1. Introduction
Ovarian malignancies can occur at any age and have a wide range of histological types, especially epithelial ovarian cancer (EOC). EOC is the most common cause of cancer-related death among all gynecological malignancies. EOC patients often present at an advanced stage with peritoneal dissemination and extensive metastases and are commonly complicated with ascites [
1]. More than 60% of cases are detected after cancer has spread into the abdomen. Previous studies have emphasized that the omentum is one of the most common regions of metastasis in EOC, and 80% of serous ovarian cancers are commonly found at the site of the omentum [
2]. However, the molecular mechanisms underlying this lipophilic metastatic characteristic remain poorly understood. In view of the rapid progression, early metastasis, and low survival rate in EOC, it is of great significance to clarify the molecular mechanisms of invasion and metastasis and identify new targets in the treatment of ovarian cancer.
The omentum, a large fat pad that serves as a rich source of adipocytes and adipose-derived mesenchymal stem cells (ADSCs), is historically known as the “policeman of the abdomen” [
3]. It is widely accepted that the omentum is involved in the immune response and fluid exchange of the human abdominal cavity [
4]. In addition, it is becoming increasingly apparent that the omentum is directly involved in the development of a series of cancers, including ovarian cancer, gastric cancer, and pancreatic cancer [
5,
6,
7]. Previous research has suggested that adipocytes provide energy for the rapid growth of ovarian cancer and enhance tumor cell metastasis [
2]. Mesenchymal stem cells (MSCs) are involved in the development of malignant tumors [
8]. The omentum is the most common metastatic site of ovarian cancer, and ADSCs are in situ MSCs. However, it seems that the specific relationships between ADSCs and ovarian cancer cells remain unknown [
9,
10]. Previous studies have reported that ADSCs can promote tumor progression and ovarian cancer metastasis by regulating several regulatory factors, such as MMPs, STAT3, and TMSB4X [
11,
12,
13]. For example, the study reported by Chu Y et al. showed that human omental adipose-derived MSCs (HO-ADSCs) enhance autophagy in ovarian carcinoma cells by activating the STAT3 signaling pathway [
12]. Evidence has shown that ADSCs exhibit an inhibitory influence on ovarian cancer aggressiveness by targeting different paracrine molecules [
14,
15]. Accordingly, it is essential to research the mechanisms underlying the contradictory roles associated with HO-ADSCs in EOC tumorigenesis and progression.
Exosomes are a special class of extracellular enclosed vesicles with small membranes secreted by various cell types [
16]. These exosomes can transfer proteins, lipids, and nucleic acids into different recipient cancer cells, thus affecting their malignant biological behavior [
17]. Xiaoli Rong revealed that human bone marrow MSC-derived exosomes (MSC-exosomes) ameliorate liver fibrosis via inhibition of hepatic stellate cell activation [
18]. Olga Kersy showed that omental tissue-derived exosomes enhanced gastric cancer growth and invasion [
19]. However, analysis concerning the involvement of exosomes secreted by ADSCs (ADSCs-exosomes) in the tumorigenesis and metastasis of ovarian cancer is limited. To the best of our knowledge, there are no reports regarding the impact of ADSC-exosomes on the interaction between the omentum and ovarian cancer cells to date.
In this paper, we explored the functional role of HO-ADSCs and their underlying mechanism in EOC growth and metastasis in vivo and in vitro. We demonstrated that HO-ADSCs promote EOC growth and metastasis through the exosome-mediated FOXM1 signaling pathway. We inferred that HO-ADSC exosomes may be secreted into ascites and are involved in ovarian cancer growth and metastasis, and these properties have potential clinical applications.
2. Materials and Methods
2.1. Omental Adipose Tissue and Ascites Specimens
Written informed consent was acquired from all participants pathologically diagnosed with ovarian cancer before inclusion in this study. None of the patients had received preoperative chemotherapy, radiotherapy, or other therapies prior to the surgical treatment. The protocol of this study was approved by the Ethics Committee of Qilu Hospital of Shandong University (approval number KYLL-202111-134). Accordingly, we followed the ethical principles for medical research involving human subjects as outlined in the Helsinki Declaration. The omental adipose tissue samples and ascites specimens were freshly collected from four middle-aged women aged who underwent surgical treatment for the first time and were diagnosed with advanced ovarian cancer with omentum metastasis at the Department of Obstetrics and Gynaecology, Qilu Hospital of Shandong University. Omental adipose tissue was directly processed for primary ADSC isolation, and the fresh ascites were centrifuged to remove cell components for further cell function studies.
2.2. ADSC Culture, Isolation, and Identification
ADSCs were isolated from omental adipose tissue samples by the explant method based on a protocol described previously [
20]. The tissue samples from the patients were washed, minced, further digested with collagenase, cleaned, centrifuged, resuspended, and cultured in DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 20% fetal bovine serum (FBS, Gibco BRL, Gaithersburg, MD, USA. The isolated cells were cultured in a common incubator at 37 °C with 5% CO
2 and 95% air, and cells from passages 3 to 5 were used in this study. Stem cell multilineage differentiation assays were used to assess the differentiation of HO-ADSCs by Oli Red O, Alizarin Red, and Alcian Blue staining. Fluorescein isothiocyanate-conjugated CD73, CD90, CD105, CD11, CD19, CD34, CD45, and HLA-DR antibodies (Proteintech, Wuhan, China)were employed to assess the phenotypic purity of HO-ADSCs by flow cytometry.
2.3. Coculture of Ovarian Cancer Cells and HO-ADSCs
EOC cell lines (SKOV3, Hey, A2780, and HO8910) were maintained in DMEM or McCoy’s 5A medium containing 10% FBS as previously described [
21]. EOC cell lines were cocultured with HO-ADSCs using Transwell chambers (0.4-μm pore size, Corning, USA) as previously described [
22]. HO-ADSCs and EOC cell lines were suspended at a density of 4 × 106 cells/mL and seeded together in plates or inserts with a 6-well transwell chamber. Subsequently, the medium was replaced half a day to ensure sufficient nutrients and HO-ADSCs were cocultured with EOC cell lines for 24 h. The single-cultured EOC cell lines were used as the control. After incubation for 24 h, ovarian cancer cells were collected and subjected to functional analysis and mechanistic studies.
2.4. EdU Staining Assay
Cell proliferation was measured using an EdU Cell Proliferation Kit (RiboBio, Guangzhou, China) according to the protocol we previously reported [
23]. Briefly, ovarian cancer cells were seeded into 24-well plates and treated with proper interventions for 24 h. Afterward, the cells were incubated with EdU reagents for 2 h and then fixed with paraformaldehyde for 10 min. Finally, the nuclei were stained with Hoechst 3342, and the EdU-positive cells in different groups were counted under a microscope in 5 random fields. Each of the above experiments was performed at least thrice.
2.5. Transwell Migration Assay
Cell migration assays were performed using a Transwell chamber (8.0 μm pore size, Corning, Glendale, AZ, USA) according to the manufacturer’s instructions. Briefly, EOC cell lines were placed into the upper chamber with an FBS-free medium, and the bottom chamber routinely contained a complete medium. After incubation at 37 °C for 24 h, cells in the upper chamber were fixed with paraformaldehyde and stained with crystal violet. Then, the cells that did not migrate were carefully removed from the upper surface of the chamber, and the cells present on the transmembrane located on the underside of the chamber were processed for further analysis. Finally, the stained cells were randomly imaged and manually counted using an inverted microscope. Each of the above experiments was performed at least thrice.
2.6. Matrigel Invasion Assay
Transwell chambers (8 µm pore size, Corning, Glendale, AZ, USA) coated with Matrigel were utilized to measure invasive cell ability, as previously reported, with some modifications. In brief, Matrigel was first added to the upper chamber (BD Biosciences, San Diego, CA, USA). Then, EOC cell lines were seeded in the inserts with serum-free medium, whereas complete medium was added into the lower chamber. After 24 h of incubation in an incubator, human EOC cell lines in the upper chamber were fixed with paraformaldehyde and stained with crystal violet, and then the cells that did not exhibit invasive properties were carefully removed from the upper surface of the chamber using a cotton swab. Subsequently, the cells that had passed through the membrane were randomly counted using an inverted microscope. Each of the above experiments was performed at least thrice.
2.7. ADSC-Exosome Extraction, Characterization, Labeling, and Uptake
To extract exosomes from HO-ADSC supernatants, cells were initially incubated with an exosome-free medium for 48 h. Then, the ADSC exosomes were isolated and purified from the cell supernatants by gradient ultracentrifugation according to a standard protocol. Briefly, the culture medium supernatant of HO-ADSCs was collected and centrifuged at 1500× g for 10 min and 10,000× g for 30 min at 4 °C to remove the cells and debris. The processed supernatant was further centrifuged at 120,000× g for 90 min at 4 °C to obtain the final exosomal pellet. The exosome protein concentration was quantified by employing a BCA Protein Assay Kit (Beyotime, China), and Western blotting was used to identify exosome-specific markers (CD63, TSG101, and HSP70). The morphology and size of exosomes were observed using a transmission electron microscope (JEOL, AICHI KEN, Japan). To detect the cellular uptake and internalization of exosomes, exosomes were labeled with PKH67 dye for 15 min and then incubated with EOC cell lines for 12 h at 37 °C. After incubation, cell nuclei were stained with DAPI for 5 min and subsequently visualized using a fluorescence microscope. The exosome pellets were resuspended in PBS and stored in a −80 °C refrigerator.
2.8. RNA Sequencing and Analysis
For RNA sequencing, total RNA from three paired HEY cells with or without HO-ADSC-exosome treatment was extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). After using the Agilent 2100 RNA nano 6000 assay kit (Agilent, San Diego, CA, USA) to analyze the quality and concentration of RNA samples, the mRNA was enriched by magnetic beads attached to oligos. Then, the first strand and the second strand of cDNA were synthesized and purified using a QIAquick PCR Purification Kit (Qiagen, Frankfurt, Germany). The purified double-stranded cDNA was amplified, and the cyclization product was qualified. The qualified cyclized products were attached to DNB, and the constructed DNB was loaded into an MGI platform sequencer for computer sequencing. The PE150 sequencing strategy was employed. The information analysis process mainly included sequencing data quality control, data comparison analysis, and in-depth transcriptome analysis. The annotated differentially expressed genes were processed based on Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis via KOBAS software. Whole transcriptome sequencing depth analysis and personalized analysis were performed at Ruizhi Bio Ltd. (Jinan, China).
2.9. In Vivo Xenograft Experiments
All nude mice (female BALB/c nude mice, 4 weeks old) were purchased from Nanjing Model Animal Research Center and maintained under special pathogen-free conditions with access to water and food. Twenty-four mice were used in the experiments to reduce the use of animal testing. We established both the subcutaneous xenograft model and peritoneal metastatic xenograft model; both of them have two subgroups, including the PBS group (n = 6 mice/group) and HO-ADSC-exosome group (n = 6 mice/group). For the subcutaneous xenograft model, the suspended EOC cell line HEY cells (1 × 105 cells in 100 µL/mouse) were subcutaneously injected into the right shoulder of mice (5 weeks old, 18–22 g). On Day 7, following cell injection, the xenograft tumors were injected with HO-ADSC exosomes or an equal volume of PBS for 7 days. For the peritoneal metastatic xenograft model, nude mice were injected with a similar number of EOC cells into the abdominal cavities, followed by intraperitoneal injection with HO-ADSC exosomes or PBS, as mentioned above. After the administration of HO-ADSC exosomes for 28 days, the mice were sacrificed via cervical dislocation, and the tumor tissues were collected. Tumors were weighed and photographed. The volumes of the tumors were calculated, and the number of metastatic nodules was counted. Thereafter, tumor tissues were stripped for further Western blotting or immunohistochemistry assays. Animal experiments were approved by the Ethical Committee for Animal Research (Permit number DWLL-2021-019) and were performed in strict compliance with the Guide for the Care and Use Laboratory Animals of Qilu Hospital of Shandong University.
2.10. Immunohistochemistry (IHC)
Immunohistochemistry was performed according to a previously described method [
23]. Briefly, all fresh ovarian cancer samples were initially incubated with paraformaldehyde, paraffin-embedded, and sliced into sections. Subsequently, the slides were deparaffinized with xylene, hydrated with alcohol, heated in EDTA solution, and blocked with BSA. Then, the slides were incubated with primary antibodies, including Ki-67, caspase-3, MMP2, MMP9, and FOXM1, for 1 h before incubation with peroxidase-labeled anti-rabbit (or anti-mouse) secondary antibodies for 30 min. Finally, the chromogenic procedures were processed with 3, 3′-diaminobenzidine solution and stained with hematoxylin reagents. Histological images were obtained using a microscope and quantitatively analyzed with ImageJ software.
2.11. Western Blot Assay
Total protein was collected from EOC cell lines or xenograft tumours of nude mice using RIPA lysis buffer (Beyotime, Beijing, China), and protein concentrations were determined by using a BCA protein assay kit. The protein samples were initially separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Beyotime, Beijing, China) and transferred onto polyvinylidene difluoride membranes (Millipore Corp, billerica, MA, USA). Then, the membrane was blocked with nonfat milk for 2 h and incubated with specific primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at 37 °C. The peroxidase signal of proteins was visualized using an enhanced chemiluminescence substrate (Millipore Corp, billerica, MA, USA), and the bands were quantified using ImageJ software. GAPDH antibody was used as a housekeeping gene for normalization.
2.12. Quantitative Real-Time PCR (qRT–PCR)
Total RNA was extracted from EOC cell lines or xenograft tumors of nude mice using TRIzol reagent (Invitrogen, Waltham, MA, USA) and quantified using a NanoDrop DS-11 spectrophotometer (NanoDrop, Gaithersburg, MD, USA). Total RNA was subsequently reverse transcribed into cDNA using a ReverTra Ace q-PCR RT Kit (Toyobo, Shiga, Japan) according to the manufacturer’s specifications. To detect RNA expression, qRT–PCR procedures were conducted by using an SYBR Premix Ex Taq II Kit (Toyobo, Shiga, Japan) and a BioRad CFX96 Real-Time PCR detection system (BioRad, Hercules, CA, USA). Each sample was tested in triplicate. GAPDH served as endogenous control, and relative messenger RNA expression levels were calculated using the 2−ΔΔCt method in the final analysis. The MIQE guidelines for real-time PCR were followed in this study.
2.13. Statistical Analysis
Data are presented as the average ± standard deviation (SD) and were analyzed using SPSS 22.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA). In all analyses, data differences were assessed for significance using the Student’s t test or one-way analysis of variance with the post Student–Newman–Keuls test, as appropriate. Differences were considered statistically significant at a p value < 0.05. Based on the consideration of the research objective and cell experimental characteristics, as well as the overall homogeneity of primary cells, the samples were freshly collected from four ovarian cancer patients. All data were obtained from three separate experiments.
4. Discussion
Ovarian cancer is the seventh most frequent malignancy worldwide, and the mortality rate ranks first among gynecological malignancies. The pathological types of ovarian cancer vary, of which EOC accounts for 85–90 percent of ovarian malignancies [
25]. Despite significant advances in the treatment of ovarian cancer, the survival rate of EOC has not improved significantly in recent years [
26]. It is generally known that EOC patients frequently develop implantation metastasis to the omentum and peritoneal organs and are commonly diagnosed at the late stage. However, the molecular basis underlying this tendency to seed the peritoneum is ambiguous [
27]. In the present study, our goal was to investigate the role of HO-ADSCs in EOC metastasis and the underlying mechanisms. We found that HO-ADSCs increase the growth, migratory and invasive capabilities of ovarian cancer cells through an exosome-mediated mechanism. In addition, we demonstrated that HO-ADSC exosomes promote the growth and metastasis of ovarian cancer cells via a mechanism dependent on the FOXM1, Cyclin F KIF20A, and MAPK signaling pathways (
Figure 10).
Increasing evidence supports that ADSCs can regulate the growth and metastasis of various malignant tumors, such as bladder cancer, cervical cancer, and ovarian cancer [
28,
29,
30]. Yijing Chu et al. illustrated that ADSCs directly or indirectly cocultured with ovarian cancer cells accelerated ovarian cancer cell proliferation, invasion, and migration [
11,
13]. In contrast, another study by C. Khalil provided convincing evidence that ADSCs appear to inhibit the aggressiveness of ovarian cancer [
31]. Thus, there are contradictory reports about the role of ADSCs in ovarian cancer development. There are many types of human EOC cell lines, such as SKOV3, CAOV3, OVCAR3, IGROV-1, CAOV-3, A2780, and IGROV-1. A2780, HO8910, SKOV3, and HEY cells are used in this study because they are typical EOC cell lines used in our laboratory. In our study, we first established that the human omentum plays a stimulatory role in promoting ovarian cancer growth and metastasis. Using ADSCs in the omentum, we further demonstrated that HO-ADSCs could increase the growth, migratory and invasive capabilities of ovarian cancer cells. These results indicated that HO-ADSCs promote EOC progression and metastasis through a paracrine mechanism. The differences in ADSCs in tumor progression reported by different groups might be due to their activation status [
32].
Over the last decade, exosomes have been regarded as the newest information carrier of intercellular communication. Exosomes package a variety of protein components and nucleic acid molecules and then change the phenotype and function of recipient cells. At present, exosomes are considered to be the key messengers of intercellular communication and participate in various processes in tumor development, including angiogenesis, metastasis, drug resistance, and immune escape [
33]. In this study, in vivo experiments indicated that exosomes are an important participant in HO-ADSC-mediated promotion of EOC cell proliferation, migration, and invasion. Using a nude mouse tumorigenesis model, we also verified that HO-ADSC exosomes promoted the tumorigenesis and progression of EOC by regulating FOXM1 signaling in vivo. However, more importantly, considering that exosomes can also be delivered to various body fluids, we further provided fresh evidence that HO-ADSC exosomes may be secreted into ascites and are potentially involved in ovarian cancer growth and metastasis. This explains why patients with malignant ovarian tumors are often complicated with ascites and frequently develop peritoneal dissemination, and this is the significance of our study. In addition, we speculate that targeting the HO-ADSC exosomes may be a potential approach to treat advanced ovarian cancer with omentum metastasis.
To understand the mechanism by which HO-ADSC exosomes increased proliferation, migration, and invasion in ovarian cancer cell lines, we focused on FOXM1, as previous investigations have shown that the FOXM1 pathway is commonly activated in various solid cancers [
34]. Recently, reports by our laboratory have demonstrated that FOXM1 is an oncogene implicated in the growth and metastasis of ovarian cancer [
24]. Moreover, our data support that Cyclin F and KIF20A are involved in FOXM1-mediated ovarian cancer formation and development, and high Cyclin F and KIF20A expression was associated with poor prognosis in patients with ovarian cancer [
20]. Consistent with the findings from a previous study of our laboratory, we provided new evidence that FOXM1 played a critical role in HO-ADSC-exosome-mediated ovarian cancer growth and metastatic progression, and FOXM1 exerts its role partially by transcriptionally regulating Cyclin F and KIF20A expression. MAPK signaling pathways are one of the most important signaling pathways that are closely related to cancer cell growth and metastasis. In addition, based on the results of the KEGG pathway enrichment analysis, we also uncovered the underlying mechanism by which HO-ADSC exosome-modulated EOC cell functions were associated with ERK1/2 and P38 MAPK signaling pathway activation. The results were generally consistent with a past study showing that exosomes derived from human bone marrow mesenchymal stem cells contributed to tumor progression through the ERK1/2 and p38 MAPK pathways [
35].
Despite promising findings, our study has some limitations. In this study, we focused on confirming the effects of HO-ADSC exosomes on EOC metastasis and the related mechanism. However, the specific key factor contained in HO-ADSC exosomes that modulates FOXM1 signaling pathways is unclear. Based on existing information, the possible regulation of FOXM1 may be active substances such as proteins, RNA, and lipids encapsulated in exosomes. In future studies, we will assess the expression patterns in HO-ADSC exosomes. In addition, in vivo animal experiments were performed using subcutaneous tumorigenesis and peritoneal metastatic xenograft models in nude mice. However, the anatomic and histological structures of the mouse and human omentum are not identical, and more clinical studies are needed.