Study of morphological and mechanical features of multinuclear and mononuclear SW480 cells by atomic force microscopy

This article studies the morphological and mechanical features of multinuclear and mononuclear SW480 colon cancer cells by atomic force microscopy to understand their drug‐resistance. The SW480 cells were incubated with the fullerenol concentrations of 1 mg/ml and 2 mg/ml. Morphological and mechanical features including the height, length, width, roughness, adhesion force and Young's modulus of three multinuclear cell groups and three mononuclear cell groups were imaged and analyzed. It was observed that the features of multinuclear cancer cells and mononuclear cancer cells were significantly different after the treatment with fullerenol. The experiment results indicated that the mononuclear SW480 cells were more sensitive to fullerenol than the multinuclear SW480 cells, and the multinuclear SW480 cells exhibited a stronger drug‐resistance than the mononuclear SW480 cells. This work provides a guideline for the treatments of multinuclear and mononuclear cancer cells with drugs.

Cancer treatment has been an important issue and great efforts have been devoted to the diagnosis and therapy of cancers (Babahosseini, Carmichael, Strobl, Mahmoodi, & Agah, 2015;Byun et al., 2013).
Because of the nanoscale resolution of atomic force microscope (AFM) in the imaging, manipulation and measurement of biological objects and structures (Lazar et al., 2013;Nawaz et al., 2012;Sokolov, 2007;Xu et al., 2012;Zhang et al., 2013;Zhang, Aslan, Besenbacher, & Dong, 2014), it has been used to investigate morphological and mechanical features of living cells (Heu, Berquand, Elie-Caille, & Nicod, 2012;Li, Mansoor, Tan, & Lim, 2006;Liu, Song, Qu, Wang, & Wang, 2015;Sikora & Iwan, 2012). Thus, AFM can be a proper tool for the manipulation and measurement of cancer cells. Studies about the changes of morphological and mechanical features of cancer cells after the treatment with anti-cancer drugs were taken. Gaspar et al. (Gaspar, Freire, Pacheco, Barata, & Castanho, 2015) studied the effect of HNP-1 on PC-3 cells and MOLT-4 cells by AFM, and the results showed that the height decrease was caused by the HNP-1 treatment. The roughness of the cell membrane is related the cell motility, and it can be used to characterize the state of cells (Antonio, Lasalvia, Perna, & Capozzi, 2012;Girasole et al., 2007). Kim et al. (Kim et al., 2012) found by AFM that the roughness of HeLa cells was increased significantly after the treatment with paclitaxel. Pelillo et al. (Pelillo et al., 2016) investigated the adhesion and morphology features of colorectal cancer cells  to study their metastatic behaviors affected by ruthenium drug NAMI-A. Hayashi et al. (Hayashi & Iwata, 2015) and Ansardamavandi et al. (Ansardamavandi, Tafazzoli-Shadpour, Omidvar, & Jahanzad, 2016) evaluated the stiffness changes of cancer cells to understand the pathophysiology. Efremov et al. (Efremov et al., 2014) characterized the cell area, focal adhesion and Young's modulus distributions of tumor cells and normal cells with AFM to study their transformational changes. Hence, morphology and mechanics are significant features for the investigation of cancer cells (Cio & Gautrot, 2016;Wirtz, Konstantopoulos, & Searson, 2011). However, little work has been carried out to investigate the difference of drug-resistance between multinuclear cancer cells and mononuclear cancer cells after the treatment with anticancer drugs.
In this work, fullerenol (C 60 (OH)x) was used as an anticancer drug to treat SW480 colon cancer cells. The morphological and mechanical features of SW480 colon cancer cells, such as the height, length, width, roughness, adhesion force and Young's modulus, were investigated after the treatment with fullerenol to understand their drug-resistance.
Different fullerenol concentrations of 1 and 2 mg/ml were used in the experiment. The details are described in the following sections.

| Sample preparation
To study the morphological features of cells by AFM, cells were immersed in 2.5% gluteraldehyde for 15 min. The samples were then rinsed three times with phosphate buffer saline (PBS) and naturally dried up at the room temperature and humidity. The prepared samples were stored at 4 8C in the dark.
To investigate the mechanical features of living cells by AFM, the cell culture glass slides were washed three times by the PBS to sweep away the suspend cells, and then immersed in RPMI-1640 media to keep the living environment. All steps were taken in a super-clean bench.

| AFM
An Agilent 5500 AFM system was used for the measurement of morphology features (height, length, width and roughness) of SW480 cells in the air, at a temperature of 23 8C and a humidity of 50%. The tapping mode was used for imaging, the scanning rang was varied from 20 lm 3 20 lm to 90 lm 3 90 lm, and the scanning speed was 0.2 line/s. The tip radius was 10 nm. The thickness of cantilever was 4 lm, and the width and length were 30 lm and 125 lm, respectively. The normal spring constant of cantilever was 40 N/m, and the resonance frequency was 300 kHz (Tap300, BudgetSensors). To analyze the changes of height, length, width and roughness of mononuclear cells and multinuclear cells after the treatment with fullerenol, and study their drugresistance, 10-28 mononuclear cells and multinuclear cells were selected from each groups and measured by AFM. The height, length, width and roughness features of each group of cells were obtained by image processing software "Pico Image Expert 6.2" from Agilent force curves were recorded for each cell. The AFM force-displacement curves were obtained from measuring the nucleus areas of cells (Cao, Sui, & Sun, 2013). Five cells were studied in each of the groups and 100 measurements were made for each cell to investigate the Young's moduli. The distributions of Young's moduli were obtained by Gaussian curve fitting. The probing positions were the nucleus areas of cells. The adhesion forces and Young's moduli were obtained by image processing software "JPKSPM Data Processing" from JPK Instruments AG (Germany).

| MTT assay
SW480 cells were planted (2 3 10 4 ) in a 96-well plate and incubated at 37 8C for 48 hr. They were then treated with 1 mg/ml and 2 mg/ml of fullerenol and incubated for another 24 hr, 48 hr, and 72 hr. After that, 20 ll of 5 mg/ml dimethyl thiazolyltetrazolium bromide (MTT) was added to each well. After the incubation at 37 8C for 4 hr, all media were aspirated and each well was treated with the 150 ll DMSO solution. Finally, the cell viability was measured using the microplate reader (Sanco, China) at an absorbance wavelength of 570 nm. Each treatment was repeated three times.

| Statistical analysis
Statistical analysis was performed by SPSS 18.0 software. One-way analysis of variance (ANOVA) was used to evaluate the data distribution. Data were shown as Mean 6 SD. The value of p < 0.05 was considered significant and p < .01 was considered highly significant. From Figure 1d, it can be seen that the viabilities of SW480 cells after the treatment with 2 mg/ml fullerenol are smaller than those of the cells treated with 1 mg/ml fullerenol.

| RE SUL TS A ND D I SCUSSION S
It is known that the morphology property change is a good way to evaluate the drug-resistance of cancer cells (Cai, Gao, Cai, Wu, & Deng, 2009). In this work, morphological images of SW480 cells were obtained by AFM.  (1) where k is the spring constant of the cantilever (k 5 0.03 N/m). ᭝L is the deflection of the cantilever. The original Hertz theory is an approximation for the contact of the very shallow indentation of two spheres in contact. For a pyramidal tip, the Hertz model can be modified as (Rico et al., 2005) F5 where F is the force.       Table 1. p < 0.01, when compared with the control group.    Fullerenol influenced the conformation and function of membranes of cells and deformed the protein in the binding residues (Grebowski et al., 2013;Qiao, Roberts, Mount, Klaine, & Ke, 2007). Thus, the skeleton and structure of cells were changed (Rungaldier, Oberwagner, Salzer, Csaszar, & Prohaska, 2013) and led to the alterations of mechanical properties (Ansardamavandi et al., 2016).

| C ONC LUSI ON
The work reported in this article shows that there are significant differ-