Six-week-old male Wistar rats weighing 250 ± 10 g were purchased from Beijing Vital River Laboratory Animal Technology Company. All rats were kept in a standardized environment (temperature 25 ± 2°C, humidity 55 ± 10%, irradiation time 8:00–20:00 h) in the Animal Resource Centre of the Faculty of Medicine, Nanjing Medical University. The entire experimental process and animal treatment adhered to the rules of the Experimental Animal Application Criteria and Institutional Animal Care and Use Committee (IACUC).

Tumors were implanted into the right caudate nucleus of Wistar rats. The rats were anesthetized and placed in a stereotaxic apparatus. C6 and 9L glioma cells (1 × 10⁶) were injected subcutaneously into the right caudate nucleus of the rats (1 mm anterior, 3 mm lateral to the bregma, depth 5 mm from dura). FTY720 (2 mg/kg) or saline was intraperitoneally injected into models daily beginning on day 2 after tumor implantation. MRI was used to examine the tumor growth 14 days after implantation.

Rat primary microglial cells were generated from 1-day-old postnatal Sprague–Dawley rats as described previously (24). Briefly, microglia were isolated from cerebral cortices by mechanical dissociation, 0.25% trypsin/EDTA (Gibco, Grand Island, NY, USA), and plated into poly-D-lysine-coated (0.1 mg/ml; Sigma Chemical, St. Louis, MO, USA) T25 culture flasks. The rat glioma cell line C6 was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS; Gibco) in a humidified 5% CO₂ and 37°C incubator. Co-culture experiments were performed as shown below.

Fingolimod (FTY720) HCl was purchased from Selleckchem (Houston, TX, USA) and anti-Iba1 antibody was purchased from FUJI FILM Wako Pure Chemicals (Japan). Anti-iNOS antibody was purchased from Santa Cruz Biotechnology, while anti-Mannose Receptor (CD206) antibodies were purchased from Abcam. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), Phospho-MEK1/2 (Ser217/221), MEK1/2, Phospho-JNK (Thr183/Tyr185), JNK, Phospho-p38MAPK (Thr180/Tyr182), p38 MAPK, and Na/K-ATPase antibodies were purchased from Cell Signaling Technology. The S1PR1 and S1PR3 antagonist VPC 23019 was purchased from Avanti Polar Lipids (Alabaster, USA).

Total RNA was extracted from cultured cells using TRIzol reagent (YIFEIXUE BIOTECH) according to the manufacturer's instructions, and a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Thermo Scientific, USA) was used to measure the purity and concentration of the total RNA. cDNA was synthesized with a PrimeScript™ RT Master Mix (Takara, Japan) according to the manufacturer's protocol.

Real-time quantitative PCR was performed using the SYBR Green mixture (Selleckchem, USA) in a QuantStudio 5 Real-Time PCR System (Applied Biosystems, USA) according to the manufacturer's method. GAPDH was used as an endogenous control and all data were assessed using the 2^−ΔΔCT method.

For Western blotting, cells were washed with phosphate-buffered saline and lysed in RIPA buffer with protease and phosphatase inhibitors (Sigma) and centrifuged at 14,000 × g at 4°C for 15 min. Cell membrane proteins were extracted using a Cell Membrane Protein and Cytoplasm Protein Extraction Kit (KeyGEN BioTECH, China) according to the manufacturer's protocols, and the concentration of each protein sample was measured using an enhanced BCA Protein Assay Kit (KeyGEN BioTECH, China). The proteins were resolved by SDS-PAGE and transferred onto PVDF membranes, which were then blocked in 10 mM Tris buffer with 5% skim milk and incubated with specific primary antibodies at 4°C overnight. After being washed, the membranes were probed with HRP-conjugated secondary antibodies, visualized by ECL, and measured with ImageJ software. All Western blots were performed at least three times.

Brain sections and cells were immunofluorescently stained as previously described (19). Briefly, brain samples and cells were fixed using 4% paraformaldehyde for 24 h or 15 min, respectively. Brains were also embedded in wax and cut into 5-μm-thick sections. The samples were then blocked with 10% normal donkey serum and 0.01% Triton X-100 in PBS for 60 min at room temperature and incubated with primary antibodies at 4°C overnight. Sections were subsequently incubated with corresponding Alexa Fluor 488-, 546-, and 555-conjugated specific secondary antibodies (Invitrogen, USA). Cell nuclei were stained with Hoechst 33258. The sections and cells were scanned with a fluorescence microscope (Olympus, Japan) by one investigator, and the staining was quantified by two independent investigators.

For histopathology, brain sections were first incubated with hematoxylin for 15 min, washed with water for 5 min, and then flushed with 1% HCl four times and then washed for 20 min. Finally, the sections were stained with eosin and photographed.

MRI was performed using Biospec 70/20 USR (Bruker, Germany) with 1H/19F circular polarized small volume coil for rat head. MSME pulse sequence (TR = 3 s and TE = 33 ms) was used to acquire multi-echo images [a field of view (FOV) 3.5 cm², data matrix = 256 × 256 × 25 slices, thickness = 1 mm].

Cell culture supernatants were collected and centrifuged for 20 min at 1,000 × g. The concentration of CXCL12, IL-6, TNF-α, and IFN-γ in the cell culture supernatants was detected according to the manufacturer's instructions.

A wound healing assay was used to examine the cell motility of C6 glioma cells. C6 glioma cells were seeded in a 24-well plate. After 12 h, a pipette tip was used to scratch the center of the well. The cells were then treated with culture medium with different concentrations of FTY720 and photographed at 0, 3, 6, 12, and 24 h.

The Transwell insert was precoated with Matrigel matrix (Corning Inc., NY, USA), and incubated at 37°C for 1 h to solidify. The insert was hydrated with 200 μl of DMEM and then 1 × 10⁴ C6 glioma cells in 200 μl of DMEM were seeded in the insert. The lower chamber was filled with 600 μl of DMEM containing 10% FBS with/without 5 × 10⁴ microglial cells to chemoattract C6 cells. After 24 h, the insert was washed twice with PBS, fixed with 5% Glutaral, and stained with 0.1% crystal violet (Sigma). A wet cotton swab was used to gently remove the cells on the top of the insert, and the cells were counted in four independent microscopic fields.

GraphPad Prism 7.0 was used for statistical analysis. Comparisons among groups were performed with one-way ANOVA, and unpaired Student's t-test was conducted between two groups. Data are expressed as mean ± SEM, and p < 0.05 was considered statistically significant.

Previous studies have shown that FTY720 possesses potent inhibitory effects in numerous cancer models, including breast cancer, multiple myeloma, and glioblastoma. In our study, we used C6 and 9L glioma allograft to evaluate the anti-tumor effects of FTY720. We found that treatment with FTY720 for 14 days did not alter the survival rate in C6 glioma-bearing Wistar rats (Figures 1A,B), while tumor volume was significantly reduced (Figure 1C), detected by MRI (Figure 1D) and H&E staining (Figure 1E), in contrast to counterparts treated with saline. Our results demonstrated the anti-glioma effect of FTY720 in C6 or 9L glioma-bearing rats.

Enormous inflammatory infiltrates, predominated by microglia and macrophages, are considered to be attracted, recruited, and subverted by glioblastoma cells for tumor growth (17, 20, 25). Therefore, we evaluated the recruitment and accumulation of GAMs in the glioma microenvironment with and without FTY720 treatment. Iba1⁺ GAMs numbers and cellular size increased and even enwrapped gliomas in C6 glioma-bearing rats, which were alleviated by FTY720 administration (Figures 2A,B). These results suggest that FTY720 could block the recruitment of GAMs.

To investigate how FTY720 mediates these changes in GAMs, we focused on various receptors, which may play significant roles in GAMs recruitment and chemoattraction. To do this, we established a C6-microglia co-culture system in vitro (Figure 5D), where we determined the expression level of different receptors expressed on the microglia. Among the chemokine receptors induced upon C6-microglia co-culture, we found specific upregulation of CCR2, CD74, and CXCR4. After treatment with FTY720, the expression of microglial CXCR4 was significantly reduced (Figures 2C,D). We then performed ELISA analysis to determine the changes of glioma-secreting chemokine responding to the reductions of microglial CXCR4. The ELISA results showed that FTY720 did not affect CXCL12 secretion by glioma cells (Figure 2E), indicating that FTY720 could regulate CXCL12–CXCR4 cross-talk between microglia and glioma via decreasing the expressions of CXCR4 on the microglia. Beider et al. demonstrated that CXCR4 can be directly targeted by FTY720, thus limiting tumor-promoting activities in multiple myeloma (26). Similarly, we found that FTY720 reduced cell-surface levels and increased intracellular levels of CXCR4. This indicates that FTY720 can also promote CXCR4 internalization by microglia (Figures 2F,G), thereby blocking the chemoattraction of GAMs.

CXCR4 receptor activation is mediated by a GPCR mechanism, coupling to an intracellular heterotrimeric G-protein associated with the inner surface of the plasma membrane (27). CXCR4 signaling is rapidly desensitized after ligand binding by receptor internalization. To explore whether the downstream pathways of CXCR4 are indeed involved in mediating the effects of FTY720, we next assessed changes to CXCR4-regulated signaling pathways. CXCR4 signaling has been shown to be involved in the MAPK signaling pathway in cancer, mediating functions that include cell migration and survival (27). Our observations suggested that FTY720 could inhibit the activation of CXCR4-dependent intracellular MAPK signaling, as there was a reduction in the phosphorylation of three members of the MAPK pathway: ERK, JNK, and p38 (Figures 3A,B).

MAPK signaling was found to play vital roles in the release of chemokines and inflammatory cytokines, which in turn sustain tumor growth, invasion, and tumor escape (14). Therefore, we tested whether microglial MAPK signaling inhibited by FTY720 could influence the secretion and release of common related cytokines and chemokines, including TNF-α, IL-6, and IFN-γ. Our analysis showed a significant decrease in the expression level of IL-6 (Figure 3C). Studies revealed that IL-6 can contribute to the proliferative and migratory abilities of glioblastoma (28). Furthermore, we found that FTY720 inhibited the migration (Figures 3D,E) and invasion (Figures 3F,G) of C6 in a glioma–microglia co-culture system. Altogether, FTY720 decreased microglial MAPK-mediated IL-6 secretion to suppress the migration and invasion of glioma.

FTY720 is known as an immunomodulator and can be phosphorylated to be a sphingosine-1-phosphate (S1P) analog in vivo and binds to all of the sphingosine-1-phosphate receptors (S1PRs) (29, 30). To examine whether S1PRs play a role in the anti-glioma effects of FTY720, we first evaluated the expressions of S1PRs in microglia. The mRNA and protein expressions of S1PR1, S1PR2, and S1PR3 were relatively high in microglia (Figures 4A,B). Then, VPC 23019, a S1PR1 and S1PR3 antagonist (Figure 4C), was used to verify the roles of S1PRs on the anti-glioma effects of FTY720. We found that VPC 23019 failed to affect the motility of C6 glioma cells treated with FTY720 in glioma–microglia co-culture system (Figure 4D). Furthermore, we examined the effects of VPC 23019 and FTY720 on the internalization of CXCR4, downstream MAPK signaling, and IL-6 secretion. The results also showed that VPC 23019 did not affect the effects of FTY720 (Figures 4E–I). Altogether, our observations indicate that FTY720 exerts anti-glioma effects independent of S1PRs.

Glioma-derived IL-6, along with other cytokines such as TGF-β, polarizes glioma-infiltrating microglia and macrophages toward a pro-tumorigenic phenotype, which, in turn, produce and secrete IL-6 (13). Therefore, we tested whether FTY720 can influence GAM phenotypes and thus offer clinical benefits.

The cell-surface marker Iba1 is commonly used to label total GAMs, while iNOS and CD206 mark pro-tumor and anti-tumor phenotypes, respectively. In our results, we observed that the co-localization of Iba1 and iNOS increased and CD206 co-localization with Iba1 decreased in the glioma core of rats after FTY720 treatment (Figures 5A–C). Additionally, we found similar results in a glioma–microglia co-culture system (Figures 5D,E). These results suggested that FTY720 could push GAMs into anti-glioma phenotypes in the glioma microenvironment.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.