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The effect of foxp3-overexpressing Treg cells on non-small cell lung cancer cells

The aim of the present study was to investigate the novel mechanisms of forkhead box protein P3 (foxp3) in T regulatory (Treg) cells in lung cancer behavior. Treg cells were isolated from the peripheral blood of healthy volunteers and then co-cultured with 95D cells. A plasmid overexpressing foxp3 was constructed and transfected into Treg cells and an MTS assay was performed to assess cell viability. Flow cytometry was performed to evaluate cell apoptosis and reverse transcription-quantitative polymerase chain reaction was used to measure mRNA expression. A Transwell assay was used to assess cell invasion. Treg cells were successfully isolated from peripheral blood with purity of 94.26%. Foxp3 expression in Treg cells was significantly increased following co-culture with 95D cells, while matrix metalloproteinase-9 expression was upregulated in 95D cells co-cultured with Treg cells. The apoptosis, invasion and migration abilities of 95D cells were suppressed by co-culture with Treg cells, whereas the adhesive ability was enhanced. Foxp3 overexpression in Treg cells enhanced the viability and invasiveness of 95D cells, whereas cell adhesion and migration were decreased. The results of the present study demonstrate that the viability and invasiveness of 95D cells are enhanced by foxp3 overexpression in Treg cells, indicating that increased levels of foxp3 in the tumor microenvironment may promote tumor cell growth.


Introduction
The escape of tumor cells from the immune system is important for the occurrence, development, recurrence, and metastasis of lung cancer (1,2). Tumor cells protect themselves from being recognized and attacked by the immune system by modifying surface antigens and altering the microenvironment of the tissue (3,4). Therefore, an imbalance between immune cells and cytokines at the tumor site and in the peripheral blood often occurs (5). The proportional and functional alterations of T cell subsets in the tumor microenvironment is a key factor that contributes to the immune escape of tumor cells, especially in cluster of differentiation (CD)4 + CD25 + forkhead box protein P3 (foxp3) + T regulatory cells (Treg cells) (6,7).
Treg cells serve an immunosuppressive function in the tumor microenvironment; as a result, the body is unable to produce an effective immune response (8), resulting in tumor deterioration (9,10). The proportion of Treg cells in the peripheral blood and tumor tissue is increased in patients with tumors. Furthermore, the number of Treg cells is correlated with the degree of tumor progression and prognosis (11,12). In a clinical study, it was demonstrated that the clinical stage of non-small-cell lung cancer (NSCLC) was positively correlated with the Treg cell ratio (13). Treg cells surround the tumor in a cluster, whereas they are scattered in normal paracarcinoma tissue (14). The results of a previous study indicated that Treg is chemoattracted to tumor tissue and promotes tumor growth via its immunosuppressive effect (15). Verma et al (16) reported that the response of neoplastic cells to neoadjuvant chemotherapy is associated with Treg levels in the peripheral circulation. The postoperative level of Treg cells was considered to be an independent of the prognosis (17).
Foxp3 is a specific transcription factor of Treg cells and serves an important role in regulating the development and function of Treg cells (18). Foxp3 is necessary to maintain the immunosuppressive effect of Treg cells (18). The role of foxp3 in tumorigenesis and tumor progression is conflicting,

The effect of foxp3-overexpressing Treg cells on non-small cell lung cancer cells
both tumor suppressive and promoting functions have been reported (19,20) It has been reported that treatment with foxp3-knockout-Treg cells reduces the incidence of tumors in animal experiments and foxp3 serves an important role in lung tumorigenesis (21). It has been demonstrated that foxp3 overexpression facilitates the proliferation and invasiveness of cervical tumor cells, resulting in the development and metastasis of cervical cancer (22). Similarly, Treg cell infiltration in tumor tissue is negatively correlated with the prognosis of NSCLC (23). In contrast, foxp3 is also known as a potential tumor suppressor gene., Foxp3 inhibition decreases cell proliferation, migration, and invasion, as well as the secretion of inhibitory cytokines, suggesting that foxp3 as inhibitor for tumor development of lung adenocarcinoma (24). It was also demonstrated that the level of foxp3+Treg cells is positively correlated with the prognosis of specific tumors (25,26).
Ladoire et al (27) reported that the expression level of foxp3 in tumor cells is positively correlated with prognosis of patients with breast cancer. Hanke et al (28) proved that the quantity of foxp3+Treg cells in the tumor is associated with the prognosis of lymph node-negative colon cancer patients. However, whether foxp3 exhibits tumor suppressive and promoting functions in NSCLC is unclear.
In order to clarify the association between the expression of foxp3 in Treg cells and NSCLC, a tumor cell and immune cell co-culture model was used to study the interaction between lung cancer cells and Treg cells in vitro.
Cell isolation. A total of 8 healthy volunteers, 4 male and 4 female, 25-30 years old, were selected from 9th December 2013 to 15th December 2013. The blood samples were collected from the cubital vein of the forearm. Peripheral blood mononuclear cells were isolated from 10 ml whole blood samples of healthy volunteers using density gradient centrifugation as described previously (29). CD4 + CD25 + CD127 dim/cells were obtained by positive magnetic cell sorting using the CD4 + CD25 + CD127 dim/-Regulatory T Cell Isolation kit II (cat. no. 130-094-775; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Briefly, the non-CD4 + and CD127 high cells were removed using magnetic beads and CD25 + cells were collected using CD25 positive selection magnetic beads. CD4 + CD25 + CD127 dim/cells were cultured for 48 h in X-VIVO15 (Biowhittaker, Inc., Walkersville, MD, USA) supplemented with 2 mM L-glutamine (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 20 mg/ml gentamicin (Gibco; Thermo Fisher Scientific, Inc.), 0.1 mg/ml rapamycin (Gibco; Thermo Fisher Scientific, Inc.), 500 U/ml interleukin (IL)-2 (Gibco; Thermo Fisher Scientific, Inc.) and 5% autologous serum (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in an atmosphere containing 5% CO 2 . The present study was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Southern Medical University (Guangzhou, China). Written informed consent was obtained from all volunteers.
Cell co-culture system. Transwell co-culture experiments were performed in 24-well plates using inner wells with 0.4-µm pores to physically separate Treg cells and 95D cells. The 95D cells (2x10 5 cells/well) were grown in the outer wells using 1.5 ml RPMI-1640 medium containing 10% FBS. Subsequently, isolated human Treg cells (4x10 5 cells/well) were added to the inner wells in 500 ml RPMI-1640 medium. Following 14 days incubation at 37˚C, Treg cells and 95D cells were harvested and washed with PBS for the following experiments. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted from the 95D and Treg cells using a TRIzol reagent kit obtained from Invitrogen (Thermo Fisher Scientific, Inc.). RNA was eluted in 50 ml RNase-free water and preserved at -70˚C. The total RNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). The conditions were as follows: 42˚C for 15 min and 85˚C for 10 min. To analyze gene expression, the qPCR mixture system containing primers, cDNA templates, and SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Inc.) was used as described previously (24). The qPCR conditions were as follows: 30 sec at 95˚C followed by 40 cycles of 15 sec at 95˚C for denaturation 30 sec at 60˚C for annealing and 25 sec at 72˚C for extension. Fold changes in gene expression were calculated using the 2 -ΔΔCq method (30). 18S was used as an internal control. The primers used in the present study were listed as follows: Foxp3, forward 5'-CGG ATT TCG ATG TTC GGT AC-3' and reverse 5'-GCC ATT GTC AGG TCC TGA GT-3'; matrix metalloproteinase (MMP)-9, forward 5'-CTA TAG CAT CAG TAA GTG GTT-3' and reverse 5'-GAG TAG GAA CTG ACC TAT-3'; 18S, forward 5'-AGA AAC GGC TAC CAC ATC-3' and reverse 5'-TAC TCA TTC CAA TTA CCA GAC TC-3'.
MTS assay. 95D cells were seeded in 96-well plates (2x10 3 cells/well) for cultivation. Cells were co-cultured with wild type Treg or Foxp3 over-expressed Treg cells for 24 h as described above. During the last 4 h of each day of culture, MTS was added to the medium. Formazan crystals were dissolved using dimethyl sulfoxide and the absorbance was measured at 450 nm using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Inc.).
Transwell assay. The invasiveness of 95D cells was assessed using a Transwell assay. The Transwell chamber (cat. no. 353097; BD Biosciences) was covered with a Matrigel (cat. no. 356234; BD Biosciences) basement membrane. The lower and upper chambers were separated with a polycarbonate membrane. The lower and upper chambers were filled with RPMI-1640 medium. The 95D cells (2x10 5 cells/well) were seeded in the upper chamber and cultured in a 5% CO 2 incubator at 37˚C for 24 h. The cell coating was removed from the upper chamber with a cotton ball 48 h later. The cells that had migrated to the reverse side of the membrane were stained with 0.05% crystal violet. The membrane was observed (magnification x200) under an inverted microscope (Olympus Corporation, Tokyo, Japan). Five fields of view were randomly selected by two independent pathologists. The cells were counted in each field of view and the mean was calculated. The experiments were performed in triplicate.
Western blotting. Total protein was extracted from Treg cells using radioimmunoprecipitation assay buffer (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 50 µg protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Subsequently, the membrane was blocked with 5% skim milk at room temperature for 1 h and incubated with primary antibodies against MMP-9 (cat. no. ab73734; 1:500; Abcam, Cambridge, MA, USA) and GAPDH (cat. no. ab9485; 1:500; Abcam) at 4˚C overnight. Following washing with PBST three times, the membrane was incubated with a horseradish peroxidase conjugated anti-rabbit IgG antibody (cat. no. ab191866; 1:500; Abcam) for 1 h at 37˚C. Finally, the protein expression was detected by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). Chemiluminescent detection was performed using Bio-Rad ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Cell adhesion assay. Fibronectin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was pre-coated on microtiter plates and 95D cells (2x10 5 /ml) were added. Non-adherent cells were washed away and the adherent cells were fixed and stained by DAPI staining as described previously (31). Finally, the cells were observed under an inverted fluorescence microscope (magnification, x100) and absorbance was measured using a microtiter plate reader.
Wound healing assay. At 24 h following cell co-culture, a wound-healing assay was performed. The 95D cells (1x10 5 cells/well) were seeded into a 24-well plate at 60-70% confluence. A lesion was created using a plastic pipette tip and cells were washed twice with PBS buffer. The monolayer was then maintained in serum-free RPMI-1640 medium and cultured at 37˚C for 24 h. Five randomly selected fields at the border of the lesion were viewed under an inverted microscope (magnification, x100; IX71; Olympus Corporation).
Statistical analysis. All data analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Differences among groups were compared using a t-test or one-way analysis of variance (ANOVA). Tukey's post hoc test was used for one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

Results
Treg cell isolation. Magnetic beads were applied to isolate Treg cells from peripheral blood CD4 + T cells were separated out and were demonstrated to account for ~21.32% of all T cells. Then CD25 beads were used to extract CD4 + CD25 + T cells (4.63%) and further isolate CD4 + CD25 + CD127 dim/-T cells (Fig. 1). The cell ratio reached 94.26% following purification, which was deemed suitable for the following experiments.

Influence of Treg and 95D cell co-culture on gene expression.
To investigate the influence of cell co-culture on foxp3 expression in Treg cells and MMP-9 expression in 95D cells, cells were incubated together for 36 h and total RNA was extracted. qPCR revealed that foxp3 gene expression was significantly increased in Treg cells following co-culture with 95D cells (P<0.05; Fig. 2A). Similarly, MMP-9 mRNA expression was significantly increased in 95D cells following co-culture with Treg cells compared with the 95D only group (P<0.05; Fig. 2B). Western blotting results revealed that the expression of foxp3 and MMP-9 proteins followed the same trend as the mRNA (Fig. 2C).
Effect of cell co-culture system on 95D cell behavior. The effect of cell co-culture on 95D cell behavior was investigated. Flow cytometry demonstrated that apoptosis was significantly reduced in 95D cells following co-culture with Treg cells (P<0.05; Fig. 3A). However, the Transwell assay revealed that co-culture failed had no significant effect on the invasive ability of 95D cells (Fig. 3B). A cell adhesion assay demonstrated that the adhesion of 95D cells was markedly enhanced following co-culture with Treg cells (Fig. 3C). In addition, a wound-healing assay revealed that 95D cell migration was attenuated by Treg cell co-culture (Fig. 3D).
Effect of foxp3 on cell co-culture. Foxp3 serves a crucial role in the regulation and development of Treg cells. Based on this, a plasmid overexpressing foxp3 was constructed and transfected into Treg cells to observe its effect on the behavior of 95D cells. A cell adhesion assay demonstrated that the adhesive ability of 95D cells decreased following co-culture with foxp3 overexpressing Treg cells (Fig. 4A). A wound healing assay demonstrated that the migration of 95D cells was decreased following co-culture with foxp3 overexpressing Treg cells (Fig. 4B). An MTS assay demonstrated that the viability of 95D cells was significantly reduced following co-culture with wild type Treg cells, while it was significantly increased following co-culture with foxp3 overexpressing Treg cells (P<0.05; Fig. 4C). In addition, the Transwell assay demonstrated that the invasive ability of 95D cells was significantly enhanced by co-culture with foxp3 overexpressing Treg cells (P<0.05; Fig. 4D).

Discussion
The tumor microenvironment influences the interactions between cells and ultimately affects the outcome of tumor (32). Treg cells are important components of the tumor immune microenvironment, which inhibits the antitumor immune function and is associated with tumor immune escape by suppressing the function of activated T cells (33). The immunosuppressive effect of Treg cells depends on intercellular contact (34) and the impact of cytokines (35). The results of the present study suggest that 95D cells promote foxp3 expression in Treg cells, indicating that Treg cells have a significant    synergistic effect with 95D cells. However, Treg cells inhibit the apoptosis of 95D cells and enhanced the adhesive ability of 95D cells. The results indicate that Treg cells promote tumor development in the NSCLC microenvironment; these results are similar to a previous study, in which it was demonstrated that Treg cell invasion was negatively correlated with prognosis in patients with esophageal cancer who received neoadjuvant radiotherapy and chemotherapy (17).
Foxp3 is a recognized marker of Treg cells and maintaining the growth and immunosuppressive function of Treg cells. Foxp3-knockout-Treg cells lost the immunosuppressive effects observed in wild type Treg cells, being altered from immunoregulatory cells to proinflammatory cells (36). The expression of foxp3 in Treg cells is regulated by transcription factor activation and chromatin molecule modification (37). Previous studies have demonstrated that T-cell receptor (TCR) signaling important for the expression of foxp3 in Treg cells (38). The expression of CD25 is upregulated by the TCR signaling pathway, which activates IL-2 signaling and the downstream signal transducer and activator of transcription 5A (39). CD28 binds with its ligand, CD80/CD86, to induce foxp3 expression (40). The effect of epigenetic regulation and nuclear factor (NF)-κB signaling pathway on foxp3 expression is also associated with the TCR signaling pathway (41). The TCR-responsive enhancer in the first intron of the foxp3 gene is dependent on a cyclic-AMP response element binding protein (CREB)/activating transcription factor site overlapping a CpG island (42). The methylation of the island inhibits CREB binding and downregulates the transcription of foxp3 (42). The TCR signaling pathway is able to upregulate the foxp3 transcription via the demethylation of CpG islands (42). Conserved noncoding sequences (CNS) 2 demethylation also serves an important role in the stabilization of foxp3; upregulation of CNS2 expression inhibits DNA methyltransferase and therefore stabilizes the Treg cell phenotype (42). The NF-κB signaling pathway regulates the CNS3 region via activation of its constituent element, c-Rel, and the TCR signaling pathway, thereby promoting foxp3 transcription and Treg cell differentiation (43). It was observed in the present study that tumor cells promote the expression of foxp3 in Treg cells, suggesting that they may be associated with cytokines secreted by tumor cells and function between cells. Tumor cells secrete a large number of cytokines, including transforming growth factor (TGF)-β, IL-10, prostaglandin-2, retinoic acid A, and indoleamine-2,3-dioxygenase (44). Cytokines induce the expression of foxp3 in Treg cells and enhance the immunosuppressive function (45). For example, TGF-β was reported to regulate the sustained expression of foxp3 and stabilize the number and immunosuppressive functions of Treg cells in vivo and in vitro (38).
In the present study, the apoptosis of 95D cell decreased following co-culture, suggesting that foxp3 may be expressed in tumor cells (46). The TCR signaling pathway is important for the regulation of foxp3 and is o key apoptotic associated pathway. Associated cell factors bind with the TNF domain on the tumor cell surface to initiate apoptosis via the TCR signaling pathway (47). Following the activation of Treg cells, the expression of the associated signal molecules is also increased, including glucocorticoid-induced TNF receptor (GITR) and cytotoxic T lymphocyte antigen 4 and TCR-inducible costimulatory receptor (48). Nocentini et al (49) demonstrated that GITR, a member of the TNF receptor family, is associated with TCR-mediated cell death, and attenuates anti-CD3 monoclonal antibody-induced apoptosis. Zhang et al (50) also revealed that co-culture of tumor cells with peripheral blood mononuclear cells upregulates GITR expression.
In the present study, Treg cell infiltration promoted tumor cell growth and reduced cell apoptosis. The influence of foxp3 expression by tumor cells remains unclear. Tan et al (51) suggested that overexpression of foxp3 in tumor cells inhibited tumor growth and promoted cell apoptosis. Studies also obtained similar results in glioma (52), gastric cancer (53), breast cancer (54) and other associated tumors (55). It was reported that endogenous foxp3 overexpression inhibited gastric cancer cell proliferation and facilitated apoptosis by upregulating microRNA-146a/b and negatively regulating the NF-κB signaling pathway (56). The role of foxp3 varies in different tumors cells and tumor microenvironments; however, mutations of foxp3 are observed in certain tumors. For example, if foxp3 loses some exons in MCF-7 cells, the variants lose the ability to suppress gene expression in cancer cells (57).
MMPs regulate the movement of hematopoietic stem cells and degrade a variety of extracellular matrix proteins (58). MMP-9 serves a crucial role in tumorigenesis and development by forming vascular endothelial growth factor receptor 2/fetal liver kinase 1 receptor in endothelial cells through remodeling the extracellular matrix and promoting germination and growth of novel vessels (59). Studies have indicated that MMP-9 expression is associated with tumor progression and prognosis (60)(61)(62). Similarly, MMP-9 expression in immunosuppressive macrophages in the tumor microenvironment also contributes to tumor invasion and metastasis (60). MMP-9 is able to promote cell migration via activating mitogen-activated protein kinase and phosphoinositide 3-kinase pathways (63). It has been demonstrated that MMP-9 expression is also associated with the activation of TGF-β (64). In dendritic cells, the MMP-9 inhibitor decreased the activation of latent TGF-β1 (65). Overexpression of MMP-9 was detected in laryngeal cancer and serves a critical role in the development of tolerogenic dendritic cells, therefore serving a key role in tumor survival (66).
The results of the present study demonstrate that the expression of MMP-9 in tumor cells, tumor invasion and foxp3 levels were increased in the co-cultured Treg cells. The effect of foxp3 on MMP-9 expression may be associated with certain cytokines [e.g., IL-17 (67)] and phosphorylation. Foxp3 downregulation reduces the expression of MMP-9 and MMP-2, thereby inhibiting tumor invasion (68). The tumor microenvironment contains a variety of cytokines, including IL-17, IL-1, IL-6, IL-10, and TGF-β. IL-17A is a proinflammatory cytokine that induces the expression of VEGF, MMPs and C-X-C motif chemokine 8 and therefore promotes neovascularization (69). IL-17A inhibition in the tumor microenvironment enhances the cytotoxicity of tumor-infiltrating lymphocytes (70). In addition, it was demonstrated that foxp3 phosphorylation downregulates MMP-9 and reduces the aggressiveness of tumor cells and affecting the NF-κB function (57). Phosphorylation of the Tyr-342 fragment in foxp3 serves an important role in downregulating the expression of MMP-9 and S-phase kinase-associated protein 2 (57). Morawski et al (71) also demonstrated that cell cycle-dependent kinase 2 has a z regulatory effect on the stability and activity of foxp3 via foxp3 phosphorylation.
In the present study, cells were seeded in a Transwell plate to detect the invasiveness of 95D cells. The results suggested that the invasive ability of 95D cells was similar following co-culture with Treg cells. However, the expression of MMP-9 in tumor cells was increased and apoptosis was decreased following co-culture. It was hypothesized that invasion may be associated with the duration of co-culture.
In the foxp3 overexpression model group, 95D cells exhibited the highest viability and it was hypothesized that this may be due to the short co-culture time and the intercellular contact inhibition (72).
In conclusion, the results of the present study demonstrate that tumor cell viability and invasiveness are enhanced by co-culture with foxp3-overexpressing Treg cells and that foxp3 in the tumor microenvironment promotes tumor cell growth. Further investigation of the foxp3 phenotype in Treg cells and the associated mechanism in tumor cells may provide a novel method for the treatment and prevention of NSCLC.

Acknowledgements
Not applicable.

Funding
No funding was received.

Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions
JP and YL designed this work. ZY and LX performed the the experiments and wrote the manuscript. JW and JL drafted statistical methods and analyzed the data. JP, DL and QY helped with data collection and data interpretation. All authors read and approved the manuscript for publication.

Ethics approval and consent to participate
The present study was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Southern Medical University (Guangzhou, China). Written informed consent was obtained from all volunteers.

Consent for publication
Not applicable.