DOI: https://doi.org/10.21203/rs.3.rs-849275/v1
The cytotoxic effect of radiation plays an important role in the treatment of head and neck cancer. However, irradiation is known to lead to the migration of various cancer cells, including those of head and neck cancer. Recently, fibroblasts in the cancer microenvironment have been reported to be involved in this mechanism. Nevertheless, the mechanism underlying the migration of head and neck cancer cells remains unclear. The purpose of this study was to elucidate this migration mechanism induced by irradiation in terms of the interaction of head and neck cancer cells with fibroblasts.
We used the head and neck squamous cell carcinoma (HNSCC) cell lines SAS and FaDu as well as fibroblast cell lines. These cells were irradiated and their viability was compared. In fibroblasts, changes in interleukin-6 (IL-6) secretion caused by irradiation were measured by enzyme-linked immunosorbent assay (ELISA). The cell migration ability of cancer cells was evaluated via a migration assay using a semipermeable membrane. HNSCC cells were cocultured with irradiated and nonirradiated fibroblasts, and their migration ability under each condition was compared. We also examined the effect of IL-6 on the migration of HNSCC cells. Furthermore, to investigate the effect of fibroblast-derived IL-6 on the migration ability of HNSCC cells, we conducted a coculture study using IL-6 neutralizing antibody.
Irradiation reduced the survival of HNSCC cells, whereas fibroblasts were resistant to irradiation. Irradiation also increased IL-6 secretion by fibroblasts. Migration of HNSCC cells was enhanced by coculture with fibroblasts and further enhanced by coculture with irradiated fibroblasts. We also confirmed that the migration of HNSCC cells was induced by IL-6. The enhanced migration of cancer cells caused by coculturing with fibroblasts was canceled by the IL-6 neutralizing antibody.
These results show that fibroblasts survive irradiation and induce the migration ability of HNSCC cells through increased secretion of IL-6.
Radiotherapy plays an important role in the treatment of head and neck cancer; indeed, it has been used at every stage of treatment [1, 2]. However, although irradiation has cytotoxic effects on cancer cells, it reportedly promotes tumorigenicity, and especially migration of cancer cells. This phenomenon results in increased local invasion, distant metastasis, and therefore poor prognosis for patients with various cancers including head and neck cancer [3, 4]. Thus, the conflicting effects of irradiation on cancer cells are a major challenge to be overcome in cancer treatment.
In recent years, it has been found that irradiation induces tumorigenic effects not only in cancer cells but also the cancer microenvironment. This alteration in the cancer microenvironment is reportedly involved in the recurrence and metastasis of cancer after radiotherapy [5, 6]. Mechanistically, the importance of various cytokines and growth factors in mediating the interaction between cancer cells and supporting cells, such as fibroblasts, in the cancer microenvironment has been confirmed [7–9]. In particular, interleukin-6 (IL-6) is known to be involved in the interaction between fibroblasts and cancer cells in the irradiated cancer microenvironment, and its role has attracted attention [10, 11].
To date, few studies have been conducted on irradiation-induced tumor progression in head and neck cancer; thus, much remains to be elucidated. The purpose of this study was therefore to elucidate the mechanism of irradiation-induced head and neck cancer cell migration in terms of the interaction of these cells with fibroblasts.
Cells and cell culture
We purchased SAS, a human tongue squamous cell carcinoma cell line, from the RIKEN Cell Bank (Tsukuba, Japan). FaDu, the cells from a human hypopharyngeal SCC cell line, were kindly gifted by the Department of Cell Biology and Morphology, Akita University Graduate School of Medicine (Akita, Japan). SF-TY, a human fibroblast cell line, was obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan).
All cells were maintained in the Dulbecco's modified Eagle's medium (DMEM; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C. For neutralization of IL-6, neutralizing mouse anti-IL-6 mAb (MAB406) and isotype control immunoglobulin G1 (IgG1) mAb (MAB005) were purchased from R & D Systems (Minneapolis, MN)
Irradiation
Cells were seeded in 60 mm dishes and cultured to 80% confluence before being irradiated at 2, 5, or 10 Gy at room temperature using a 160 kVp cabinet X-ray system filtered with 0.5 mm Cu by the Faxitron CP-160 (Faxitron X-Ray Corp., Wheeling, IL, USA). After irradiation, cells were cultured for 24 h prior to being harvested and subjected to an IL-6 enzyme-linked immunosorbent assay (ELISA) assay or cell migration assay. To conduct a cell survival assay, cells were cultured for 6 days after irradiation before being fixed and stained with crystal violet and then being observed under a light microscope (Olympus, Tokyo, Japan).
Cell survival assay (crystal violet staining)
The cells were fixed in 4% paraformaldehyde for 20 min at room temperature and then stained with 0.04% crystal violet in 1% ethanol (20 min at room temperature). The plates were subsequently washed extensively under running tap water and air dried. After solubilization of the samples in 1% sodium dodecyl sulfate (SDS), the optical density values were read by plate reader at 550 nm.
Migration assay
We evaluated cell migration in vitro using semipermeable modified Boyden inserts with a pore size of 8 µm (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). In total, 3 × 104 SAS and FaDu cells were plated in the inserts. For the coculture assay, 5 × 104 fibroblast cells were plated in a holding well. Plating was conducted on serum‑free DMEM. We plated the same number of cells from the inserts in 96-well plates to serve as loading controls. Both the inserts and holding wells were filled with serum-free medium composition. Depending on the needs of experiment, 10 ng/ml of IL-6, 20 ng/ml of IL-6 neutralizing antibody, or 20 ng/ml of control IgG were added to the medium. After 24 h of treatment at 37°C in a 5% CO2 incubator, we gently wiped away the cells in the insert using a cotton swab. Cells on the reverse side of the insert were fixed and stained with Diff‑Quik® (Sysmex, Kobe, Japan) according to the manufacturer's instructions. We counted the invading cells in four representative fields using light microscopy at a magnification of 200×. Cells plated in 96-well plates were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and we normalized the cell numbers across the groups. We also adjusted the number of migrating cells accordingly.
Western blotting
We detected protein expression using western blot analysis with actin used as an internal control. We lysed cell lines in detergent containing 1% NP-40, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin and then determined the protein levels using the Bio-Rad Protein Assay method (Bio-Rad Laboratories Inc., Hercules, CA, USA). We separated 40 µg of the total protein on 8% SDS-PAGE gels and transferred these to nitrocellulose membranes using a semidry transfer machine (Bio-Rad Laboratories). Next, we blocked membranes with 5% skimmed milk/TBS with Tween-20 solution for 1 h at room temperature, before incubating with primary antibodies in 5% skimmed milk in TBS‑T overnight at 4°C. After washing with TBS-T three times, we incubated the membranes for 1 h with horseradish-peroxidase-conjugated secondary antibody (Bio-Rad Laboratories) at 1:3,000 diluted in 5% skimmed milk in TBS‑T. We then rinsed the filters with TBS‑T three times and developed the blot using Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by autoradiography. The band intensities were analyzed using ImageJ (U. S. National Institutes of Health). We used the following primary antibodies: mouse anti-IL-6Rα (1:1,000; Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-gp130 (1:1,000; Santa Cruz Biotechnology), and mouse anti-actin (1:3,000; Santa Cruz Biotechnology).
ELISA
The serum levels of IL-6 were measured by an ELISA using an ELISA kit (Proteintech, Rosemont, IL, USA.). To evaluate changes in IL-6 expression by irradiation in fibroblasts, 3 × 106 fibroblasts were seeded on 6-well plates in DMEM with 10% fetal bovine serum (FBS) overnight. The medium was changed to serum-free DMEM and irradiated at 10 Gy. Nonirradiated fibroblasts were also prepared as a control. At 24 h after irradiation, supernatants were collected, and subjected to an ELISA assay.
Statistical analysis
Statistical analyses were performed using Statcel 3 (OMS Publishing, Tokorozawa, Japan). A Wilcoxon–Mann–Whitney two-tailed exact test was used to assess the statistically significant differences in cell survival, migration studies, and IL-6 expression. Data are presented as means ± standard deviation (SD) from experiments that were repeated at least three times. We considered differences with P < 0.05 as statistically significant.
Fibroblast resistance to irradiation compared with HNSCC cells
Radiotherapy utilizes the difference in radiosensitivity between target cancer cells and healthy cells; during radiotherapy, cancer cells are killed by irradiation, whereas other cells in the surrounding microenvironment survive. In particular, the major cells in the microenvironment, fibroblasts, survive and are involved in tissue remodeling after radiotherapy [12]. In the present study, we first assessed whether there was a difference in radiosensitivity between HNSCC cells and fibroblasts. When two HNSCC cell lines, SAS and FaDu, as well as a healthy fibroblast cell line were irradiated with 2, 5, and 10 Gy the survival rate of HNSCC cells decreased with irradiation in a dose-dependent manner relative to the survival of cells under nonirradiated conditions; it contrast, the fibroblasts did not show a significant decrease in survival rate (Fig. 1). This result suggests that fibroblasts are more resistant to radiation than HNSCC cells and may survive even after radiotherapy.
Irradiation enhances the ability of fibroblasts to promote HNSCC cell migration
Fibroblasts are known to contribute to cancer progression and to be important factors in cancer control in various solid cancers [13]. In addition, fibroblasts that survive after radiotherapy serve as a scaffold and contribute to cancer recurrence and metastasis [5, 6, 14]. In other words, irradiation enhances the ability of fibroblasts to promote the activity of cancer cells. In the present study, we investigated how irradiation affects the HNSCC-activating effect of fibroblasts. In a migration assay, HNSCC cells were seeded into semipermeable inserts and cocultured with or without irradiated or nonirradiated fibroblasts, which were seeded into the lower chamber. Our aim was to determine whether fibroblasts influence the migration of HNSCC cells and, if so, how irradiation might affect this ability. As shown in Fig. 2, the migration of HNSCC cells was enhanced even when they were cultured with nonirradiated fibroblasts, but this migration ability was further enhanced when cocultured with irradiated fibroblasts. Thus, irradiation seems to extend the ability of fibroblasts to promote HNSCC cell migration. Furthermore, the result suggests that the factor promoting the migration of HNSCC cells might be a liquid component secreted from fibroblasts.
Irradiation increases the expression of IL-6 from fibroblasts
The results shown in Fig. 2 suggest that fibroblasts use some humoral factor to induce migration of HNSCC cells and that the expression of this factor is increased by irradiation. Although various humoral factors are known to be involved in cancer progression, IL-6 has recently been associated with various carcinomas including HNSCC. Because IL-6 elicits several tumor growth-promoting effects including promotion of cancer cell migration, it is also a promising therapeutic target [15, 16](Johnson DE 2018; Choy 2020). Therefore, we investigated IL-6 as the potential factor secreted from fibroblasts; specifically, we assessed whether IL-6 secretion was increased by irradiation. According to an ELISA, expression of IL-6 in the culture media of irradiated (10 Gy) fibroblasts increased compared with its expression in media from nonirradiated fibroblasts (Fig. 3). This result indicates that irradiation induces IL-6 secretion from fibroblasts; it also suggests the possibility that IL-6 is the factor that caused migration of HNSCC cells (see Fig. 2).
IL-6 induces HNSCC cell migration
To determine whether IL-6 induces migration of HNSCC cells, we first confirmed whether HNSCC cells can be activated by IL-6. For IL-6 to demonstrate its biological activities, the target cell must express IL-6 receptor (IL-6R) and gp130 [17]. We confirmed that both SAS and FaDu HNSCC cell lines express IL-6R and gp130 via western blotting (Fig. 4A). This result suggests that HNSCC cells can be activated by an intracellular signal transduction pathway via exogenous IL-6. In addition, we conducted a migration assay using HNSCC cells with or without IL-6 and found that HNSCC cell migration was increased in the presence of IL-6 (Fig. 4B).
IL-6 plays a crucial role in the ability of fibroblasts to promote HNSCC migration
We confirmed whether fibroblasts promote HNSCC cell migration using IL-6 secretion. For this purpose, we performed a HNSCC cell migration assay including coculturing with fibroblasts and with or without an IL-6 neutralizing antibody. Results showed that HNSCC cell migration was induced by coculturing with fibroblasts; however, this induction of HNSCC migration was significantly suppressed by the IL-6 neutralizing antibody (Fig. 5). Thus, IL-6 secreted from fibroblasts is apparently involved in the enhancement of HNSCC cell migration.
Invasion and distant metastasis are major characteristics of cancer that are associated with the progression of the disease and poor prognosis [18]. This is true for HNSCC, especially of distant metastases that are difficult to treat despite the development of therapeutic agents [19–21]. Thus, metastasis is clinically important in cancer treatment. It has been reported that the migration of cancer cells is essential in the process of metastasis. When cancer cells invade surrounding tissues, the cells migrate between tissues while destroying the surrounding tissues with enzymes. The migrating cells then infiltrate the vessels and become tumor cells that circulate throughout the body, creating metastatic lesions at distant locations. Thus, cell migration is an important first step in distant metastasis [22].
Previous studies have shown that irradiation contributes to increased distant metastases in many cancers, including head and neck cancers [23–25]. Therefore, the enhancement of cell migration by irradiation has attracted the attention of researchers. Various studies have shown hypoxia, epithelial-to-mesenchymal transition, and specific cytokines as causes of irradiation-induced migration enhancement [3, 4]. In particular, cytokines are secreted not only from cancer cells but also from the cells in the environment surrounding the cancer, and they are important for various tumor progression phenomena such as cancer cell invasion and the increase in circulating tumor cells [8, 10]. Fibroblasts in the cancer microenvironment are known as cancer-associated fibroblasts; these are strongly associated with cancer cells and contribute to cancer progression including the progression of head and neck cancers [26, 27]. Under irradiation, fibroblasts are also important for cancer progression [28]. For example, in pancreatic and liver cancers under irradiation, fibroblasts increase the secretion of various humoral factors including cytokines such as bFGF, TNF-a, VEGF, IL-6, EGF, MMP-2, and MMP-9. These humoral factors contribute to cancer progression, including the migration and invasion of cancer cells after irradiation [7, 9]. Thus, cytokines induced from fibroblasts by irradiation have important effects on the enhanced migration ability of cancer cells after irradiation.
In the present study, we showed that IL-6 secreted from fibroblasts is an important factor in the induction of HNSCC cell migration. IL-6 has been primarily reported as a factor in inflammation, but it is also known to be involved in tumor progression in many cancers [29, 30]. Therefore, its potential as a therapeutic target for cancer has been indicated; furthermore, the antitumor effect of inhibiting the JAK/STAT3 pathway, which is the main signaling pathway of IL-6, has been reported [30, 31]. In addition, IL-6/JAK/STAT3 pathway inhibition has a synergistic antitumor effect with conventional cancer treatments, e.g., its effect is enhanced in combination with chemotherapy and radiotherapy [30, 32]. In particular, recent studies have reported that IL-6 is associated with the effects of immune checkpoint inhibitor in the treatment of head and neck cancer. Consequently, IL-6 is a factor expected to become increasingly important in the treatment of HNSCC in the future.
The results of this study showed that fibroblasts survive irradiation and that irradiation induces expression of IL-6 from fibroblasts. It was also found that irradiation enhances the ability of fibroblasts to promote the migration of HNSCC cells.
Moreover, it was confirmed that fibroblasts induce HNSCC cell migration through IL-6 expression. Taken together, these results suggest the possibility that fibroblasts survive irradiation and enhance the promotion of HNSCC cell migration through IL-6 secretion. To the best of our knowledge, we are the first to speculate on the mechanism by which irradiation promotes HNSCC cell migration. Thus, our results could contribute to the future development of therapeutics in HNSCC including the treatment of recurrence and metastasis after radiotherapy.
DMEM Dulbecco's modified Eagle's medium
ELISA Enzyme-linked immunosorbent assay
FBS Fetal bovine serum
HNSCC Head and neck squamous cell carcinoma
SD Standard deviation
SDS Sodium dodecyl sulfate
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and material
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Competing interest
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.
Funding
The present study was supported by a Grant-in-Aid for Scientific Research (C) (grant no. 18K09337) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Author Contributions
SS: conceptualization, design, and writing-original draft preparation. ST, YA, TE, and TK: data acquisition and interpretation. TY: supervision. All authors have read and approved the final manuscript.
Acknowledgements
Not applicable.