, Fukusumi T, Sakai D, Kudo T, Haraguchi N, et al: Aldehyde dehydrogenase high gastric cancer stem cells are resistant to chemotherapy. Int J Oncol. 42:1437–1442. 2013.PubMed/NCBI

32 

Katsuno Y, Ehata S, Yashiro M, Yanagihara K, Hirakawa K and Miyazono K: Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-β. J Pathol. 228:391–404. 2012. View Article : Google Scholar : PubMed/NCBI

33 

Wakamatsu Y, Sakamoto N, Oo HZ, Naito Y, Uraoka N, Anami K, Sentani K, Oue N and Yasui W: Expression of cancer stem cell markers ALDH1, CD44 and CD133 in primary tumor and lymph node metastasis of gastric cancer. Pathol Int. 62:112–119. 2012. View Article : Google Scholar : PubMed/NCBI

34 

Marcato P, Dean CA, Giacomantonio CA and Lee PW: Aldehyde dehydrogenase: Its role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle. 10:1378–1384. 2011. View Article : Google Scholar : PubMed/NCBI

35 

Storms RW, Trujillo AP, Springer JB, Shah L, Colvin OM, Ludeman SM and Smith C: Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci USA. 96:9118–9123. 1999. View Article : Google Scholar : PubMed/NCBI

36 

Kastan MB, Schlaffer E, Russo JE, Colvin OM, Civin CI and Hilton J: Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells. Blood. 75:1947–1950. 1990.PubMed/NCBI

37 

Collins SJ: Retinoic acid receptors, hematopoiesis and leukemogenesis. Curr Opin Hematol. 15:346–351. 2008. View Article : Google Scholar : PubMed/NCBI

38 

Bosch FX, Lorincz A, Muñoz N, Meijer CJ and Shah KV: The causal relation between human papillomavirus and cervical cancer. J Clin Pathol. 55:244–265. 2002. View Article : Google Scholar : PubMed/NCBI

39 

Liu SY and Zheng PS: High aldehyde dehydrogenase activity identifies cancer stem cells in human cervical cancer. Oncotarget. 4:2462–2475. 2013. View Article : Google Scholar : PubMed/NCBI

40 

Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L, Wang H, Liu Z, Su Y, Stass SA, et al: Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res. 7:330–338. 2009. View Article : Google Scholar : PubMed/NCBI

41 

Charafe-Jauffret E, Ginestier C, Iovino F, Tarpin C, Diebel M, Esterni B, Houvenaeghel G, Extra JM, Bertucci F, Jacquemier J, et al: Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin Cancer Res. 16:45–55. 2010. View Article : Google Scholar

42 

Deng S, Yang X, Lassus H, Liang S, Kaur S, Ye Q, Li C, Wang LP, Roby KF, Orsulic S, et al: Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1), in human epithelial cancers. PLoS One. 5:e102772010. View Article : Google Scholar :

43 

Yang L, Ren Y, Yu X, Qian F, Bian BS, Xiao HL, Wang WG, Xu SL, Yang J, Cui W, et al: ALDH1A1 defines invasive cancer stem-like cells and predicts poor prognosis in patients with esophageal squamous cell carcinoma. Mod Pathol. 27:775–783. 2014. View Article : Google Scholar

44 

Zhou W, Lv R, Qi W, Wu D, Xu Y, Liu W, Mou Y and Wang L: Snail contributes to the maintenance of stem cell-like phenotype cells in human pancreatic cancer. PLoS One. 9:e874092014. View Article : Google Scholar : PubMed/NCBI

45 

van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Pelger RC and van der Pluijm G: The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis. Clin Exp Metastasis. 28:615–625. 2011. View Article : Google Scholar : PubMed/NCBI

46 

Yan J, De Melo J, Cutz JC, Aziz T and Tang D: Aldehyde dehydrogenase 3A1 associates with prostate tumorigenesis. Br J Cancer. 110:2593–2603. 2014. View Article : Google Scholar :

47 

Singh S, Brocker C, Koppaka V, Chen Y, Jackson BC, Matsumoto A, Thompson DC and Vasiliou V: Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress. Free Radic Biol Med. 56:89–101. 2013. View Article : Google Scholar :

48 

Gasparetto M, Sekulovic S, Brocker C, Tang P, Zakaryan A, Xiang P, Kuchenbauer F, Wen M, Kasaian K, Witty MF, et al: Aldehyde dehydrogenases are regulators of hematopoietic stem cell numbers and B-cell development. Exp Hematol. 40:318–329.e2. 2012. View Article : Google Scholar

49 

Moreb JS, Mohuczy D, Ostmark B and Zucali JR: RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide. Cancer Chemother Pharmacol. 59:127–136. 2007. View Article : Google Scholar

50 

Muzio G, Trombetta A, Maggiora M, Martinasso G, Vasiliou V, Lassen N and Canuto RA: Arachidonic acid suppresses growth of human lung tumor A549 cells through down-regulation of ALDH3A1 expression. Free Radic Biol Med. 40:1929–1938. 2006. View Article : Google Scholar : PubMed/NCBI

51 

Pappa A, Brown D, Koutalos Y, DeGregori J, White C and Vasiliou V: Human aldehyde dehydrogenase 3A1 inhibits proliferation and promotes survival of human corneal epithelial cells. J Biol Chem. 280:27998–28006. 2005. View Article : Google Scholar : PubMed/NCBI

52 

Muzio G, Trombetta A, Martinasso G, Canuto RA and Maggiora M: Antisense oligonucleotides against aldehyde dehydrogenase 3 inhibit hepatoma cell proliferation by affecting MAP kinases. Chem Biol Interact. 143–144:37–43. 2003. View Article : Google Scholar

53 

Wang JS, Fang Q, Sun DJ, Chen J, Zhou XL, Lin GW, Lu HZ and Fei J: Genetic modification of hematopoietic progenitor cells for combined resistance to 4-hydroperoxycyclophosphamide, vincristine, and daunorubicin. Acta Pharmacol Sin. 22:949–955. 2001.PubMed/NCBI

54 

Canuto RA, Muzio G, Ferro M, Maggiora M, Federa R, Bassi AM, Lindahl R and Dianzani MU: Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines. Free Radic Biol Med. 26:333–340. 1999. View Article : Google Scholar : PubMed/NCBI

55 

Calderaro J, Nault JC, Bioulac-Sage P, Laurent A, Blanc JF, Decaens T and Zucman-Rossi J: ALDH3A1 is overexpressed in a subset of hepatocellular carcinoma characterised by activation of the Wnt/β-catenin pathway. Virchows Arch. 464:53–60. 2014. View Article : Google Scholar

56 

Vesuna F, Lisok A, Kimble B and Raman V: Twist modulates breast cancer stem cells by transcriptional regulation of CD24 expression. Neoplasia. 11:1318–1328. 2009. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

August-2016
Volume 49 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Wu D, Mou Y, Chen K, Cai J, Zhou Y, Pan Y, Xu X, Zhou W, Gao J, Chen D, Chen D, et al: Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis. Int J Oncol 49: 611-622, 2016
APA
Wu, D., Mou, Y., Chen, K., Cai, J., Zhou, Y., Pan, Y. ... Zhang, R. (2016). Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis. International Journal of Oncology, 49, 611-622. https://doi.org/10.3892/ijo.2016.3551
MLA
Wu, D., Mou, Y., Chen, K., Cai, J., Zhou, Y., Pan, Y., Xu, X., Zhou, W., Gao, J., Chen, D., Zhang, R."Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis". International Journal of Oncology 49.2 (2016): 611-622.
Chicago
Wu, D., Mou, Y., Chen, K., Cai, J., Zhou, Y., Pan, Y., Xu, X., Zhou, W., Gao, J., Chen, D., Zhang, R."Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis". International Journal of Oncology 49, no. 2 (2016): 611-622. https://doi.org/10.3892/ijo.2016.3551