Human endometrial cancer cell line JEC was purchased from the Cell Bank of Chinese Academy of Sciences (CAS). JEC cells were cultured in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a humidified incubator with 5% CO₂.

Conventional 2-D cultures was performed for the self-renewal and successive spheriod generation experiments according to related references (29–33). Spheroid culture conditions: parental cell lines or the floating spheres were plated in serum-free sphere formation media (SFM): low-glucose (1 g/l) DMEM and supplemented with L-glutamine, sodium pyruvate, penicillin/streptomycin (Wisent Inc.), 20 ng/ml basic FGF, 20 ng/ml EGF, and B27 (Invitrogen, Grand Island, NY, USA), 100 mg/ml gentamycin using 6-well ultra-low attachment plates (Corning Inc., Tewksbury, MA, USA) (31). Spheres were dissociated with trypsin every 7 days and split at 1:3 ratio for the next passage generation. For sphere formation assay, 5×10⁴ cells were seeded in ultra-low attachment 6-well plate (Corning Inc.) in 2 ml SFM/well for 2 weeks. For self-renewal assay, cells were diluted into ~1–3 cells/well in 96-well ultra-low attachment plates (Corning Inc.) using SFM for 2 weeks, both the media and the drugs were replaced every 3 days. Then the floating spheres were photographed at ×100 magnification (Nikon Eclipse TS100). Sphere number was determined by ImageJ program.

Microbead sorting was performed using CD133 and CD44 microbead kit (Miltenyi Biotec, Germany). JEC cells were adjusted into 1×10⁸/ml and centrifuged at 1,300 rpm for 5 min, the supernatant was discarded and cells were resuspened in 300 µl cold buffer, then mixed softly. The remaining steps were performed according to the manufacturer's instructions. After CD133 sorting, the appropriate number of CD133⁺ cells were expanded for 7 days by culture and then harvested, sorted with CD44 microbeads for the enrichment of CD133⁺/CD44⁺ cells. This two-step isolation enabled us to obtain a suffcient number of CD133⁺/CD44⁺ CSCs for the experiment.

To verify the purity of the isolated CD133⁺/CD44⁺ CSCs or spheres of different generations, flow cytometric analysis of CD133⁺/CD44⁺ cells was performed with CD133-FITC and CD44-PE antibodies (Miltenyi Biotec) according to the manufacturer's instructions using a FACSCalibur instrument (Beckman Coulter Life Sciences, USA).

For EIG121 knockdown, lentiviral constructs containing EIG121 shRNA (TRCN0000263309 and TRCN0000263310) were purchased from Sigma. JEC cells stably expressing Lv-con-shRNA or EIG121 shRNA were generated by lentiviral infection at MOI 25 and selected with 2 mg/ml puromycin.

Total RNA was extracted with the TRIzol reagent (Invitrogen, USA) and cDNA was reverse-transcribed using a reverse transcription kit (Fermentans, Germany) according to the manufacturer's protocol. Quantitative detection of RT products was performed using SYBR Green qRCR Mix (Toyobo, Japan). The PCR was performed using Fluorescence quantitative PCR instrument 7300 (ABI PRISM, USA). The PCR reaction condition was as follows: 95°C for 3 min, 95°C for 10 sec, 58°C for 30 sec, and 72°C for 30 sec, 40 cycles, to obtain fluorescence intensity. β-actin was used as an internal control. The specific primers are listed in Table I. Relative mRNA expression levels were determined by the 2^−ΔΔCt method in comparison with control group.

Cells were lysed in RIPA buffer and cleared by centrifugation at 13,000 rpm for 10 min. The proteins (100 mg) were resolved in a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Primary antibodies against the LC3B antibody (2 µg/ml, ab81785, Abcam, USA), EIG121 (1:1,000, ab156275, Abcam), Beclin1 (1:1,000, ab14071, Abcam), CD133 (1:500, 18470-1-AP, Proteintech, China), CD44 (1:500, 15675-1-AP, Proteintech), OCT4 (1:1,000, ab181557, Abcam), c-Myc (1:500,10828-1-AP, Proteintech), CK4 (1:500, ab155406, Abcam), and ABCG2 (1:1,000, ab108312, Abcam), or β-actin (1:2,000, LCA01, Auragene, Changsha, China) were added to the membrane in 8% non-fat milk and incubated at 4°C overnight. After extensive washing, the membrane was incubated in 8% non-fat milk containing HRP conjugated anti-rabbit secondary antibody (1:2,000, SA009, Auragene) or anti-mouse secondary antibody (1:2,000, SA002, Auragene) for 30 min at room temperature. The AuraECL detection kit (Auragene) was used to visualize the target bands.

Cells were cultured on cover glasses coated with 0.1% gelatin in PBS in 6-well tissue culture plates with DMEM. The cells were incubated overnight then washed with PBS, and fixed in 4% paraformaldehyde for 15 min. They were then permeabilized in 0.2% Triton X-100 for 10 min prior to blocking in 6% bovine serum albumin (BSA) for 30 min. For H&E staining, cells were washed using PBS 3 times then stained with hematoxylin for 10 min. After washing, stained with eosin for 15 min, dehydrated by anhydrous alcohol for 10 sec and dried. Subsequently, stained cells were photographed at ×400 magnifcation (Nikon Eclipse TS100). For immunofluorescence staining, the cells were incubated overnight with anti-LC3B (1:200, 12741S, Cell Signaling, USA) at 4°C, followed by incubation with goat anti-rabbit IgG (H+L)-Cy3 (1:200, SA012, Auragene) for 1 h at room temperature in dark. Nuclei were counterstained with DAPI (10 µg/ml, C0065, Solarbio, China). Finally, cells were analyzed using a confocal fluorescence microscope (BX50; Olympus, Japan) and the percentage of cells with LC3 puncta was calculated with the help of Image-Pro Plus 6.0.

ECSCs were dispersed and seeded into 96-well plates at 5×10³ cells/well and incubated with DMEM containing PTX (20 µg/ml) alone or with CQ (10 µM)/3-MA (1 mM) respectively for 24 h. MTT solution (10 µl) was added to each well and incubated at 37°C for 4 h, then 150 µl DMSO was added to each well. Ten minutes later, the absorbance at 570 nm was monitored using a microplate reader (BioTek) to assess cell viability. Cell growth inhibition was normalized and expressed relative to the absorbance of cells treated with DMSO alone.

It was known that when the culture environment is restored to normal culture condition of tumor cells, the spheres would differentiate to normal adherent tumor cells and proliferation. So, to some extent, the extension of cells in 6-well plates reflect the cell viability. The phenotypic recovery assay was performed to determine the re-differentiation ability of different cells. P3 JEC spheres or CD133⁺/CD44⁺ CSCs were digested with 0.05% trypsinase to obtain single cell suspension, then the cells were inoculated in 6-well normal cell culture plate filled with complete DMEM medium containing CQ or 3-MA, respectively. When the cell was completely adherent, the phenotypic change was observed and photographed at ×100 magnification (Nikon Eclipse TS100). The cell-free areas were filled with black using Photoshop and measured by Image-Pro Plus 6.0.

Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of The Affiliated Tumour Hospital of Xiangya Medical School of Central South University. The protocol was approved by the Committee on the Ethics of Animal Experiments of The Affiliated Tumour Hospital. NOD/SCID mice at age of 3–5 weeks, male, were maintained in pathogen-free conditions at animal facility. The JEC, Lv-Con, and Lv-EIG121-shRNA cells were resuspended in serum-free medium and mixed with Matrigel at the ratio of 1:1. NOD/SCID mice were randomly divided into 3 groups (n=4 per group). Cells (1×10⁶) were inoculated subcutaneously into the inguinal folds of NOD/SCID mice. Tumor formation was evaluated regularly after injection by palpation of injection sites. At the end of experiment, the mice were sacrificed under deep anesthesia with pentobarbital. The tumors were then dissected and captured. The primary tumors were immunostained for PCNA and active caspase-3 as previously described (34).

The data are presented as mean ± standard deviation. The means of groups were compared with one-way analysis of variance, and after checking for equal variance, comparisons between two means were performed using the least significant difference (LSD) method. Student's t-test was used for two group's comparison. Analysis of variance was used for statistical analyses. P<0.05 was considered as statistically significant.

It has been reported that CD133⁺ cells exhibit properties of CSCs and CD44 is another important marker of ECSCs (35–37). To better understand the underlying mechanism responsible for low chemosensitivity in EC, CD133⁺/CD44⁺ cells were sorted from the JEC cell lines by CD133 and CD44 microBeads (38). The purity of CD133⁺/CD44⁺ cells before and after sorting from JEC was 8.27 and 90.1%, respectively (Fig. 1A). The QPCR was performed to detect the autophagy markers in JEC control, CD133⁺/CD44⁻, CD133⁺/CD44⁺ and CD133⁻/CD44⁺ cells. The results showed that Beclin1, Atg5, Atg7 mRNA levels were significantly enhanced but the P62 was obviously reduced in CD133⁺/CD44⁺ cells compared to JEC cells (Fig. 1B). Besides, the mRNA alteration of Atg5 and P62 exhibited significance between CD133⁺/CD44⁻ and CD133⁺/CD44⁺ groups. The results demonstrated that the CD133⁺/CD44⁺ cells which exhibit CSC properties possess higher autophagy.

Firstly, it was found that bead sorted cells (CD133⁺/CD44⁺ cells) are enriched for autophagosomic LC3B II over parental cells (Con) alone which suggeted the autophagy level of ECSCs would be higher than normal cancer cells (Fig. 2A).

Spheroid culture is a mainstream method to obtain cells exhibiting properties of CSCs. In order to observe the autophagy dynamic changes in the process of stemness dynamic changing, successive spheroid cultures were performed from passage 1 (P1) to passage 4 (P4). QPCR was performed to detect the relative mRNA level of CSC markers and autophagy markers. The results showed that the relative expression of CSC marker CD133, CD44, Oct4, c-Myc and drug resistance marker ABCG2 were increased but the differentiation marker CK4 decreased obviously from P0 (Con) to P3. Besides, the mRNA level of ABCG2, c-Myc and CK4 were decreased from the relative peak value when the spheroid generations have undergone P4 culture but the CD133 and CD44 are still maintained at peak level. On the other hand, flow cytometric staining was used to detect the purity of CD133⁺/CD44⁺ cells in spheres at different passages, the percentage of CD133⁺/CD44⁺ cells increased significantly from P1 to P3, but to the P4 generation, the increasement was not obvious compared to P3 (Fig. 2B and C). We thought the JEC P3 spheres could be partially considered as ECSCs with strongest stemness using the successive spheroid culture method. It was of great interest that the expression of classic autophagy marker Beclin1 and LC3Π/I kept in line with the stemness changing, in other words, autophagy was enhanced gradually from P0 to P4 (Fig. 2D). These results allowed to conclude that stemness kept in line with autophagy in successive culture of JEC spheres and the synchronized level suggested the inherent link between ECSCs and JEC autophagy.

It has been reported that autophagy plays an important role in CSC maintenance, plasticity and survival (17–19) and takes part in the dynamic transformation between normal cancer cells and CSC in pancreatic cancer (27,28). In order to clarify if autophagy inhibition could influence the dynamic transformation of ECSCs, conventional autophagy inhibitors 3-MA and CQ were added during the successive spheroid culture, respectivly, then the relative expression of CSC markers was detected by QPCR and western blotting. Results showed when 3-MA or CQ was added, the CSC markers (CD133, CD44, OCT4 and ABCG2 decreased compared to the control group of the same sphere passage (Fig. 3A and B). To further confirm the effect of autophagy inhibitors, western blotting was performed to detect expression of P62 and LC3II/I in P3 and P4 spheres. The experimental results showed that autophagy inhibitor 3-MA addition could increase P62 while decrease the ratio of LC3 II/I and CQ addition could increase both P62 and the ratio of LC3 II/I compared to spheres without autophagy inhibitor in P3 and P4 JEC spheres (Fig. 3C). Re-differentiation ability is one of the important characteristics of CSCs. For the sake of detecting the re-differentiation ability of JEC spheres, P3 spheres were used to perform phenotypic recovery assay which aims to show whether the addition of autophagy inhibitor could decrease the re-differentiation of JEC spheres. The extension of cells in 6-well plates reflect the cell viability. It was observed that when the P3 spheres were treated with 3-MA or CQ, the recovery was more difficult to adherent phenotype and proliferation under routine culture conditions (Fig. 3D and E).

We obtained CD133⁺/CD44⁺ cells by microBead sorting and CSC markers were detected by QPCR and western blotting. The relative expression of CD133, CD44, ABCG2, OCT4 and ALDH was significantly enhanced in CD133⁺/CD44⁺ cells, so that CD133⁺/CD44⁺ cells would be considered as endometrial carcinoma stem cells (ECSCs) to some extent. Besides, the expression levels of EC autophagy marker EIG121, Beclin1, Atg5, Atg7 and LC3Π/I were obviously increased dependent on VS JEC cells (Con). The above demonstrated that the ECSC cells exhibited higher level of autophagy (Fig. 4A). For evaluating the role of autophagy in the PTX chemoresis-tance of ECSCs, cell viability, self-renewal and phenotypic recovery assays were performed using PTX treatment with or without autophagy inhibitors. The results showed when the ECSCs were treated in combination with 3-MA or CQ, the cell viability attenuated significantly (Fig. 4C). Self-renewal capacity reduced both in sphere size and number (Fig. 4B and D). Phenotypic recovery assays were performed to imitate recurrence after chemotherapy in vitro aiming to show that the addition of autophagy inhibitor could enhance the growth inhibition of PTX of JEC cells, and the extension of cells in 6-well plates reflect the cell viability (Fig. 4E and F) compared to ECSCs treated with PTX only, autophagy inhibition attenuated the cell growth capacity to some extent after removal of drugs and return to normal condition (Fig. 4E and F). Thus, the lower self-renewal capacity was not only embodied in propagating clone numbers but also the offspring sphere size.

It was reported previously that the novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under EC stress (39). In this experiment, it was found that EIG121 was increased in ECSCs similarly to other autophagy-associated proteins (Fig. 4A). EIG121 loss-of-function stem cell model was constructed to explore whether the EIG121 could mediate the autophagy in JEC ECSCs. Lv-EIG121-shRNA lentivirus infection of ECSCs was performed. When the relative expression of EIG121 was effectively knocked down by Lv-EIG121-shRNA in ECSCs, the expression of Beclin1, ATG5 and Atg7 were also down-regulated in ECSCs (Fig. 5A and B). Overexpression EIG121 would greatly increase cytoplasmic vacuole accumulation in MDA-MB-213 (39). As cytoplasmic vacuolation is a hallmark of autophagy, the H&E staining was performed on JEC ECSCs at 12 h after lentivirus infection and it was shown that the JEC-obtained ECSCs contained numerous cytoplasmic vacuoles. However, when the EIG121 was knocked down, cytoplasmic vacuoles became blurred (Fig. 5C). On the other hand, autophagy induction is associated with LC3, conjugated LC3 moves into autophagosomes and tightly binds to the autophagosome membrane. Thus, LC3 translocation is a reliable biomarker of autophagy (1,40). Immunofluorescence staining was performed to detect the LC3 translocation. The results showed that in ECSCs, LC3 expression occurred predominantly in punctuate dot-like structures, consistent with autophagy induction. Whereas in Lv-EIG121-shRNA infected ECSCs, LC3 puncta decreased obviously and LC3 was uniformly distributed in the cytoplasm, the percentage of cells with LC3 puncta was calculated with the help of Image-Pro Plus 6.0 (Fig. 5D and E). These results demonstrated that EIG121 played an autophagy-induced role not only in EC normal cells, but also in JEC-obtained ECSCs.

We considered that autophagy could play an important role in the transformation between JEC cells and JEC-obtained cancer stem cells. Besides, EC novel autophagy-induction protein EIG121 was found to exert autophagy-induction function in ECSCs. Based on this information, it was hypothesized that the novel autophagy-inducing gene EIG121 would mediate the stemness of JEC cells. To explore whether EIG121 expression could affect stemness and tumorigenesis, EIG121 loss-of-function cell model was constructed in JEC cells. Lentivirus (Lv-EIG121-shRNA) infected JEC cells exhibited lower relative expression of stemness marker genes of CD133, CD44, ABCG2, OCT4 and ALDH at both mRNA and protein levels (Fig. 6A and B). Besides, Lv-EIG121-shRNA infected JEC cells showed reduced self-renewal capacity in both colony size and number (Fig. 6C and D). Moreover, cells were inoculated into nude mice and all mice developed xenograft tumors at the injection site 20 days after injection. It was found that the tumors formed of the Lv-EIG121-shRNA group were generally smaller than JEC and Lv-Con group (Fig. 6E). Additionally, the average tumor weight was obviously lower in the Lv-EIG121-shRNA group compared to other groups (Fig. 6H). The tumors developed from Lv-EIG121-shRNA cells displayed weaker PCNA staining and stronger active caspase-3 staining than that in JEC and Lv-Con cells (Fig. 6F and G). These results indicate that EIG121 knockdown in JEC cells resulted in reduction of stemness and tumorigenesis.