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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 1  |  Page : 9-16

Suppressive effect of mesenchymal stromal cells on interferon-γ-producing capability of spleen cells was specifically enhanced through humoral mediator(s) from mouse oral squamous cell carcinoma Sq-1979 Cells In Vitro


1 Department of Oral Biochemistry, Division of Oral Structure, Function and Development, Asahi University School of Dentistry, Gifu; Department of Oral and Maxillofacial Surgery, Division of Reparative and Regenerative Medicine, Institute of Medical Science, Mie University Graduate School of Medicine, Mie, Japan
2 Department of Management and Information Studies, Chemistry Laboratory, Asahi University School of Business Administration, Gifu, Japan
3 Department of Oral Biochemistry, Division of Oral Structure, Function and Development, Asahi University School of Dentistry, Gifu, Japan
4 Department of Anesthesiology, Division of General Medicine, Asahi University School of Dentistry, Gifu, Japan
5 Department of Oral and Maxillofacial Surgery, Division of Oral Pathogenesis and Disease Control, Asahi University School of Dentistry, Gifu, Japan

Date of Submission08-Aug-2017
Date of Acceptance16-Jan-2018
Date of Web Publication26-Feb-2018

Correspondence Address:
Dr. Masako Mizuno-Kamiya
Department of Management and Information Studies, Chemistry Laboratory, Asahi University School of Business Administration, 1851 Hozumi, Mizuho, Gifu 501-0296
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ctm.ctm_34_17

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  Abstract 

Aim: The aim of this study was to compare the immunomodulatory effects in the tumor milieu of mouse oral squamous cell carcinoma (OSCC) cells harboring primary and advanced phenotypes. We established an in vitro co-culture system using mouse OSCC cells, spleen cells, and mesenchymal stromal cells.
Methods: Sq-1979 is an OSCC cell line derived from C3H mice; 233-11 cells were established from a primary Sq-1979 tumor; L3–5, L5–11, and L6–8 cells were established from lymph node-metastasized Sq-1979 cells. 10T1/2 is a fibroblast line derived from C3H mice. The OSCC cells were co-cultured with anti-CD3 antibody-stimulated mouse spleen cells in the presence or absence of 10T1/2 cells, and the producing capability of interferon (IFN)-γ and interleukin (IL)-10 was evaluated using enzyme-linked immunosorbent assay.
Results: The production of IFN-γ by the stimulated spleen cells was specifically enhanced in the presence of co-cultured L-cells. The production of IL-10 was conversely reduced in the co-culture with all the OSCC cell lines used. The production of IFN-γ was significantly reduced in the co-culture with directly contacted 10T1/2 cells, and further reduction was observed in the presence of Sq-1979 and 233-11 cells but remained unchanged in the presence of L-cells. The reduction of IFN-γ production was also observed by the addition of conditioned medium from the Sq-1979-1 cells.
Conclusion: Sq-1979 cells specifically enhanced the immune-suppressive activity of mesenchymal stromal cells through humoral factor (s).

Keywords: Immune suppression, interferon-γ, OSCC, reciprocal interaction, spleen cells, stromal cells


How to cite this article:
Inagaki T, Mizuno-Kamiya M, Takayama E, Kawaki H, Chihara E, Muramatsu Y, Sumitomo S, Kondoh N. Suppressive effect of mesenchymal stromal cells on interferon-γ-producing capability of spleen cells was specifically enhanced through humoral mediator(s) from mouse oral squamous cell carcinoma Sq-1979 Cells In Vitro. Cancer Transl Med 2018;4:9-16

How to cite this URL:
Inagaki T, Mizuno-Kamiya M, Takayama E, Kawaki H, Chihara E, Muramatsu Y, Sumitomo S, Kondoh N. Suppressive effect of mesenchymal stromal cells on interferon-γ-producing capability of spleen cells was specifically enhanced through humoral mediator(s) from mouse oral squamous cell carcinoma Sq-1979 Cells In Vitro. Cancer Transl Med [serial online] 2018 [cited 2018 Dec 14];4:9-16. Available from: http://www.cancertm.com/text.asp?2018/4/1/9/226170




  Introduction Top


To evaluate the prognosis of cancer patients, merely observing phenotypes of cancer cells is insufficient to completely estimate the malignancy. Cancer malignancy is also affected by the immunological response, in part contributed by the tumor microenvironments (TMEs) which are composed of fibroblasts, extracellular matrix, immune competent cells, and secreted soluble factors.[1],[2] The most important components of TMEs that play active roles for the development of malignancy are cancer-associated fibroblasts (CAFs).[3],[4],[5],[6] CAFs have reciprocal interaction not only with tumor cells but also with immune cells, which can lead to tumor progression.[7],[8],[9]

We have previously developed a model of oral malignancy using a mouse oral squamous cell carcinoma (OSCC) cell line, Sq-1979, and several subclones, including L-cells that were established from metastasized lymph node tissues of Sq-1979-implanted mice. The majority of the L-cells showed highly malignant phenotypes.[10] Compared to parental Sq-1979 and 233 cells, most L-cells represent a higher growth rate and transplantability and also confer lower survival rates in the implanted mice.[10]

These OSCC models enable us to analyze the development of the tumor immunogenicity, especially in the myeloid-derived suppressor cells (MDSCs). Our results demonstrated that the population of intratumoral polymorphonuclear morphology (PMN)-MDSCs are significantly increased in metastasized subclone (L5-11)-implanted mice but not in the mice implanted with the primary OSCC cell line (Sq-1979).[11] Our model, therefore, demonstrates that potential immunomodulatory mechanisms of the OSCC cells are developed depending on their malignancy; in L-cell-implanted mice, PMN-MDSCs may be the major mediator of immune suppression.[11] However, the mechanism of immune modulation in Sq-1979-cell-bearing mice has not been elucidated.

We have also demonstrated that a Th1 cytokine, interferon (IFN)-γ-producing capability of LPS-stimulated peripheral blood is significantly decreased; by contrast, a Th2 cytokine, interleukin (IL)-10-producing capability is increased in the patients carrying higher grade (stage III) hepatocellular carcinoma,[12] suggesting that the Th1 and Th2 cytokines are reciprocally regulated in the development of tumors. A similar observation was reported by studying a marine solid tumor model implanted with colon carcinoma CT26 cells.[13] These results suggest that humoral factors interacting with TME could be important mediators and potential markers for immunomodulatory activities of the OSCC cells.

In this study, we attempted to evaluate immunomodulatory mechanisms of Sq-1979 cells in TMEs by establishing a unique in vitro system, in which mesenchymal stromal cells, activated lymphocytes, and tumor cells are co-cultured.


  Methods Top


Experimental animals

Five-week- or twenty-four-week-old male C3H/HeN mice were purchased from Chubu Kagaku Shizai Co., Ltd. (Nagoya, Japan) and maintained ad libitum on Oriental MF solid chow (Oriental Yeast Co., Tokyo, Japan) for more than 1 week before the start of experiments. This study was approved by the Animal Ethics Committee of Asahi University (No. 14-019).

OSCC cells and subclones

The C3H mouse OSCC cell line, Sq-1979, was obtained from the RIKEN BioResource Center (Ibaraki, Japan). Cells were grown in Eagle's minimum essential medium (E-MEM; Wako, Osaka, Japan), supplemented with 10% fetal bovine serum (FBS, NICHIREI BIOSCIENCES INC., Tokyo, Japan), and 1% Pen–Strep (penicillin 10,000 unit/mL, streptomycin 10,000 μg/mL; Gibco®, Life Technologies, Grand Island, NY, USA). This medium composition is recommended for Sq-1979 in the RIKEN BioResource Center. Establishments of Sq-1979 subclones are as described.[10] Briefly, ten million of Sq-1979 cells suspended in 0.1 mL of saline were subcutaneously inoculated in the posterior neck area of 6-week-old male C3H/HeN mice. After 3 months, metastasized regional lymph nodes were dissected to isolate the attached metastasized cells. Subclones of such, including L3–5, L5–11, and L6–8 cells, were isolated by a serial limiting dilution method. Using the same procedure, 233-11 cells were isolated from primary OSCC tissues. Sq-1979-1 and Sq-1979-2 cells were isolated from parental Sq-1979.

C3H mouse embryonic fibroblasts, 10T1/2 cells, were obtained from the RIKEN BioResource Center. Cells were grown in Basal Medium Eagle (BME; Gibco®, Life Technologies) supplemented with 10% FBS (NICHIREI BIOSCIENCES INC.) and 1% Pen–Strep (Gibco®, Life Technologies). This medium composition is recommended for 10T1/2 cells in the RIKEN BioResource Center.

Before using for co-culture, OSCC and 10T1/2 cells were harvested at 80%–90% confluence with 0.25% trypsin/0.1% ethylenediaminetetraacetic acid (EDTA) (Invitrogen, Carlsbad, CA, USA) and collected by centrifugation at 1,000 rpm for 5 min at room temperature. Then, the cells were washed and resuspended with the medium for co-culture, namely, Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Nichirei Biosciences Inc.), 50 μM 2-mercaptoethanol (Nacalai Tesque, Kyoto, Japan) and 1% antibiotic-antimycotic solution (penicillin 10,000 U/mL, streptomycin 10,000 μg/mL, amphotericin B 25 μg/mL; Gibco®, Life Technologies). This RPMI-1640 basal medium is suitable for culturing spleen cells.

Preparation of spleen cells from mice

Spleens were surgically removed from more than twenty-four-week-old male C3H/HeN mice. Then, spleen cells were isolated as described.[14] Briefly, the spleen tissue was smashed with a stainless steel mesh in the RPMI-1640 basal medium, containing 10% FBS, 50 μM 2-mercaptoethanol, and 1% antibiotic-antimycotic solution. Cells were collected by centrifugation at 1,500 rpm for 5 min, and then, red blood cells in the collected cells were removed using a red blood cell lysis buffer (10 mM Tris-HCl [pH 7.3] containing 140 mM NH4 Cl and 1 mM Na2 EDTA). The spleen cells were washed and resuspended with the RPMI-1640 basal medium and filtered using a cell strainer (Falcon®, Corning Inc., NY, USA) to remove the residue.

Analysis of cytokine production from spleen cells which were co-cultured with OSCC cell and/or 10T1/2 cells

The spleen cell suspension (4 × 105/well) was added to a 96-well plate (3599, Corning), on which 1 μg/mL of anti-CD3 monoclonal antibody (mAb) (145-2C11, BD Biosciences, San Diego, CA, USA) had been immobilized (0.1 mL/well) at 4°C overnight. Then, OSCC and/or 10T1/2 cells were added to the wells and co-cultured with the spleen cells in the RPMI-1640 basal medium for 48 h in 5% CO2 at 37°C.

Cell viability, morphology, and growth rate were monitored during culturing of OSCC cell lines (Sq-1979 and L cells) as well as 10T1/2 cells in RPMI medium supplemented with or without 10% FBS.

To examine effects of humoral factors from OSCC or 10T1/2 cells, Transwell® (0.4 μm pore size, Corning, Inc., NY, USA) 24-well plates were used. The culture density is maintained from the 96-well plate and scaled up 6 times proportionally. The spleen cell suspension (2.4 × 106/well) was added to lower wells of the Transwell on which 1 μg/mL of anti-CD3 mAb (145-2C11) had been immobilized (0.6 mL/well) at 4°C overnight. OSCC or 10T1/2 cells were added to the upper Transwell inserts and were co-cultured for 48 h in 5% CO2 at 37°C.

Forty-eight hours later, the supernatant was harvested by centrifugation at 3,000 rpm for 5 min and stored at − 80°C. The concentration of IFN-γ and IL-10 in the supernatant of the cell culture was assayed by enzyme-linked immunosorbent assay (ELISA) using BD OptE1A set (BD Biosciences).

OSCC cell-conditioned medium

To confirm humoral factor(s) from Sq-1979 cells, conditioned medium (CM) was prepared from cultured Sq-1979-1 cells. One million OSCC (Sq-1979-1) cells were cultured in 10 mL of serum-free RPMI-1640 medium in 5% CO2 at 37°C. The OSCC-CM was collected after 48 h by centrifugation at 1,000 rpm for 5 min and then diluted to a final concentration of 12.5% in the culture medium.

Statistics

Data are expressed as means ± standard deviation (SD). For ELISA, Student's t-test was applied to determine the significance of differences between the two groups. P < 0.05 was considered to be statistically significant.


  Results Top


Interferon-γ-producing capability of spleen cells co-cultured with Sq-1979, 233, and L cells

In a preliminary experiment, we investigated the relationship between numbers of individual cells and the effects on cytokine production by spleen cells and have decided the optimal condition. The number of each cell types used in each assay was shown in figure legends. As shown in [Figure 1]a, a 1.8-, 2.1-, and 1.6-fold increase in the production of IFN-γ was observed in the co-culture of spleen cells with L3–5, L5–11, and L6–8 cells, respectively. However, the increase was abolished when cell-cell contacts between spleen cells and L-cells were eliminated using Transwell system [Figure 1]b.
Figure 1: The modulatory effects of OSCC cells on interferon-γ production by spleen cells stimulated with anti-CD3 monoclonal antibody. (a) Direct effects of L-cells on stimulated spleen cells: Spleen cells and L-cells were mixed and co-cultured in the same lower well of Transwell. (b) Indirect effects of L-cells on stimulated spleen cells: Spleen cells in the lower well of Transwell were co-cultured with L-cells in the upper Transwell insert. (c) Direct effects of Sq-1979- and 233 cells on stimulated spleen cells: Spleen cells and Sq-1979- and 233 cells were mixed and co-cultured in the same lower well of Transwell. (d) Indirect effects of Sq-1979- and 233 cells on stimulated spleen cells: Spleen cells in the lower well of Transwell were co-cultured with Sq-1979-and 233 cells in the upper Transwell insert. In all assay, spleen cells (2.4 × 106/well) were co-cultured with OSCC cells (6 × 104/well). The interferon-γ production was expressed as % of control, in which only spleen cells were cultured alone. Values were described as means ± standard deviation (n = 4). Student's t-test (*P < 0.05)

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The increase of IFN-γ production was not observed in the presence of Sq-1979-1, Sq-1979-2, and 233-11 cells irrespective of the direct cell contact to the spleen cells [Figure 1]c and [Figure 1]d.

The production of IFN-γ was hardly detected in the culture of the OSCC alone (data not shown). Therefore, these results indicate that the production of IFN-γ from spleen cells was specifically enhanced in the presence of L-cells by mechanism(s) dependent on the direct cell contact to the spleen cells.

Interleukin-10- and interleukin-4-producing capability of spleen cells co-cultured with Sq-1979, 233, and L cells

As shown in [Figure 2], in the presence of all OSCC cells examined, the production of IL-10 from the anti-CD3-stimulated spleen cells was significantly reduced by 30%–50% compared to that from the antibody-stimulated spleen cells only. Cell contacts between spleen cells and co-cultured cells are not required for the observed IL-10 reduction [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d.
Figure 2: The modulatory effects of OSCC on interleukin-10 production by spleen cells stimulated with anti-CD3 monoclonal antibody. Direct (a and c) or indirect (b and d) effects of OSCC on stimulated spleen cells were compared using Transwell chambers. All the experimental conditions are the same as in Figure 1. Values were described as means ± standard deviation (n = 4). Student's t-test (*P < 0.05; **P < 0.01)

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The effect of OSCC cells on the production of IL-4, another Th2 cytokine, was also examined [Figure 3]. In all of the OSCC cells examined, the production of IL-4 was reduced by 20%–50% compared to the antibody-stimulated spleen cells only, independent of the cell contacts between spleen cells and co-cultured cells [Figure 3]a,[Figure 3]b,[Figure 3]c,[Figure 3]d. Thus, IL-4 and IL-10 production was similarly downregulated by soluble factor(s) from OSCC cells.
Figure 3: The modulatory effects of OSCC cells on interleukin-4 production by spleen cells stimulated with anti-CD3 monoclonal antibody. Direct (a and c) or indirect (b and d) effects of OSCC cells on stimulated spleen cells were compared using Transwell chambers. All the experimental conditions are the same as in Figure 1. Values were described as means ± standard deviation (n = 4). Student's t-test (*P < 0.05; **P < 0.01)

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Interferon-γ-producing capability of spleen cells co-cultured with mesenchymal stromal cells

Next, we examined the effect of co-culturing mesenchymal stromal cells upon the production of Th1 cytokine from the anti-CD3-stimulated spleen cells. As shown in [Figure 4], the production of IFN-γ from the stimulated spleen cells significantly decreased to 40% upon co-culturing with 10T1/2 cells compared to control (lane 2 compared to lane 1). However, no IFN-g reduction was observed when the direct cell contact was interrupted by Transwell (lane 3). Therefore, the suppressive effect by 10T1/2 cells requires direct cell-cell contact with the spleen cells.
Figure 4: The suppressive effect of 10T1/2 cells on the interferon-g production by spleen cells stimulated with anti-CD3 monoclonal antibody. Spleen cells (Sp cells, 2.4 × 106/well) were added to lower well of Transwell, and then, 10T1/2 cells (1.8 × 104/well) were added to the lower well (Lane 2) or the upper insert (Lane 3). Cells were co-cultured as described above in “Materials and Methods.” The interferon-γ production was expressed as % of control, in which only spleen cells were cultured alone (Lane 1). Values were described as means ± standard deviation (n = 3). Student's t-test (*P < 0.05; **P < 0.01)

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Interferon-γ-producing capability of spleen cells co-cultured with mesenchymal stromal cells and OSCC cell

Our results so far revealed that the IFN-γ-producing capability of the stimulated spleen cells was significantly suppressed by the direct contact of mesenchymal stromal cells in our co-culture system. Next, we examined if the OSCC cells could further regulate the immunological suppression. As shown in [Figure 5]a and [Figure 5]b, the IFN-γ-producing capability of the stimulated spleen cells was significantly reduced in the presence of both 10T1/2 and Sq-1979-1 or Sq-1979-2 cells. The reduction shown in the presence of Sq-1979-1 or Sq-1979-2 cells was more than the levels in the presence of 10T1/2 cells only. Similarly, a significant reduction was also observed in the co-culture with 233-11 and 10T1/2 cells [Figure 5]c. By contrast, the IFN-γ-producing capability of the stimulated spleen cells was not reduced even in the presence of both 10T1/2 and L cells [Figure 5]d,[Figure 5]e,[Figure 5]f. Therefore, the suppressive activity of 10T1/2 was specifically enhanced by Sq-1979 cells, including Sq-1979-1,-2 and 233 cells but not by L-cells.
Figure 5: The effects of OSCC cells on the immune-suppressive activity of 10T1/2 cells. Spleen cells (4 × 105/well) were cultured with 10T1/2 cells (3 × 103/well) in the presence or absence of OSCC cells (3 × 103/well), (a-c) Sq-1979-and 233 cells, (d-f) and L cells. Cells were co-cultured as described above in “Materials and Methods.” The interferon-γ-production was expressed as % of control, in which spleen cells and 10T1/2 cells were co-cultured in the absence of OSCC cells. Values were described as means ± standard deviation (n = 4). Student's t-test (*P < 0.05; **P < 0.01 vs. control)

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Direct or indirect efficacy of Sq-1979 cells upon the immune-suppressive activity of 10T1/2 cells

We demonstrated that the immune suppression by 10T1/2 cells requires direct cell-cell contact with the stimulated spleen cells. Therefore, we also examined whether or not the suppressive effect by Sq-1979 cells, in the co-culture with 10T1/2 cells, also requires cell contacts. As shown in [Figure 6], the reduction of IFN-γ by Sq-1979-1 cells (lane 2) also occurred when the direct cell contact was eliminated by the Transwell filter (lane 3).
Figure 6: The direct or indirect effects of Sq-1979-1 cells upon the immune-suppressive activity of 10T1/2 cells in the presence (lanes 2, 3) or absence (lane 1) of Sq-1979-1 cells (3 × 103/well). In all experiments, spleen cells and 10T1/2 cells were in the same lower well of Transwell. Lane 1; Sq-1979-1 cells were absence. Lane 2; Sq-1979-1 cells were also in the lower well. Lane 3; Sq-1979-1 cells were in the upper Transwell insert. The interferon-g production was expressed as % of control (lane 1). Values were described as means ± standard deviation (n = 4). Student's t-test (*P < 0.05 vs. control)

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Effect of conditioned medium prepared from Sq-1979-1 cells

Our results suggest that humoral factor(s) secreted from Sq-1979-1 cells could specifically transmit a suppressive function against spleen cells in the co-culture system. To examine the hypothesis, we treated co-cultured spleen cells and 10T1/2 cells with CM prepared from Sq-1979-1 cells. As shown in [Figure 7]b, a comparable reduction of IFN-γ, as in the presence of Sq-1979-1 cells [[Figure 6] lane 2], was observed even in the absence of the cells. The reduction of IFN-γ production by CM was not observed in the absence of 10T1/2 cells [Figure 7]a.
Figure 7: The effects of conditioned medium prepared from Sq-1979-1 on the immune-suppressive activity of 10T1/2 cells. (a) Spleen cells (4 × 105/well) were cultured alone in the presence or absence of conditioned medium of Sq-1979-1. (b) Spleen cells (4 × 105/well) were cultured with 10T1/2 cells (3 × 103/well) in the presence or absence of conditioned medium of Sq-1979-1.The interferon-γ production was expressed as % of control, in which spleen cells and 10T1/2 cells were co-cultured in the absence of OSCC cells. Values were described as means ± standard deviation (n > 3). Student's t-test (*P < 0.05)

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  Discussion Top


Our results demonstrated that the production of a Th1 cytokine, IFN-γ, from the stimulated spleen cells was specifically enhanced in the presence of L-cells. On the other hand, the production of IFN-γ was unchanged even in the presence of Sq-1979 and 233-11 cells. The enhancement of immunological reaction against contacting L-cells was probably due to immunogenic variants obtained by mutagenesis.[15]

We demonstrated that direct contact of 10T1/2 cells exerted immunosuppressive effects upon antibody-stimulated spleen cells. In line with our observation, mesenchymal stromal cells have a broad range of immunosuppressive effects.[16],[17] We further demonstrate that the immunosuppressive effect of 10T1/2 cells was significantly enhanced in the presence of Sq-1979 cells; by contrast, the effect was not observed in the presence of L-cells. The results of our experiments using Transwell and CM from the Sq-1979 cells further revealed that this enhancement of immunosuppressive effect was mediated by potential humoral factor(s) secreted from the Sq-1979 cells. L-cells may not secrete such humoral factors in this co-culture system. As mentioned above, our in vivo model demonstrates that immunomodulatory mechanisms of the OSCC cells are developed depending on their malignancy;[18] however, in the presence of a co-culture system, the role of Th2 cytokines, IL-10 and IL-4, may not be essential since the producing capability was equally suppressed in the presence of all OSCC subclones. Our present results may, at least in part, explain an early event in a series of immune-suppressive mechanisms in the Sq-1979 cell-bearing mice, in which IFN-γ-producing capability of the spleen cells is markedly suppressed. By contrast, the mechanisms in the L-cell-bearing mice seem to be quite different.[11]

In squamous cell carcinoma tissues, tumor derived-transforming growth factor (TGF)-β1 modulates myofibroblasts to secrete hepatocyte growth factor, which, in turn, promotes tumor growth.[19] CAFs can promote regulatory T-cells through the expression of several cytokines including TGF-β, IL-6, CXCL8, and VEGF.[20] CAFs also promote an immune suppression through induction of protumoral macrophage.[9] Reciprocal interaction between OSCCs and CAFs mediated by IL-1 promotes cancer progression.[8],[21] However, most of these events seem to be associated with more progressive phenotypes with advanced tumors compared to those that we have observed in the Sq-1979 cells.[10] Even in the normal epidermal tissue, keratinocytes proliferate and differentiate in the interaction between stromal fibroblasts using specific cytokines.[22] In our preliminary experiment, an active form of TGF-β was not produced by all of the cells tested in our co-culture system.

In conclusion, our results using a mouse OSCC model suggest that immune-modulatory functions of OSCC depending on mesenchymal stromal cells could be uniquely regulated according to their malignant stages. The immune-suppressive function of 10T1/2 cells was specifically enhanced by humoral factor(s) from primary Sq-1979 cells. We will further evaluate the factor(s) essential for this activity of the non-advanced OSCC cells.

Acknowledgment

The authors would like to express their deepest thanks to Ms. Masako Sawada for her excellent secretarial assistance.

Financial support and sponsorship

This study was financially supported by the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science KAKENHI Grant Numbers (JP26463055 and JP15K11331).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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