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Abstract

This study investigated the metastatic potential of tongue squamous cell carcinoma (TSCC) cells after X-ray irradiation as well as radiation-induced changes in the biomechanical properties and cytoskeletal structure that are relevant to metastasis. Tca-8113 TSCC cells were X-ray-irradiated at increasing doses (0, 1, 2, or 4 Gy), and 24 h later, migration was evaluated with the wound healing and transwell migration assays, while invasion was assessed with the Matrigel invasion assay. Confocal and atomic force microscopy were used to examine changes in the structure of the actin cytoskeleton and Young’s modulus (cell stiffness), respectively. X-ray radiation induced dose-dependent increases in invasive and migratory potentials of cells relative to unirradiated control cells (p < 0.05). The Young’s modulus of irradiated cells was decreased by radiation exposure (p < 0.05), which was accompanied by alterations in the integrity and organization of the cytoskeletal network, as evidenced by a decrease in the signal intensity of actin fibers (p < 0.05). X-ray irradiation enhanced migration and invasiveness in Tca-8113 TSCC cells by altering their biomechanical properties and the organization of the actin cytoskeleton. A biomechanics-based analysis can provide an additional platform for assessing tumor response to radiation and optimization of cancer therapies.

Keywords

X-ray irradiation tongue squamous cell carcinoma metastasis atomic force microscopy biomechanical property actin cytoskeleton

Introduction

Tongue squamous cell carcinoma (TSCC) is the most common type of head and neck squamous cell carcinoma and is the sixth leading cause of cancer deaths worldwide.¹ In clinical oncology, radiation therapy is a standard treatment used for tumors; yet, despite significant advances in therapeutics, the prognosis for TSCC patients has not improved over the past three decades.^2,3 Numerous studies have shown that conventional radiotherapy used in cancer treatment may increase the risk of metastasis of malignant tumor cells,^4
–6 which is associated with the likelihood of disease recurrence.⁷ To improve patient survival, the potential for metastasis of tumors in response to radiation needs to be addressed.

Recent studies have indicated that the structural and mechanical properties of cells, including stiffness—which is calculated by Young’s modulus—is the basis of many cellular features and processes such as motility, transformation, morphology, and metastasis.^8
–10 Advances in bioengineering have enabled the investigation of mechanical properties of individual cells and molecules and physical forces acting on biological structures using probes that offer direct, real-time measurements.^10,11 Atomic force microscopy (AFM) is a widely used method that provides information on cellular ultrastructure (Figure S1).^12,13 AFM offers high-resolution image capturing, can be used in a range of imaging environments and for quantitative analyses,^14,15 and has been increasingly applied to cancer research.^16,17

The migratory capability and invasiveness of cells is dependent on the cytoskeleton, which governs the mechanical properties of cells.^10,18 Filamentous (F)-actin is a major component of the cytoskeleton, and plays a crucial role in cell shape changes during developmental and pathogenic processes.¹⁰ Alterations in the actin cytoskeletal network can thus provide important information regarding the status or behavior of a cell and is amenable to examination by AFM.¹⁹

Cancer metastasis induced by radiation is based on changes in various biological parameters.^20,21 To date, the effects of radiation on the metastatic process of tumor cells have not been investigated in the context of the biomechanical characteristics of tumor cells.²² This study investigated the consequences of X-ray irradiation on migration and invasiveness of TSCC cells and the role of biomechanical properties and cytoskeletal organization in irradiation-induced changes in metastatic potential.

Materials and methods

Reagents and materials

Phalloidin–fluorescein isothiocyanate (FITC) conjugate solution (F7250) and 4′,6-diamidino-2-phenolindole (DAPI; D9564) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Transwell inserts (PIEP12R48) and membrane matrices (356234) were from Millipore (Danvers, Massachusetts, USA) and Becton Dickinson Bioscience (Billerica, Massachusetts, USA), respectively. Roswell Park Memorial Institute (RPMI)-1640 medium (11875093) and fetal bovine serum (FBS) (10099141) were purchased from Life Technologies (Carlsbad, California, USA). The human TSCC cell line Tca-8113(GDC17) was obtained from the China Center for Type Culture Collection (Wuhan, China). Plastic Petri dishes (35 mm) (430165), 24-well plates (3524), and cell culture flasks (T-75) (156472) were purchased from Corning Incorporated (Corning, New York, USA).

Cell culture and sample preparation

Cells were maintained in RPMI-1640 medium (11875093, Life Technologies, Carlsbad, California, USA) containing 10% FBS (10099141, Life Technologies), 1% penicillin–streptomycin solution (15140122, Life Technologies), and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (15630080, Life Technologies) in culture flasks (156472, Corning Incorporated, Corning, New York, USA) at 37°C in a humidified atmosphere of 5% carbon dioxide (CO₂) and 95% air and grown to 70%–90% confluence. Cells were passaged every 3 days and were incubated in 0.25% trypsin–ethylenediaminetetraacetic acid (15400054, Life Technologies) for 1 min at 37°C before use. Trypsinization was terminated by adding fresh supplemented medium to the culture. Cells were counted with a hemocytometer. Prior to migration and invasion assays, the monolayer of cells was serum-starved in RPMI-1640 containing 0.5% FBS and 1% penicillin/streptomycin for 24 h. Cells were prepared for confocal or AFM by plating in 35 mm Plastic Petri dishes (430165, Corning Incorporated) with 2 ml media/dish at a density of 5 × 10⁴ cells/ml or inoculating on sterilized 14 mm² glass coverslips (021-54090204, WHB, Shanghai, China), respectively, for 24 h prior to X-ray irradiation.

Irradiation

Cells grown in culture flasks and Petri dishes (Corning Incorporated) were treated with 100 kVp X-ray at doses of 1, 2, and 4 Gy and a rate of 1.027 Gy/min using a 43885D X-ray machine (Faxitron, Lanzhou, China). Cells were irradiated from a vertical direction at room temperature in the absence of CO₂. Unirradiated cells (0 Gy) served as the control. Cells were incubated at 37°C for 24 h before analysis.

Wound healing assay

The assay was performed as previously described,^23
–25 with some modifications. Briefly, cells were grown to confluence and manually wounded by scratching the culture flask/dish with a 200 μl pipette tip, then washed with phosphate-buffered saline (PBS; pH 7.4; 20012027, Life Technologies) to remove cellular debris. After irradiation, cells were allowed to migrate in serum-free medium for 24 h and then monitored using a TS100 inverted light microscope (Nikon, Tokyo, Japan). The distance from the wound site (0 h) to the position 24 h later was measured using Image-Pro Plus v6.0 software (Media Cybernetics, Inc., Rockville, Maryland, USA).

Transwell migration assay

In addition to the scratch test, cell migration was assessed using a 24-well microchemotaxis chamber with upper and lower sections separated using a polycarbonate filter with 8-μm pores (Millipore).⁴ Irradiated and control cells were trypsinized, resuspended in serum-free medium with 0.1% bovine serum albumin (BSA; ZLI-9023, ZSGB-BIO, Beijing, China), and the concentration was adjusted to 5 × 10⁵ cells/ml; 200 μl of the cell suspensions (1 × 10⁵ cells) were transferred to the upper half of a well. After adding 500 μl medium supplemented with 10% FBS (10099141, Life Technologies) and 1% penicillin/streptomycin (15140122, Life Technologies) to the bottom half, the chamber was incubated at 37°C in humidified air with 5% CO₂. After 24 h, the chamber was disassembled, and the cells that had not migrated were removed with a cotton drill, while migrated cells on the underside of the membrane were fixed in methanol (Boster, Wuhan, China) and stained with 0.04% crystal violet (C6158-50G, Sigma-Aldrich). Migrated cells were counted from four random fields under an inverted light microscope (TS100, Nikon, Tokyo, Japan) (20×).

Matrigel invasion assay

Cell invasion was assessed by the penetration of cells through transwell inserts (PIEP12R48, Millipore, Danvers, Massachusetts, USA) placed on a 24-well plate (3524, Corning Incorporated) and coated with Matrigel (50 μg/ml; 356234, Becton Dickinson Bioscience) overnight at 4°C.²⁵ Irradiated cells were trypsinized, resuspended in medium containing 0.1% BSA (ZLI-9023, ZSGB-BIO, Beijing, China), and adjusted to a final concentration of 1 × 10⁶ cells/ml; 200 μl (2 × 10⁵ cells) of cell suspension were added to the upper half of the chamber. Medium supplemented with 10% FBS (500 μl) was added as a chemoattractant to the lower half. After incubation at 37°C for 24 h, cells remaining on the upper side of the membrane were scraped off with a cotton swab, and cells that had passed through the insert to the lower side were fixed with 100% methanol (Boster) and stained with 0.04% crystal violet (C6158-50G, Sigma-Aldrich). Cells that had invaded through the Matrigel-coated membrane were counted in four random fields under an inverted light microscope (TS100, Nikon; 20×).

AFM imaging

The ultrastructure of single cells was examined and Young’s modulus was calculated by AFM.^26,27 Before imaging, cells cultured on glass coverslips were washed three times with PBS (20012027, Life Technologies) to remove dead or nonadherent cells, and serum-free medium was added to Petri dishes (35 mm; 430165, Corning Incorporated). A JPK NanoWizard III (JPK Instruments AG, Berlin, Germany) AFM was used in combination with an inverted optical microscope (Axiovert 200 MAT; Zeiss, Göttingen, Germany). Measurements were performed in cell culture medium under ambient conditions. A sharpened silicon nitride cantilever tip with a nominal spring constant calibrated to 0.03 N/m was used for cell scanning.²⁸ The cantilever sensitivity was calibrated by indenting the glass substrate in the presence of cell culture medium. An indentation depth of at least 1 μm was selected in order to better simulate deformations that occur physiologically.²⁹ A representative optical microscope screenshot obtained during the AFM experiment is shown in Figure S2. All indentations were performed on randomly selected single cells. The Young’s modulus data acquisition and analysis were carried out with JPK data processing software (Version spm_4.2.50, JPK, Germany). The Young’s modulus of individual cells was obtained by applying a Hertz’s contact model^30,31 to the force–distance curves:

F = \frac{4}{3} \frac{E}{(1 - v^{2})} \sqrt{R} δ^{\frac{3}{2}},

where F is the indentation force, E is the Young’s modulus, ν is the Poisson ratio (assumed to be 0.5 for soft biological materials³¹), R is the radius of the spherical structure, and δ is indentation depth.

Actin cytoskeleton imaging by confocal microscopy

The organization of the actin cytoskeleton (i.e. the F-actin network) was examined by direct fluorescence labeling.³² Cells grown on glass coverslips were irradiated and were fixed 24 h later with 1 ml 3.7% formaldehyde solution (F8775-500ML, Sigma-Aldrich) in PBS for 30 min at room temperature, washed three times with PBS, permeabilized with 1 ml 0.1% Triton X-100 (T9284-100ML, Sigma-Aldrich), and blocked with 1% BSA (ZLI-9023, ZSGB-BIO, Beijing, China) in PBS for 25 min. To label F-actin, cells were incubated with phalloidin–FITC solution (F7250, Sigma-Aldrich) at a 1:34 dilution for 1 h, and nuclei were stained with 1:500 DAPI (D9564, Sigma-Aldrich) for 15 min. The staining was performed at 37°C in a dark room with three PBS washes between steps. Coverslips with stained cells were placed face down on a glass slide and sealed with nail polish. Fluorescence images were acquired using a confocal microscope (LSM700; Zeiss, Germany; 40×). The emission wavelengths for phalloidin–FITC and DAPI were 405–488 nm and 559–639 nm, respectively. The mean staining intensity of the actin cytoskeleton in each cell was analyzed using Image-Pro Plus v6.0 software (Media Cybernetics, Inc., Rockville, Maryland, USA).

Statistical analysis

All experiments were performed at least in triplicate. Data are shown as the mean ± SD. Mean differences were evaluated using Student’s t test (two-sided). A p value < 0.05 was considered statistically significant.

Results

Increase in metastatic potential of TSCC cells after X-ray irradiation

Cell migration and invasion are fundamental to the metastatic process of tumor cells. To examine the effect of radiation on the metastatic potential of Tca-8113 cells, migration and invasiveness were assayed 24 h after exposure to a range of doses of X-ray radiation (0, 1, 2, and 4 Gy), with untreated controls set as 100%.

In the wound-healing assay, X-ray irradiation at 1 Gy caused an increase in the percentage of migrated cells to 134% of the control group, while migration increased to 185% with a dose of 2 Gy. At the highest dose of 4 Gy, cell migration was at 237% of the control value (Figure 1). These results were confirmed by the transwell migration assay, that is, the percentages of cells that migrated through the membrane to the lower part of the chamber after exposure to 1, 2, or 4 Gy of X-ray radiation were 173%, 230%, and 309%, respectively, relative to unirradiated control cells, showing a dose-dependent effect of radiation on cell migration (Figure 2). The average number of migrated cells counted from four random fields was listed accurately (Table S1).

Figure 1.

Migratory potential of Tca-8113 TSCC cells left untreated (control) or treated with indicated doses of X-ray radiation, as assessed by the wound-healing assay. Results are expressed as a percentage of control cells (100%). Data are means ± SD (n = 3; t test; *p < 0.05). TSCC: tongue squamous cell carcinoma.

Figure 2.

Migratory potential of Tca-8113 TSCC cells left untreated (control) or treated with indicated doses of X-ray radiation, as assessed by the transwell migration assay. Results are expressed as a percentage of control cells (100%). Data are means ± SD (n = 3; t test; *p < 0.05). TSCC: tongue squamous cell carcinoma.

The Matrigel assay was used to assess changes in invasive potential. Tca-8113 cells had markedly higher invasiveness after irradiation at doses of 1, 2, or 4 Gy (140%, 198%, and 285%, respectively, relative to the control; Figure 3). The average number of invaded cells counted from four random fields was shown accurately (Table S2). Taken together, these results indicate that exposure to X-ray radiation enhances migratory and invasive potentials in TSCC cells in a dose-dependent manner.

Figure 3.

Invasive potential of Tca-8113 TSCC cells left untreated (control) or treated with indicated doses of X-ray radiation, as assessed by the Matrigel invasion assay. Results are expressed as a percentage of control cells (100%). Data are means ± SD (n = 3; t test; *p < 0.05). TSCC: tongue squamous cell carcinoma.

Decrease in Young’s modulus and increase in morphological distortion in X-ray-irradiated TSCC cells

In AFM, different scan modes (e.g. contact or tapping) are selected according to specific characteristics of the sample.³³ Given the relatively soft cell surface, the contact mode was used to examine the central region of live Tca-8113 cells, which allowed a large number of images to be acquired. AFM vertical deflection images (Figure 4(a), (d), (g) and (j)) showed a variety of changes in cell surface morphology. Under normal culture conditions, cells had a regular shape, with cell surface molecules intact and uniformly distributed (Figure 4(a)). In contrast, in irradiated cells dose-dependent changes in the surface morphology were observed, with cells becoming irregular and misshapen (Figure 4(d), (g) and (j)), resulting in decreased cell height (Figure 4(b), (e), (h) and (k)). The AFM data integrated into three-dimensional images are shown in Figure 4(c), (f), (i) and (l). Force–distance curves were obtained by mechanically distorting individual cells.

Figure 4.

Images obtained by AFM of Tca-8113 TSCC cells before and after exposure to indicated doses of X-ray radiation. (a) to (c) Unirradiated cells (0 Gy) served as the control; cells were exposed to ((d) to (f) 1 Gy, ((g) to (i)) 2 Gy, and ((j) to (l)) 4 Gy of radiation. Representative images of vertical deflection ((a), (d), (g) and (j)) and height ((b), (e), (h) and (k)) and in three dimensions ((c), (f), (i) and (l)) are shown (×40). AFM: atomic force microscopy; TSCC: tongue squamous cell carcinoma.

The histograms of Young’s modulus values for individual cells are shown in Figure 5. Irradiation reduced the Young’s modulus in TSSC cells relative to nonirradiated control cells, and a minimum value was attained at 4 Gy (0.950 ± 0.025 kPa), suggesting that cells were more deformable at higher doses of radiation.

Figure 5.

Histograms of changes in Young’s modulus measurements from individual Tca-8113 TSCC cells treated with indicated doses of X-ray radiation determined by AFM analysis. Data are means ± SD (n = 3; t test; *p < 0.05). AFM: atomic force microscopy; TSCC: tongue squamous cell carcinoma.

Decrease in Young’s modulus associated with changes in the actin cytoskeleton

To explore the relationship between mechanical properties of tumor cells and the cytoskeletal network, the distribution of F-actin was examined in phalloidin-treated cells by confocal microscopy. The nucleus was labeled with DAPI to confirm cell viability. Confocal images of cell nuclei showed that the cells were not dead or apoptotic (Figure 6(a), (d), (g) and (j)). In unirradiated control cells, actin fibers formed an isotropic network throughout the cell body, organized into parallel filamentous structures (Figure 6(b)). Treatment with increasing doses of X-ray radiation led to a decrease in the intensity of the actin signal and progressive disorganization of the actin F network (Figure 6(e), (h) and (k)). Thus, the dose-dependent decrease in Young’s modulus and higher metastatic potential observed upon irradiation was associated with a loss of actin fibers and cytoskeletal organization relative to control cells (Figure S3). Taken together, these results indicate that exposure to X-ray radiation induces changes in the biomechanical properties of TSCC cells that are likely responsible for the corresponding increase in metastatic potential, including greater capacities for migration and invasiveness.

Figure 6.

X-ray-induced morphological changes in F-actin organization in Tca-8113 TSCC cells. Images of (a) DAPI-stained cell nuclei (blue) and (b) phalloidin–FITC-stained actin fibers (green) after a 24 h radiation treatment at a dose of 0 Gy. (c) Merged images of (a) and (b). Representative images of cell nuclei and the actin cytoskeleton, as well as merged images are shown for cells treated with doses of ((d) to (f)) 1 Gy, ((g) to (i)) 2 Gy, and ((j) to (l)) 4 Gy (×40). F-actin: filamentous actin; TSCC: tongue squamous cell carcinoma; DAPI: 4′,6-diamidino-2-phenolindole; FITC: fluorescein isothiocyanate.

Discussion

Conventional radiotherapy is generally considered to be effective for the treatment of cancer, including for squamous cell carcinomas of the oral cavity. However, radiation exposure carries the risk of undesirable secondary effects, such as the potential for tumor cell metastasis,^4,34,35 which is a major factor in cancer recurrence.^36,37 Thus, while radiation therapy is one of few available treatment options, the chances of patient survival are diminished due to the possibility of radiation-induced metastasis.³⁸ X-ray irradiation increases the metastatic potential of glioma cells;^39,40 given the poor prognosis of TSCC, it is essential to determine whether similar risks exist for this cancer so that effective and safer treatment methods can be developed.

The majority of studies have examined the effects of X-ray radiation on the metastatic potential of cancer cells by evaluating changes in the expression or status of migration-related proteins and signaling pathways.^39,41,42 However, less is known about how radiation affects the biomechanics of the cell and thereby alters cell behavior. Several reports have demonstrated that biomechanical properties are a fundamental determinant of metastatic potential.^32,43 For instance, low migratory and invasive potentials are associated with a high elastic modulus (i.e., greater stiffness).⁴³ In this study, the role of biomechanical properties in radiation-induced metastatic potential were investigated by AFM. A nanoindentation technique was used to determine Young’s modulus and analyze force curves generated for irradiated, single TSCC cells relative to nonirradiated control cells based on Hertz’s contact model.

Consistent with the findings of other studies, the AFM scanning analysis showed that cellular ultrastructure changed markedly upon radiation exposure (Figure 4), corresponding to lower Young’s modulus values (Figure 5) and increased migratory and invasive capacities (Figures (1) to (3)) compared with the control cells. The Hertz contact model assumes a homogeneous, continuous, and incompressible sample surface, which is only an approximation for living cells.⁴⁴ Nonetheless, this model is widely accepted as a means of characterizing cell stiffness and is particularly useful in studies using cell lines, which are assumed to be more homogeneous than biological samples.¹⁷

The shape of a cell is determined by the cytoskeleton, which confers tensile strength and resistance to deformation.³ According to one study, cells with lower Young’s modulus values also have greater disorganization of the cytoskeletal structure.⁴⁵ In accordance with these findings, in this study, the observed decrease in Young’s modulus in cells exposed to X-ray radiation was associated with a reduction in the organization of the actin cytoskeleton and decrease in signal intensity of actin fibers stained with a phalloidin–FITC conjugate. These data indicate a decrease in cell stiffness, an alteration that would favor cell movement and, by extension, metastasis.

In summary, the results presented here demonstrate that the metastatic behavior of TSCC cells—including migration and invasion—increases dose dependently with exposure to X-ray radiation. This was due to a reduction in cell stiffness wrought by changes in the actin cytoskeleton network. Thus, alterations in the biomechanical properties of tumor cells underlie behavioral changes that promote metastasis and ultimately reduce patient survival. These findings can inform clinical decisions of appropriate therapies for cancer treatment and also provide a basis for the development of new drugs that target biomechanical characteristics of tumor cells, which could diminish the risk of metastasis and improve patient prognosis.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by the National Basic Research Program of China (973 Program) (2010CB834202), the State Ethnic Affairs Commission Research Project (12XB12), and the Fundamental Research Funds for the Central Universities (31920130029, lzujbky-2014-160).

Supplemental Material

The online data supplements are available at .

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X-ray radiation promotes the metastatic potential of tongue squamous cell carcinoma cells via modulation of biomechanical and cytoskeletal properties