Human placentas were obtained at term pregnancy during uncomplicated Caesarean sections with written and informed consent from women who were negative for HIV-I, and hepatitis B and C. This study has been approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital, and written informed consent was obtained from all participants. The protocol of hAECs isolation was described previously (11). hAECs was cultured in DMEM/F12 (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), streptomycin (100 U/ml; Thermo Fisher Scientific), penicillin (100 U/ml; Thermo Fisher Scientific), 1× non-essential amino acids (Thermo Fisher Scientific), and 1 mM of sodium pyruvate (Thermo Fisher Scientific), and incubated at 37°C in an incubator containing 5% CO₂. Once the density of cells reached 80–90% confluency, cells were collected for subsequent experiments. hAECs (5×10⁶) were seeded in 100 mm diameter dishes (Corning, USA) in complete culture medium for 72 h. The hAEC-CM were then collected, 0.22 mm filtered, and used in subsequent experiments.

hAECs were identified by using the indirect immunofluorescent labeling technique to detect the expression of CK-7 and vimentin. Cells were fixed with 4% paraformaldehyde (PFA), and then incubated with the following primary antibodies at 4°C overnight: CK-7 (1:200 dilution; Boster, Wuhan, China) and vimentin (1:200 dilution; Boster). After that, cells were incubated with secondary antibody conjugated with Alexa Fluor^® 488 (1:200; Thermo Fisher Scientific). Cells were counterstained with DAPI (Thermo Fisher Scientific) and examined under the fluorescence microscope (Leica, Germany).

The EOC cell lines, SK-OV-3 and A2780 were obtained from Shanghai Cell Bank of Chinese Academy of Sciences and cultured in DMEM/high glucose (Thermo Fisher Scientific) medium supplemented with 10% FBS, streptomycin (100 U/ml) and penicillin (100 U/ml). Cells were incubated at 37°C in an incubator containing 5% CO₂. Once the density of cells reached 80–90% confluency, cells were collected for subsequent experiments.

Cell viability was detected with cell counting kit-8 (CCK8; Dojindo Molecular Technologies, Japan) according to the manufacturer's protocol. EOC cells (7,000 cells per well) were seeded in 96-well plate with complete culture medium or hAEC-CM or mixture of complete culture medium and hAEC-CM at 1:1 ratio or different concentration of rhTGF-β1 for 48 h.

Transwell co-culture system (Corning) was used to evaluate the effects of hAEC-secreted factors on EOC cells. hAECs (1×10⁶ cells) were seeded on Transwell inserts and EOC cells (2×10⁶ cells) were cultured in 6-well culture plates at the lower compartment. Cells were cultured in DMEM/high glucose medium supplemented with 10% FBS, streptomycin (100 U/ml) and penicillin (100 U/ml). All groups were incubated at 37°C in a 5% CO₂ incubator for 4 days.

Total RNA was extracted from cultured EOC cells by TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions. Real-time quantitative PCR reactions were performed in triplicate, using the SYBR Green Real-time PCR Master Mix (Takara, Japan). PCR primers were designed according to cDNA sequences in the NCBI database. The following primers were used: GAPDH, 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-GGGGTCGTTGATGGCAACA-3′; PCNA, 5′-TTCCTGTGCAAAAGACGGAG-3′ and 5′-TCACCGTTGAAGAGAGTGGA-3′; Ki-67, 5′-GAGGCAAATCATCCGAACCC-3′ and 5′-TTATTTTGGCGTCTGGAGCG-3′. Cycling conditions for the PCR machine were as follows: 95°C 5 sec, 60°C 30 sec and 72°C 30 sec for 40 cycles. All reactions were performed in a 10-µl volume. Gene expression levels were evaluated using the ΔΔCt method, standardized to levels of GAPDH amplification.

Twelve female BALB/c nude mice (4-week-old) were obtained from Shanghai Jiao Tong University School of Medicine (Shanghai, China). All experimental protocols were approved by the Ethics Committee of the School of Medicine of Shanghai Jiao Tong University, and were in accordance with the approved guidelines set by the Institutional Animal Care and Use Committee. The mice were housed under a laminar flow hood in an isolated room and were maintained under pathogen-free conditions. Five mice per group were subcutaneously injected with 2×10⁶ SK-OV-3 or SK-OV-3/hAECs (2×10⁶:1×10⁶). Tumor volume was measured every two weeks and tumor weight was measured at the endpoint of the experiment. Animals were sacrificed by cervical dislocation under anesthetic status after 28 days. Tumor volume was calculated using the formula: tumor volume (mm³) = 0.52 × [width (mm²)] × [length (mm)] (12). To localize the hAECs in the tumor microenvironment, we transfected hAECs with green fluorescent protein (GFP) by lentivirus which was kindly provided by the Professor Lijian Hui (Chinese Academic of Sciences, Shanghai, China). Two mice were used to establish SK-OV-3/hAECs^GFP (2×10⁶:1×10⁶) xenografts and were sacrificed after 28 days.

Tissue sections were deparaffinized and dehydrated. The slides were then incubated in a 3% H₂O₂ solution to block the endogenous peroxidase (IF staining can skip this procedure), followed by rinsing in PBS. To retrieve the antigenicity, sections were then treated with heated antigen retrieval solution containing ethylene diamine tetraacetic acid (EDTA; Wuhan Goodbio Technology, Wuhan, China). After being incubated with 5% bull serum albumin for 30 min to block the non-specific antibody binding sites, the samples were then incubated with the following primary antibodies at 4°C overnight: PCNA (1:6,000 dilution; CST, USA), Ki-67 (1:200 dilution; Arigo Biolaboratories, Taiwan, China), p16^INK4A (1:300 dilution; Wanleibio, China), p21 (1:300 dilution; Wanleibio), phospho-JNK (1:300 dilution; Wanleibio), phospho-pRB (Ser807) (1:500 dilution; Abcam, UK). For IHC analysis, horseradish peroxidase (HRP)-labelled anti-mouse/rabbit second antibodies and diaminobenzidine were used according to the manufacturer's instructions (Wuhan Goodbio Technology). Slides were counterstained with hematoxylin, differentiated with 0.1% hydrochloric acid alcohol, and washed by water. They were then dehydrated through a graded ethanol series, immersed in xylene and mounted with permount™ mounting medium. For IF analysis for GFP, tissues were stained with secondary antibody conjugated with Alexa Fluor 488 (1:200; Thermo Fisher Scientific). Then tissues were counterstained with DAPI (Thermo Fisher Scientific) and examined under the fluorescence microscope (Leica).

For the semi-quantitative evaluation of p21, p16^INK4A and phospho-JNK expressions, we used a scoring method described by Budwit-Novotny et al (13). The evaluation of PCNA, Ki-67 and phospho-pRB (Ser807) were measured by the percentage of cells with positive signals in the nucleus.

A human antibody array 1000 (a combination of human L-507 and human L-493) (RayBiotech, Inc., USA) was performed to detect the expression of anticancer-associated cytokines in hAECs. Samples of hAEC-CM were collected as previously described. Fresh DMEM/F12 culture medium was used as the control. The cytokine array was performed according to the instructions. The intensities of signals were quantified by densitometry.

EOC cells were cultured alone or in the presence of hAEC-CM. After 48 h, cancer cells were harvested by 0.25% trypsin/ethylene diamine tetraacetic acid, then being washed with phosphate-buffered solution, and fixed with 70% cold-ethanol for 24 h at −20°C. The cells were treated with 50 µg/ml RNase A (Tiangen, China) for 30 min at 37°C and then stained with 100 µg/ml propidium iodide (PI; Thermo Fisher Scientific) for 30 min at 4°C. The cells were analyzed using a Cytomics™ FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using Beckman Coulter CXP software.

The ELISA kit was used to test the efficiency of TGF-β1 antibody added to Transwell system to neutralize hAEC-secreted TGF-β1 at 48 h (R&D Systems, USA) and was conducted according to the manufacturer's instructions.

Results from three independent experiments are reported as the mean ± SEM. Kolmogorov-Smirnov test was used to test whether data accorded with Gaussian distribution. Homogeneity of variance test was conducted by Bartlett's test. Two-tail Student's t-test or ordinary one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test or Mann-Whitney U test (non-parametric test) or Kruskal-Wallis test (non-parametric test) was used to evaluate statistical differences via GraphPad Prism version 6 (GraphPad Software). Differences were considered significant at p-value <0.05.

The hAECs represented a cobblestone-like morphology. These cells were positive for epithelial marker CK7, and negative for mesenchymal marker vimentin using IF analysis (Fig. 1A).

CCK-8 assay was conducted to test whether hAEC-secreted factors could influence the viability of EOC cells. Results showed that significant vitality inhibition in EOC cells was induced by hAEC-CM in different concentration compared with the control group at 48 h (n=6). Moreover, the growth inhibition induced by hAEC-CM on EOC cells was significantly dose-dependent (Fig. 1B).

Results from real-time PCR showed that the expression levels of PCNA and Ki-67, two genes relating to cellular proliferation, were significantly downregulated within EOC cells co-cultured with hAECs compared with the control group (Fig. 1C; n=3).

To explore the effects of hAECs on EOC cells in vivo, we established a tumor-bearing nude mouse model by subcutaneously co-injecting SK-OV-3 cells and hAECs at 2:1 ratio (2×10⁶:1×10⁶ cells; five mouse per group) at both sides of the scapular region. Four weeks after injection, we observed that hAECs significantly decreased the average weight and average volume of xenografted tumors compared with the control group (Fig. 2A–C; n=10). To further trace the location of hAECs in the tumor tissues, we labelled hAECs with GFP (Fig. 2D–a) and established a xenografted mouse model by subcutaneously co-injecting SK-OV-3 and hAEC^GFP+ cells at 2:1 ratio (2×10⁶:1×10⁶ cells; two mice were used) at both sides of the scapular region. After 28 days, hAEC^GFP+ cells were found in the stromal area of xenografted tumor tissues and no positive signal was observed in the negative control using IF assay (Fig. 2D–b and –c).

Results from IHC assay showed that hAECs significantly decreased the positive rates of both PCNA and Ki-67 in the SK-OV-3/hAECs co-injected tumor tissues compared with that in the only SK-OV-3 injected tumor tissues (Fig. 2E and F; n=5), which was consistent with the ex vivo results.

Cell count assay showed that hAEC-CM significantly decreased the cell numbers of both SK-OV-3 and A2780 cells at 48 h, indicating hAEC-secreted factors influenced the division of EOC cells (Fig. 3A; n=3).

By using Transwell system, we observed that hAECs significantly induced G0/G1 cell cycle arrest in both SK-OV-3 and A2780 cells with a significant decrease in S phase using flow cytometry analysis (Fig. 3B; n=3).

We further tested the effects of hAECs on the expression levels of three negative regulators of cell cycle progression, p16^INK4A and p21, and phospho-JNK, which was associated with TGF-β1-induced growth inhibition, in the tumor tissues (14–16). IHC assay revealed that SK-OV-3/hAECs co-injected tumor tissues exhibited significantly higher expressions of p16^INK4A, p21 and phospho-JNK compared with tumor tissues obtained from SK-OV-3 injected group (Fig. 4A–C; n=5). Furthermore, the level of phospho-pRB (Ser807), a major regulator controlling cell division cycle from G1 to S phase, was detected in the tumor tissues using IHC. Results showed that hAECs significantly inhibited phosphorylation of pRB (Ser807) within tumor tissues obtained from xenografts compared with control group (Fig. 4D; n=5).

The results from cytokine array revealed that hAECs express various anticancer-related cytokines, including TGF-β1, granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), IL-6, IL-1, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), TGF-β2 and Smad 7 (Fig. 5A–a). The relative expression of cytokines was defined as the ratio of certain cytokine to Smad 7. We found that the level of hAEC-secreted TGF-β1 was the most abundant among these cytokines which were reported to be able to negatively regulate cell cycle progression (Fig. 5A–b).

To illustrate the effects of TGF-β1 on cell cycle progression within EOC cells, we conducted CCK-8 cell viability assay and found that the proliferative inhibition mediated by rhTGF-β1 was dose-dependent (Fig. 5B; n=6). Further we added rhTGF-β1 to treat EOC cells and then cell cycle progression was evaluated by flow cytometry. Results showed that rhTGF-β1 induced a significant increase in G0/G1 phase and a decrease in S phase in both cell lines (Fig. 5C; n=3).

To evaluate the neutralization efficiency of antibody used to attenuate the function of hAEC-secreted TGF-β1, an ELISA kit was used. Results indicated that 10 ng/ml TGF-β1 antibody showed the best efficiency of reducing the amount of hAEC-secreted TGF-β1 in the supernatant (Fig. 5D).

Finally, we added TGF-β1 antibody into the Transwell system and evaluated the cell cycle progression of EOC cells at 48 h. We observed that TGF-β1 antibody reversed hAEC-induced G0/G1 cell cycle arrest and increased the population in S phase and G2/M phase (Fig. 5E; n=3).