Human omental adipose-derived mesenchymal stem cells enhance autophagy in ovarian carcinoma cells through the STAT3 signalling pathway

Journal Pre-proof Human omental adipose-derived mesenchymal stem cells enhance autophagy in ovarian carcinoma cells through the STAT3 signalling pathway

Yijing Chu, Yan Wang, Kun Li, Meixin Liu, Yan Zhang, Yan Li, Xiaoyu Hu, Chong Liu, Huansheng Zhou, Jianxin Zuo, Wei Peng PII:

S0898-6568(20)30026-7

DOI:

https://doi.org/10.1016/j.cellsig.2020.109549

Reference:

CLS 109549

To appear in:

Cellular Signalling

Received date:

17 October 2019

Revised date:

23 January 2020

Accepted date:

23 January 2020

Please cite this article as: Y. Chu, Y. Wang, K. Li, et al., Human omental adiposederived mesenchymal stem cells enhance autophagy in ovarian carcinoma cells through the STAT3 signalling pathway, Cellular Signalling(2019), https://doi.org/10.1016/ j.cellsig.2020.109549

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© 2019 Published by Elsevier.

Journal Pre-proof

Human omental adipose-derived mesenchymal stem cells enhance autophagy in ovarian carcinoma cells through the STAT3 signalling pathway Yijing Chu1# , Yan Wang2# , Kun Li3 , Meixin Liu1 , Yan Zhang1 , Yan Li1 , Xiaoyu Hu1 , Chong Liu1 , Huansheng Zhou1 , Jianxin Zuo1* , Wei Peng1* 1

Department of Obstetrics and Gynecology, the Affiliated Hospital of Qingdao

University, Qingdao, China.

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Department of Orthopaedic Surgery, the Affiliated Hospital of Qingdao University,

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2

Qingdao, China.

Department of Hepatobiliary and Pancreatic Surgery, the Affiliated Hospital of

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3

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Qingdao University, Qingdao, China.

#These authors contributed equally to this work.

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*Corresponding authors:

Wei Peng, Department of Obstetrics and Gynecology, the Affiliated Hospital of University,

16

Jiangsu

Road,

Qingdao

266061,

China.

Tel.:

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Qingdao

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+86-18661808067. E-mail: [email protected]. Jianxin Zuo, Department of Obstetrics and Gynecologythe Affiliated Hospital of University,

16

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Qingdao

Jiangsu

Road,

Qingdao

+86-18661807263. E-mail: [email protected].

266061,

China.

Tel.:

Journal Pre-proof Abstract Background: Our previous study showed that human omental adipose-derived stem cells (ADSCs) promote ovarian cancer growth and metastasis. In this study, the role of autophagy in the ovarian cancer-promoting effects of omental ADSCs was further determined. Methods: The growth and invasion of ovarian cancer cells were detected by CCK-8 and Transwell assays, respectively. The autophagy of ovarian cancer cells transfected with MRFP-GFP-LC3 adenoviral vectors was evaluated by confocal microscopy and

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western blot assay. Transfection of STAT3 siRNA was used to inhibit the expression of STAT3.

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Results: Our results show that autophagy plays a vital role in ovarian cancer and is

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promoted by ADSCs. Specifically, we show that proliferation and invasion are correlated with autophagy induction by ADSCs in two ovarian cancer cell lines under

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hypoxic conditions. Mechanistically, ADSCs activate the STAT3 signalling pathway, thereby promoting autophagy. Knockdown of STAT3 expression using siRNA

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of ovarian cancer cells.

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decreased hypoxia- induced autophagy and decreased the proliferation and metastasis

Conclusion: Taken together, our data indicate that STAT3- mediated autophagy

Key words

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induced by ADSCs promotes ovarian cancer growth and metastasis.

Ovarian cancer; adipose mesenchymal stem cells; autophagy; STAT3 signalling pathway

Background Ovarian cancer is one of the most common types of gynaecological malignancy worldwide1 . Metastasis is a complex, multistep process2 . After decades of study, tumour progression and metastasis remain the primary causes of mortality in patients with ovarian cancer. Epithelial ovarian cancer (EOC) most commonly metastasizes to peritoneum, and peritoneal metastasis is associated with a five- year survival rate of

Journal Pre-proof 30%3 . We currently know little about omental metastasis mechanisms in ovarian cancer. Therefore, the study of this topic is crucial for ovarian cancer treatment. Mesenchymal stromal cells, immune cells, the vasculature, and non-cellular components around tumour tissue comprise the tumour microenvironment (TME)4 . In tumour development, the TME plays an important role in the interactions between host components and tumour cells5 . Recent evidence has suggested that the TME significantly contributes to ovarian cancer metastasis6 . As an important TME component, mesenchymal stem cells (MSCs) have been suggested to play important

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roles in many steps throughout metastasis development7 . Identifying the interactions between tumour cells and MSCs may offer insight into potential therapy that can be

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utilized to treat peritoneal metastasis in ovarian cancer.

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Adipose-derived stem cells (ADSCs), an important mesenchymal stem cell type, can be isolated from human lipo-aspirates. Accumulating evidence indicates that

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ADSCs may play a positive role in tumour progression, especially metastasis. MSCs can participate in tumour progression by impacting cancer cell biology or by

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modulating immune status and angiogenic processes; thus, MSCs affect tumour

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progression in a complicated manner, especially tumour metastasis8 . Our previous study found that ADSCs in the omentum can be activated by ovarian cancer cells to

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promote ovarian cancer growth and metastasis by increasing matrix metalloproteinase (MMP) expression9 . However, the mechanisms by which ADSCs promote cancer metastasis remain controversial. Autophagy is a highly conserved self-degradation process and plays an important role in maintaining metabolic sufficiency and survival10 . Due to rapid growth, metabolic changes and hypoxic conditions, tumour cells are more reliant than normal cells on autophagy for survival11 . Autophagy is important in the motility and invasion of metastatic tumour cells in vitro and tumour metastasis in vivo12 . Previous studies have demonstrated that autophagy induction is mediated by interleukin-6 (IL-6) signalling in metabolically stressed cancer cells, and autophagy induction promotes tumour growth and progression13 . In this study, we found that omental ADSCs may induce increased autophagy through the STAT3 pathway to promote ovarian cancer

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Methods

Cell culture Human omental tissues were collected from adult female donors undergoing abdominal surgery to treat benign gynaecologic disease who did not have other diseases9 . All procedures were performed according to the ethical guidelines of the

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Qingdao University Affiliated Hospital in Qingdao, China. Briefly, fresh omentum tissues were collected and cleaned using phosphate-buffered saline (PBS). Omentum

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tissues were cut into small pieces with scissors and incubated with 0.1% collagenase I

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(Sigma, St. Louis, USA) in DMEM/F12 (hyClone, Thermo scientific, USA) for one hour at 37°C. After gentle agitation, the tissues were added to DMEM/F12 containing

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10% foetal bovine serum (FBS, Gibco, Australia) to terminate digestion. After passing through a 100-micron screen filter, the filtrate was centrifuged, and the cells were

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plated on culture plates in DMEM/F12 with 10% FBS. To investigate the effects of

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hypoxia on the autophagy of ovarian cancer cells, we used a low-oxygen incubator with a 0.5% oxygen concentration.

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SKOV3 and A2780 human ovarian cancer cells were purchased from the China Center for Type Culture Collection. These two cell lines were both cultured in DMEM containing 10% FBS. The SKOV3 and A2780 cells had been identified by Short Tandem Repeat profiling assays within 2 months before the experiments and tested for mycoplasma contamination within 2 weeks before the experiments. All cells were cultured in an incubator with 5% CO 2 at 37°C.

ADSC conditioned medium preparation When the ADSCs from omental tissues had grown to 80% confluence (approximately 5×106 cells/10 cm well), the medium was removed, and DMEM/F12 without FBS (10 ml) was added to the cells for another 24 h. For the next experiments, the medium was collected after centrifugation at 1200 × g for 12 min and filtering

Journal Pre-proof through a 0.22- micron screen filter (Millipore, Billerica, MA). To further understand the effect of ADSC CM, the prepared CM was diluted to different concentrations (1 : 5, 10, 100, 1000) in normal growth medium.

CCK-8 cell proliferation assay Three ovarian cancer cell lines were seeded at a density of 5,000 cells/well was in the 96-well plates and cultured. The CCK-8 reagent (Thermo Fisher Scientific, MA, USA) was used to measure ovarian cancer cell proliferation daily. We added the

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CCK-8 reagent to each well, and the cancer cells were cultured for an additional1.5 hours. Then, colourimetric assays were performed by measuring the absorbance (OD

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value) of each well at a wavelength of 450 nm in a microplate reader. The growth

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curves were determined for three independent experiments.

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EOC cell invasion assay

Two lines of ovarian cancer cells (5×105 ) were labelled with Hoechst 33342

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(Invitrogen, Carlsbad, CA) and covered on 24-well Transwell plates (Corning, NY,

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USA). The membranes with 8-μm pores in the 24-well Transwell plates were coated with 50 μL of BD Matrigel™ matrix (1:10 dilution). The lower chamber was filled

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with 600 μL of one of 2 types culture medium: medium containing 10% FBS (control) or medium containing 10% FBS and ADSC conditioned medium (CM). Next, the cancer cells were cultured for 6 h and then fixed in 4% paraformaldehyde. The penetrated cells located on the lower surface of the membrane were retained. The ovarian cancer cells were cultured without FBS for 24 h before the Transwell assays. The number of penetrated cells per high-power field was counted to represent the invasive capability of the ovarian cancer cells. The experiments were performed three times.

Transfection of siRNA SKOV3 or A2780 cells (5×105 ) were cultured in 6-cm plates for 24 h. When the cells reached subconfluence, they were then transfected with a STAT3-specific siRNA

Journal Pre-proof or control scrambled siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The STAT3 siRNA was a mix of 3 target-specific 20- to 25-nucleotide siRNAs designed to knock down gene expression.

Confocal microscopy SKOV3 and A2780 cells were grown to 60%–80% confluence for transfection of MRFP-GFP-LC3 adenoviral vectors. According to the manufacturer’s instructions, MRFP-GFP-LC3 adenoviral vector (HanBio, Shanghai, China) infection was

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performed as previously described14 . Since the green and red fluorescent proteins have different pH stabilities, inside lysosomes, the EGFP fluorescent signal of ovarian

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cancer cells is quenched under acidic conditions (pH < 5), and the mRFP fluorescent

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signal remains under acidic conditions. In merged images, yellow spots in cells show autophagosomes, and increases in both yellow and red spots indicate increased

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autophagic flux. In our study, ovarian cancer cells were treated with adenovirus in growth medium at 37°C for 2 h and then with CM under hypoxic conditions at 37°C

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Real-time PCR

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Tokyo, Japan).

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for 24 h. LC3 spots were counted via fluorescence microscopy (200×, Olympus,

After transfection of STAT3 siRNA, SKOV3 and A2780 cells were collected. Subsequently, total RNA was extracted from transfected ovarian cancer cells using TRIzol reagent (Invitrogen), and the complementary DNA was synthesized using a reverse transcription kit (Toyobo, Osaka, Japan). According to the manufacturer ’s protocols, real-time PCR (qPCR) was performed using an ABI 7500 Sequencing Detection System and SYBR Premix Ex Taq (Takara, Japan). The following primer sequences were used: GAPDH, 5’-ATGGGGAAGGTGAAGGTCG-3’ (forward) and 5’-GGGGTCATTGATGGCAACAATA-3’ 5’-CACCTTGGATTGAGAGTCAAGAC-3’

(reverse); (forward)

STAT3, and

5’-AGGAATCGGCTATATTGCTGGT-3’ (reverse). All reactions were performed in three times.

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Western blotting RIPA buffer (Sigma, St. Louis, USA) was used to lyse ovarian cancer cells on ice for 12 min. To remove cell debris, the cell lysates were centrifuged at 12,000 g. Protein sample mixtures were treated with LDS Sample Buffer and separated by SDS-PAGE. Then, proteins were transferred from the gel to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA), and the membranes were blocked with 5% skim milk. The membranes were incubated with primary rabbit monoclonal

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antibodies against human STAT3, pSTAT3 (1:1000 dilution; R&D Systems), LC-3, p62 and β-actin (1:1000 dilution; CST, Danvers, MA). The membranes were then

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incubated with peroxidase-conjugated AffiniPure secondary IgG antibodies (H+L) (1:1000; CST). In a chemiluminescence detection system, protein-antibody complexes

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were detected and then quantitated using Image Lab™ version 5.1 software (Bio-Rad,

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Hercules, CA).

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Enzyme-linked immunosorbent assay

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Ovarian cancer cell lines (0.2×105 cells per well) were cultured in six-well plates overnight with culture medium containing 10% FBS. The cancer cell supernatants

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were then placed in fresh culture medium without FBS and cocultured indirectly with ADSCs in transwell plates with 0.4-μm pores for 2, 3 and 4 d. Then, IL-1 and IL-6 expression in the supernatant was measured via enzyme- linked immunosorbent assay (ELISA) (Raybiotech, GA, USA), according to the manufacturer’s instructions.

Statistical Analysis All data are expressed as the mean ± standard deviation from at least three independent experiments. Statistical analysis was performed with a two-tailed Student’s t-test or one-way analysis of variance. A P value <0.05 was considered to indicate significance. A P value < 0.05 indicated that a difference was statistically significant.

Journal Pre-proof Results ADSCs promote EOC cell growth and invasion under hypoxic conditions To understand the ovarian cancer promotion effect of ADSCs during hypoxia, we tested the effects of ADSC CM on the growth and invasion of ovarian cancer cells using CCK-8 and Transwell assays (Fig. 1A-B). Cell proliferation activity increased significantly after treatment with ADSC CM under hypoxic conditions (both P<0.001). Similarly, the number of penetrated SKOV3 and A2780 cells increased approximately

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6-fold after treatment with ADSC CM under hypoxic conditions.

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ADSCs induce autophagy in EOC cells

Many studies have shown that autophagy is an important part of tumour

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progression; however, the involvement of autophagy in the interrelation between

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ADSCs and ovarian cancer cells has not been tested. Thus, the SKOV3 and A2780 ovarian cancer cells were treated with ADSC CM for 24 h under hypoxic conditions, and LC3-II expression levels were assessed by western blot analysis. LC3 is a

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microtubule-associated structural protein, and LC3-II-enriched lysate is associated

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with autophagosomes, which are required for autophagy15 . The expression of LC3-II

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in ovarian cancer cells treated with ADSC CM increased significantly under hypoxic conditions (Fig. 1C). To further confirm the effects of ADSC CM on the autophagy of ovarian cancer cells under hypoxia, we examined p-STAT3, STAT3, LC3-II and P62 expression levels in ovarian cancer cells treated with an ADSC CM concentration gradient (Fig. S1). The results showed that the ADSC CM could activate STAT3 phosphorylation at a 1 : 1000 dilution and increase LC3-II expression significantly at a 1 : 100 dilution. Since the increase of in LC3-II levels in ovarian cancer cells after ADSC CM treatment indicates either blockage of autophagy or increased autophagy flux, we examined the LC3 and pSTAT3 expression in the presence or absence of leupeptin, a membrane-permeable thiol protease inhibitor that blocks autophagy and mediates LC3-II accumulation. In ovarian cancer cells, leupeptin administration increased

Journal Pre-proof ADSC-induced LC3-II levels, which confirmed that autophagy flux increased after ADSC treatment (Fig. 1D). To further confirm the role of ADSCs in inducing EOC cell autophagy under hypoxic conditions, we used direct fluorescence to monitor LC3 spot formation as an index for autophagosome accumulation in ovarian cancer cells. We transduced SKOV3 and A2780 cells with MRFP-GFP-LC3 adenoviral vectors with or without CM treatment. A punctate LC3 pattern was detected in ADSC CM-treated cancer cells but not in untreated cells (Fig. 1E). These data show that ADSC treatment activates an

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autophagic response in human ovarian cancer cells, which may promote ovarian

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cancer cell growth and invasion.

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ADSCs promote ovarian cancer cell autophagy through activation of STAT3 pathways

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Emerging evidence suggests that the STAT3 signalling pathway may regulate autophagy16,17 . To understand whether STAT3 mediates ADSC-induced autophagy, we

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tested the effect of ADSC CM treatment on STAT3 activation. SKOV3 and A2780

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cells were cultured in CM for 24 h, and IL-1 and IL-6 concentrations were assessed by enzyme- linked immunosorbent assays (ELISAs). IL-1 and IL-6 levels were

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significantly increased in the supernatants of ADSCs treated with SKOV3 and A2780 CM (Fig. 2A). Activation of the STAT3 pathway was then determined by western blotting and qPCR. ADSC CM treatment increased STAT3 expression and phosphorylation, confirming that ADSCs activate STAT3 pathways (Fig. 2B-C). Next, we determined whether STAT3 activation was responsible for ADSC- induced autophagy. SKOV3 and A2780 cells were left untreated or were treated with ADSC CM in the presence or absence of the autophagy inhibitor 3-MA (10 mM) for 24 h, and LC3-II expression levels and STAT3 pathway activation were examined by western blotting. As shown in Fig. 2B, ADSC CM treatment increased the STAT3 level in ovarian cancer cells, and this increase was not inhibited by 3-MA. Furthermore, the increased level of LC3-II induced by ADSC CM was inhibited by 3-MA. To further understand the mechanism of autophagy regulation by ADSCs, we

Journal Pre-proof knocked down ATG5 and BECN1 expression (autophagy-associated proteins) with specific siRNA, and LC3-II expression levels and STAT3 pathway activation were examined (Fig. S2A-B). ATG5 and BECN1 knockdown could attenuate the autophagy induction effect of ADSCs, which indicated that ADSCs increased LC3-II levels by increasing autophagy flux. We analysed ovarian cancer cell proliferation using a CCK8 assay and found significantly increased ovarian cancer cell proliferation after ADSC CM treatment, and this increase was reduced when the cells were treated with 3-MA (Fig. 2D). We

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also tested the invasion of ovarian cancer cells using a Transwell assay and found that ADSC CM increased cancer cell invasion, but this effect was inhibited by 3-MA (Fig.

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2E). Since autophagy is considered a cell survival mechanism, these data indicate that

of

STAT3

decreases

ADSC-mediated autophagy

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Knockdown

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ADSC-mediated autophagy may enhance ovarian cancer cell survival and invasion.

and

the

tumour-promoting effect of ADSCs

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To understand the direct role of the STAT3 pathway in autophagy induction in

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ovarian cancer cells, the effects of STAT3 knockdown on LC3-I/II and STAT3 expression were examined by western blotting and qRT-PCR. SKOV3 and A2780

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cells were transfected with nontarget (siControl) or STAT3 siRNAs (siSTAT3), and the total STAT3 expression level in ovarian cancer cells transfected with STAT3 siRNA was significantly decreased compared with that in cells transfected with siControl (Fig. 3A-B). As expected, ovarian cancer cells transfected with siControl cultured in ADSC CM expressed a higher level of LC3-II. By contrast, knockdown of STAT3 by transfection of siRNA significantly decreased the expression of LC3-II in ovarian cancer cells but increased p62 (Fig. 3A). To further confirm the role of STAT3 in ADSC-mediated ovarian cancer cell autophagy, we used fluorescence to monitor LC3 puncta formation. A punctate LC3 pattern was observed in ADSC CM-treated cancer cells transfected with siControl (Fig. 3C). These assays suggest that knockdown of STAT3 expression in ovarian cancer cells reduces ADSC- mediated autophagy.

Journal Pre-proof To confirm the effect of autophagy reduction induced by STAT3 knockdown on ovarian cancer cell growth and invasion, SKOV3 and A2780 cells were transfected with STAT3 siRNA or siControl. CCK8 and Transwell assays were used to test ovarian cancer cell growth and invasion. Fig. 3D shows that STAT3 knockdown significantly decreased ADSC-induced growth. A similar inhibition effect on invasion is shown in Fig. 3E.

Pharmacological inhibition of autophagy decreases the tumour-promoting effect

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of ADSCs without affecting STAT3

To understand the role of autophagy in the tumour-promoting effect of ADSCs,

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we evaluated the effect of pharmacological autophagy inhibition on ovarian cancer

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cells cultured in ADSC CM. A pharmacological inhibitor of class III-PI3K, 3-MA, which acts at the pre-autophagosome formation level, was used to inhibit autophagy

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in ovarian cancer cells. Specifically, SKOV3 and A2780 cells were cultured in normal growth medium or ADSC CM for 24 h in the presence or absence of 3-MA, and the

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levels of LC3-II and STAT3 were determined by western blotting. The results show

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that 3-MA significantly decreased ADSC-induced LC3-II expression in ovarian cancer cells compared with that in untreated cells (Fig. 4A). In addition, 3-MA did not

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affect ADSC-induced STAT3 activation (Fig. 4A), suggesting that pharmacological inhibition of autophagy inhibits the ADSC-induced tumour-promoting effect without regulating the STAT3 signalling pathway.

STAT3 activation increases ovarian cancer cell autophagy, viability, and invasion To examine if STAT3 activation increases ovarian cancer cell autophagy, viability, and invasion, we transfected ovarian cancer cells with a STAT3 plasmid (GeneChem, Shanghai, China) to overexpress STAT3, and we examined STAT3 mRNA and protein expression in trophoblasts (Fig. 4B-C, P<0.001). A CCK-8 assay indicated that the viability of cells was significantly increased by STAT3 plasmid transfection compared to empty control vector transfection (Fig. 4D, P<0.001). Consistent with the CCK-8 assay results, the transwell assay results suggested that

Journal Pre-proof STAT3-overexpressing ovarian cancer cells showed higher invasion ability than control cancer cells, as determined by the number of penetrated cells (Fig. 4E, P<0.001). These results suggest that ADSCs contribute to ovarian cancer cell growth and invasion by modulating autophagy through the STAT3 signalling pathway.

Discussion Tumour progression depends on the interaction between tumour cells and cells in

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the surrounding microenvironment18 . Adipose tissue is a complex organ with metabolic, immune regulatory and endocrine functions. ADSCs are widely present in

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adipose tissue, secreting many growth factors to regulate angiogenesis and cell

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differentiation19,20 . Our previous study found that ADSCs in the omentum can promote ovarian cancer cell growth and invasion and may play a role in formation of

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the TME. However, the underlying mechanisms of the ovarian cancer-promoting effects of ADSCs remain unclear9 . In the present study, noticeable autophagy was

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observed after exposing ovarian cancer cells to ADSC CM. Therefore, we tested the

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function of the STAT3 signalling pathway in the interaction between ADSCs and autophagy in ovarian cancer cells. Our research suggests that ADSCs can induce

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ovarian cancer cell autophagy through activation of the STAT3 signalling pathway to promote tumour cell growth and invasion. Autophagy can help cells preserve metabolic energy and survive under nutrient stress by recycling metabolites, such as fatty acids, amino acids, and nucleotides 21 . Autophagy has been thought to serve as a tumour-suppressive mechanism under physiological conditions, which can help remove and mitigate harmful stresses, such as oxidative stress, genomic instability and inflammation22 . However, advanced tumours are constantly exposed to mutations, radiation, chemotherapy and anoxic-ischaemia, and autophagy may act as an important strategy for survival23 . Although the exact role of autophagy is debatable in different tumo ur models, crosstalk occurs between autophagy and the TME11 . Various studies have shown the role of autophagy in the interaction between

Journal Pre-proof tumour cells and the TME, where stromal cells promote tumour cell survival and metastasis by regulating tumour cell autophagy24-27 . For example, autophagy is implicated in the regulation of inflammatory responses in the TME through fibroblasts and macrophages28,29 . Moreover, autophagy controls the differentiation of fibroblasts into cancer-associated fibroblasts, which is a crucial event in the immune response in the TME30 . In our study, ADSC CM treatment increased autophagy in EOC cells, elevating proliferation and invasion ability under hypoxic conditions, suggesting that the paracrine action of ADSCs in the TME regulates autophagy in

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tumour cells. However, the molecular mechanisms involved in these processes require further study.

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Constitutive STAT3 activation in cancer tissues plays an important role in cancer

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development31 . The STAT3 signalling pathway and its downstream effector molecules, such as Bcl-2, have been found to participate in autophagy induction in ovarian cancer

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progression32 . Consistent with previous studies, our results showed that IL-6 secreted by ADSCs activated STAT3 expression and autophagy in ovarian cancer cells.

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Furthermore, the knockdown of STAT3 weakened the promoting effect of ADSC CM

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on autophagy in ovarian cancer cells, resulting in decreased cancer cell growth and

Conclusions

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metastasis.

Taken together, the results presented here show that the tumour-promoting effects of ADSCs are mainly mediated by paracrine regulation of autophagy in ovarian cancer cells through the STAT3 signalling pathway. However, our knowledge of how ADSCs in the TME influence ovarian cancer metastasis and progression remains limited. Cell-cell interactions between stromal cells and tumour cells potentially play important roles in tumour progression and should be a focus of further oncology research.

List of Abbreviations ADSCs : Adipose-derived mesenchymal stem cells; EOC: Epithelial ovarian cancer; TME: Tumour

Journal Pre-proof microenvironment; MSCs: Mesenchymal stem cells; MMPs: Matrix metalloproteinases; IL-6: Interleukin-6; PBS: Phosphate-buffered saline; FBS: Foetal bovine serum; CM: Conditioned medium.

Declarations

Ethics approval and consent to participate

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Hospital. All participants provided written informed consent.

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The study was approved by the Ethics Committee of the Qingdao University Affiliated

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Consent for publication

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Availability of data and materials

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Not applicable.

The datasets used and/or analysed during the current study are available from the

Competing interests

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corresponding author on reasonable request.

Funding

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The authors declare no potential conflicts of interest.

This work was supported by grants from Qingdao postdoctoral application research project, Youth fund Project and Clinical medicine + X Project of the Affiliated Hospital of Qingdao University. All funders had no role in the design of the study, collection, analysis, and interpretation of the data, or in writing the manuscript.

Authors’ contributions

Study conception and design: Chu, Peng, Wang; Acquisition of data: Liu M, Li K, Peng, Li Y, Hu; Analysis and interpretation of data: Zuo, Liu C, Zhou; Drafting of manuscript: Chu, Wang; Critical revision: Zhang, Peng, Zuo

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Acknowledgements We thank the Department of Obstetrics and Gynecology, the Affiliated Hospital of Qingdao University, Qingdao, China, for providing tissue samples.

References 1.

Siegel, R.L., Miller, K.D. & Jemal, A. Cancer Statistics, 2017. CA: a cancer journal for Stuelten, C.H., Parent, C.A. & Montell, D.J. Cell mot ility in cancer invasion and metastasis:

oo

2.

f

clinicians 67, 7-30 (2017). insights from simple model organisms. Nature reviews. Cancer 18, 296-312 (2018). 3.

Nakayama, K., Nakayama, N., Katagiri, H. & M iyazaki, K. Mechanisms of ovarian cancer

pr

metastasis: biochemical pathways. International journal of molecular sciences 13, 11705-11717 (2012).

Lopez-Soto, A., Gonzalez, S., Smyth, M.J. & Gallu zzi, L. Control of Metastasis by NK Cells.

e-

4.

Cancer cell 32, 135-154 (2017).

Sleeboom, J.J.F., Eslami A mirabadi, H., Nair, P., Sah lgren, C.M . & den Toonder, J.M.J.

Pr

5.

Metastasis in context: modeling the tumo r microenviron ment with cancer-on-a-chip approaches. Disease models & mechanisms 11(2018). 6.

Luo, Z., et al. Tumor microenviron ment: The culprit fo r ovarian cancer metastasis? Cancer Ridge, S.M., Su llivan, F.J. & Glynn, S.A. Mesenchymal stem cells: key players in cancer

rn

7.

al

letters 377, 174-182 (2016).

progression. Mol Cancer 16, 31 (2017). 8.

McDonald, M.M., Fairfield, H., Falank, C. & Reagan, M.R. Adipose, Bone, and Myeloma:

9.

Jo u

Contributions from the Microenvironment. Calcified tissue international 100, 433-448 (2017). Chu, Y., et al. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer. Experimental cell research 337, 16-27 (2015). 10.

Go mes, L.R., Vessoni, A.T. & Menck, C.F. Microenvironment and autophagy cross -talk: Implications in cancer therapy. Pharmacological research 107, 300-307 (2016).

11.

Mowers, E.E., Sharifi, M.N. & Macleod, K.F. Functions of autophagy in the tu mor microenvironment and cancer metastasis. The FEBS journal (2018).

12.

Sharifi, M .N., et al. Autophagy Promotes Focal Adhesion Disassembly and Cell Motility of Metastatic Tumor Cells through the Direct Interaction of Paxillin with LC3. Cell reports 15, 1660-1672 (2016).

13.

Katheder, N.S., et al. Microenviron mental autophagy promotes tumour growth. Nature 541, 417-420 (2017).

14.

Yu, T., et al. Fusobacterium nucleatum Pro motes Chemoresistance to Colorectal Cancer by Modulating Autophagy. Cell 170, 548-563.e516 (2017).

15.

Levy, J.M.M., Towers, C.G. & Thorburn, A. Targeting autophagy in cancer. Nature reviews. Cancer 17, 528-542 (2017).

16.

Jingjing Gong, Amanda R. Muñoz, Dan iel Chan, Rita Ghosh & Ku mar, A.P. STAT3 down

Journal Pre-proof regulates LC3 to inhib it autophagy and pancreatic cancer cell growth. oncotarget 5, 2529-2541 (2014). 17.

Hu, F., et al. Docetaxel-mediated autophagy promotes chemoresistance in castration -resistant prostate cancer cells by inhibiting STAT3. Cancer letters 416, 24-30 (2018).

18.

Cao, Y., Luo, X., Ding, X., Cui, S. & Guo, C. LncRNA ATB pro motes proliferat ion and metastasis in A549 cells by down-regulation of microRNA-494. J Cell Biochem (2018).

19.

Yeh, W.L., Tsai, C.F. & Chen, D.R. Peri-foci adipose-derived stem cells pro mote chemoresistance in breast cancer. Stem cell research & therapy 8, 177 (2017).

20.

Rezaeeyan, H., Shirzad, R., McKee, T.D. & Saki, N. Role of chemo kines in metastatic niche: new insights along with a diagnostic and prognostic approach. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 126, 359-370 (2018).

21.

Guo, J.Y., Xia, B. & White, E. Autophagy-mediated tu mor p ro motion. Cell 155, 1216-1219 Janji B, V.E., Moussay E, Paggetti J, Arakelian T, Mgrditchian T, Messai Y, No man MZ, Van

oo

22.

f

(2013).

Moer K, Has mim M, Mami-Chouaib F, Berchem G, Chouaib S. The mult ifaceted role of

pr

autophagy in tumor evasion from immune surveillance. Oncotarget 2016.2.20, Epub ahead of print (2016). 23.

Maycotte, P., et al. STAT3-mediated autophagy dependence identifies subtypes of breast

e-

cancer where autophagy inhibition can be efficacious. Cancer research 74, 2579-2590 (2014). 24.

Dower, C.M., Wills, C.A., Frisch, S.M. & Wang, H.G. Mechanisms and context u nderlying

25.

Pr

the role of autophagy in cancer metastasis. Autophagy, 1-19 (2018). Vera-Ramirez, L., Vodnala, S.K., Nini, R., Hunter, K.W. & Green, J.E. Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence. Nature 26.

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communications 9, 1944 (2018).

Sousa CM, B.D., Wang X,Halb rook CJ,Sherman M H,Zhang

L,Kremer D,Hwang

rn

RF,Wit kiewicz AK,Ying H,Asara JM. Pancreat ic stellate cells support tumour metabolis m through autophagic alanine secretion. Nature 536, 479-483 (2016). Pavlides, S., et al. Warburg meets autophagy: cancer-associated fibroblasts accelerate tu mor

Jo u

27.

growth and metastasis via o xidative stress, mitophagy, and aerobic glycolysis. Antioxidants & redox signaling 16, 1264-1284 (2012). 28.

Ko mohara, Y. & Takeya, M. CAFs and TAMs: maestros of the tumour microenviron ment. The Journal of pathology 241, 313-315 (2017).

29.

Ngabire, D. & Kim, G.D. Autophagy and Inflammatory Response in the Tu mor Microenvironment. International journal of molecular sciences 18(2017).

30.

Ishii, G., Ochiai, A. & Neri, S. Phenotypic and functional heterogeneity of cancer-associated fibroblast within the tumor microenviron ment. Advanced drug delivery reviews 99, 186-196 (2016).

31.

Mali, S.B. Rev iew of STAT3 (Signal Transducers and Activato rs of Transcription) in head and neck cancer. Oral oncology 51, 565-569 (2015).

32.

Zhu, L., et al. Autophagy is a pro-survival mechanism in ovarian cancer against the apoptotic effects of euxanthone. Biomedicine & pharmacotherapy = Biomedecine & pharmacot herapie 103, 708-718 (2018).

Figure legends

Journal Pre-proof Figure 1. ADSCs promote EOC cell prolife ration, invasion and autophagy under hypoxic conditions (A): The effects of ADSCs on ovarian cancer cell proliferation under hypoxic conditions were evaluated with a CCK8 assay. Ovarian cancer cells were treated with ADSC CM, and the cell densities of both groups, measured at 450 nm, were determined and analysed. The data were obtained from three separate experiments. (B): The ovarian cancer cells that migrated through the 8-μm Transwell membrane pores were counted to determine the changes in ovarian cancer cell invasiveness after

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treatment with ADSC CM. (C): LC3 and P62 expression in ovarian cancer cells treated with ADSCs under normoxia and hypoxia was examined by western blotting.

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(D): LC3 and pSTAT3 expression in ovarian cancer cells treated with ADSCs or

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leupeptin (100μM) was showed by western blotting. (E): Representative fluorescence images of mRFP-LC3 (autolysosomes) and merged RFP-GFP-LC3 (autophagosomes).

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SKOV3 and A2780 cells were transduced with MRFP-GFP-LC3 adenoviral vectors and exposed or not exposed to ADSC CM under normoxic and hypoxic conditions for

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24 h. Image analysis of yellow puncta (autophagosomes) for three independent

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experiments were showed. *** P<0.001.

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Figure 2. ADSCs activate the STAT3 signalling pathway, which promotes

(A): ELISA results of IL-1 and IL-6 in ovarian cancer cell supernatants treated with or without ADSC CM and in ADSC CM. Supernatants from ADSC and ovarian cancer cell cultured alone were used as controls. (B): Western blotting analysis of LC3, STAT3 and pSTAT3 expression in ovarian cancer cells under hypoxic conditions. Ovarian cancer cells were treated with ADSC CM with or without the autophagy inhibitor 3-MA (10 mM) for 24 h. Ovarian cancer cells cultured alone under hypoxic or normal conditions served as negative and blank controls, respectively. (C): qPCR analysis of STAT3 mRNA expression in ovarian cancer cells under hypoxic conditions. Ovarian cancer cells were treated with ADSC CM with or without the autophagy inhibitor 3-MA (10 mM) for 24 h. (D-E): Cell proliferation was evaluated by the

Journal Pre-proof CCK8 assay. The invasion of ovarian cancer cells was determined by Transwell assays. Ovarian cancer cells were treated with 3-MA and ADSC CM under hypoxic conditions. Ovarian cancer cells treated with ADSC CM alone served as the positive control, and ovarian cancer cells cultured alone served as the negative control. * P<0.05; ** P<0.01; *** P<0.001.

Figure 3. The knockdown of STAT3 by siRNA decreases ADSC-induced autophagy while decreasing the tumour-promoting effect of ADSCs

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(A): Ovarian cancer cells were pre-treated with STAT3 siRNA (50 nM) for 48 h before exposure to ADSC CM for another 12 h. LC3, STAT3 and p62 expression

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levels were detected by western blotting. Ovarian cancer cells treated with siControl

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or cultured alone served as negative controls. (B): The relative STAT3 mRNA expression levels in ovarian cancer cells were determined by qPCR. (C):

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Representative fluorescence images of ovarian cancer cells transduced with MRFP-GFP-LC3 adenoviral vectors to monitor autophagic flux. (D-E): Cell

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** P<0.01; *** P<0.001.

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proliferation and invasion were evaluated by CCK8 and Transwell assays. * P<0.05;

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Figure 4. Autophagy inhibition decreases the tumour-promoting effect of ADSCs without STAT3 regulation. (A): SKOV3 and A2780 cells were left untreated or were treated with ADSC CM in the presence or absence of 3-MA for 24 h. LC3, STAT3 and p62 expression levels were detected by western blotting. Ovarian cancer cells cultured alone served as the negative control. (B): Ovarian cancer cells were transfected with a STAT3 overexpression plasmid, and STAT3 mRNA expression was tested by qPCR. Empty vector (Vec) cells served as controls. (C): The expression of STAT3, P62 and LC3 in ovarian cancer cells transfected with the STAT3 plasmid and treated with or without ADSC CM under hypoxia was tested by western blotting. (D-E): Cell proliferation and invasion were evaluated by CCK-8 and transwell assays. Two ovarian cancer cell lines were transfected with the STAT3 overexpression plasmid and then treated with

Journal Pre-proof or without ADSC CM under hypoxic conditions. Cancer cells transfected with the vector served as a control. * P<0.05, ** P<0.01, *** P<0.001.

Figure S1. ADSC-mediated autophagy induction and STAT3 activation in ovarian cancer cells were dose dependent. Western blot analysis shows exogenous ADSC CM at 10-fold serial dilution (1 × 10~ (-3)) induces STAT3, p-STAT3, LC3 and P62 expression in SKOV3 and A2780 cells.

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Figure S2. BECN1 and ATG5 knockdown by siRNA decreases ADSC-induced autophagy.

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(A): SKOV3 and A2780 cells were transfected with BECN1-specific siRNA and

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treated with ADSC CM under hypoxic conditions. LC-3, STAT3, pSTAT3 and BECN1 expression was detected by western blot analysis. Cells cultured in normoxic

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conditions and treated with or without ADSC CM were used as controls. (B): SKOV3 and A2780 cells were transfected with ATG5-specific siRNA and treated with ADSC

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CM under hypoxic conditions. LC-3, STAT3, pSTAT3 and ATG5 expression was

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detected by western blot analysis. Cells cultured in normoxic conditions and treated

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with or without ADSC CM were used as controls.

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Journal Pre-proof Authors’ contributions

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Study conception and design: Chu, Peng, Wang Acquisition of data: Liu M, Li K, Peng, Li Y, Hu Analysis and interpretation of data: Zuo, Liu C, Zhou, Drafting of manuscript: Chu, Wang Critical revision: Zhang, Peng, Zuo

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Highlights In the present study, we demonstrated that adipose derived stem cells from normal human omentum enhanced autophagy in ovarian carcinoma cells with elevated growth and metastasis properties, which may be at least partially due to the activation of STAT3.

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These findings provide a novel insight into the mechanism how the

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adipose derived stem cells fuels the spread of ovarian carcinoma.

Journal Pre-proof Abstract Background: Our previous study showed that human omental adipose-derived stem cells (ADSCs) promote ovarian cancer growth and metastasis. In this study, the role of autophagy in the ovarian cancer-promoting effects of omental ADSCs was further determined. Methods: The growth and invasion of ovarian cancer cells were detected by CCK-8 and Transwell assays, respectively. The autophagy of ovarian cancer cells transfected with MRFP-GFP-LC3 adenoviral vectors was evaluated by confocal microscopy and

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western blot assay. Transfection of STAT3 siRNA was used to inhibit the expression of STAT3.

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Results: Our results show that autophagy plays a vital role in ovarian cancer and is

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promoted by ADSCs. Specifically, we show that proliferation and invasion are correlated with autophagy induction by ADSCs in two ovarian cancer cell lines under

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hypoxic conditions. Mechanistically, ADSCs activate the STAT3 signalling pathway, thereby promoting autophagy. Knockdown of STAT3 expression using siRNA

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of ovarian cancer cells.

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decreased hypoxia- induced autophagy and decreased the proliferation and metastasis

Conclusion: Taken together, our data indicate that STAT3- mediated autophagy

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induced by ADSCs promotes ovarian cancer growth and metastasis.