Real-time monitoring regulatory volume decrease of cancer cells: A

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Real-time monitoring regulatory volume decrease of cancer cells: A new model for the evaluation of cell migration Bin Zhou, Xinxin Lu, Yan Hao, and Peihui Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00004 • Publication Date (Web): 03 Jun 2019 Downloaded from http://pubs.acs.org on June 5, 2019

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Analytical Chemistry

Real-time monitoring regulatory volume decrease of cancer cells: A new model for the evaluation of cell migration

Bin Zhou§, Xinxin Lu§, Yan Hao, Peihui Yang* Department of Chemistry, Jinan University Guangzhou 510632, P. R. China

*Corresponding

author:

Peihui Yang, Ph.D, Professor Department of Chemistry, Jinan University Guangzhou 510632, P. R. China E-mail: [email protected] Tel/Fax: +86-20-85223039

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Cell migration plays a vital role in carcinoma invasion and metastasis. Cell regulatory volume decrease (RVD), a mechanism of adjusting cell volume, is a basic physiological function of cells, which is closely related to cell migration. In this work, A quartz crystal microbalance (QCM) cytosensor was firstly developed for real-time monitoring of cell RVD to evaluate the migration of human breast cancer cells. While stimulating the immobilized cells on the chip with hypotonic solutions, the temporal dynamics of RVD can be tracked by QCM sensor via analyzing frequency shifts during the cell swelling and shrinkage. The results showed that, due to the difference in cell migration capability, the level of RVD for MCF-7 cells and MDA-MB-231 cells was 32.8±2.9% and 49.7±4.2% (n=3), respectively. Furthermore, tamoxifen, a chloride channels blocker, was used to suppress cell RVD, indicating concentration dependence and inhibition difference in both types of cells. Combining QCM measurement with cell migration assay, the results showed that the blockage of RVD was positively correlated to the inhibition of cell migration with tamoxifen concentration ranging from 5 μM to 60 μM, which revealed the relation between cell RVD and cell migration. The study provided a non-invasive and real-time strategy for monitoring cell RVD as well as assessing cell migration, which was expected to supply a new diagnostic tool for metastatic cancers.

KEYWORDS: Quartz crystal microbalance, cytosensor; Real-time monitoring; Regulatory volume decrease; Cell migration


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INTRODUCTION Tumor metastasis has been the dominating factor of high mortality for a long period, responsible for nearly 90% of all cancer deaths, even though techniques for diagnosis and treatment of cancer have been improved significantly.1,2 Cancer cell migration is one of the critical steps during tumor metastasis, which can drive cells into blood or lymphatic vessels as well as penetrate into neighboring tissues and even distant organs.3-5 The process of cell migration is exceedingly complicated with cell polarization, protrusion, adhesion and rear retraction, which is accompanied with the changes of cell volume and shape, leading to the activities of cytomembrane involving transporters and ion channels.6-8 To maintain normal volume under osmotic fluctuation, cells can usually regulate their volume to offset osmotic swelling or shrinkage by mainly transporting Cl− , K+ ions through the ion channels and driving water in or out of cells, among which the regulation mechanism for hypotonic stimulation is referred to as regulatory volume decrease (RVD).9,10 And cell volume regulation is fundamentally important for various cell functions.11 Specifically, cellular RVD plays a significant role in cell migration because precise regulation of cell volume indispensably guarantees the correct direction of cell movement.12,13 In addition, the pharmacological properties of cells in migration and RVD process have been widely investigated by evaluating the inhibitory effects of drug on Cl- and K+ ion channels with patch clamp techniques.8,14 For example, tamoxifen (TAM), an antagonist of estrogen receptor as well as a potent blocker of chloride channels, has been applied to regulate chloride channels and affect RVD for most of cells including breast cancer cells.14 In view of the limitation of patch clamp techniques, such as invasive assay and cell content disturbance, it is very essential and meaningful to find a highly sensitive and non-invasive measurement which is capable of assessing the ability of cell migration by analyzing cellular RVD. Currently, a number of different technologies have been utilized to measure the size and volume of cells, including coulter device,15 atomic force microscopy,16 flow cytometry.17 Although these techniques are able to provide accurate detection of cell volume, most of them are performed on end-point measurement, which cannot



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continuously track dynamic information of cell volume changes. Fluorescence microscopy18 and phase contrast microscopyError!

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have been

employed to fix this problem. But fluorescence microscopy used for analyzing cell volume may suffer from photobleaching and fluorescent dye losses over long measurement periods, leading to ambiguous results. As for phase contrast microscopy, the imaging of cells is heavily affected by phase shift and optical artifacts which reduce the accuracy of measurement results. Moreover, cell volume is easily influenced by water content and osmotic forces, which could potentially result in irregular cell shape making accurate size determination difficult.19 Thus, a novel measurement for cell volume that allows highly sensitive, noninvasive and real-time monitoring has currently become a prominent demand. Over the past few years, quartz crystal microbalance (QCM) has rapidly developed from a primary mass sensing device to a label-free, noninvasive, highly sensitive bio-sensing platform which is able to dynamically evaluate cellular behavior, such as cell adhesion, growth and proliferation.20-22 By monitoring frequency or dissipation, QCM can provide dynamic information about the structure, mass and viscoelastic properties of surface-adsorbed layers.23-25 Cell size is usually presented as either cell volume or mass, and cell volume is inextricably linked to its mass which may be influenced by osmotic forces and water content. Thus, tracking cell mass can factually reflect the changes of cell volume.19,26 Given that, the cellular RVD processes can be tracked by monitoring the response frequency of QCM in real time. Based on the correlation between cell migration and RVD, it is innovative to assess the capability of cell migration with the analysis of cellular RVD through a highly sensitive, non-invasive, real-time QCM sensor. To date, there is still no report concerning the application of QCM to study the role of RVD in the migration of human breast cancer cells. Herein, we firstly proposed a real-time QCM sensor for monitoring cellular RVD under hypotonic stimulation and evaluating the potency of cell migration. The breast cancer cells MCF-7 and MDA-MB-231 with different migratory potential were used as the research model and respectively adhered on gold chip modified with


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poly-L-Lysine (PLL). Cellular RVD process was tracked in real time by monitoring frequency changes in hypotonic solutions with QCM, and the graphs of frequency response of cellular RVD were firstly obtained (Scheme 1). It was founded that the two breast cancer cells differed from each other on RVD process due to their different migration capability. Furthermore, tamoxifen, a chloride channel blocker, was used to inhibit the cellular RVD which exhibited significant inhibition differences between MCF-7 and MDA-MB-231 cells. Cell migration assay was further utilized to confirm the results, and the correlation between RVD and cell migration was subsequently extrapolated since both RVD and cell migration were dependent on tamoxifen concentration. Therefore, this work presented a novel approach to evaluate cell migration capability by monitoring their RVD, facilitating further understanding of cell functions and mechanisms of cancer metastasis on the aspect of physiology and pathology.

EXPERIMENTAL SECTION Reagents. Mercaptosuccinic acid (MSA) and tamoxifen (TAM) were purchased from Adamas Reagent

Co.,

Ltd.

(Shanghai,

China).

Adenosine

5′-triphosphate

(ATP),

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), poly-L-Lysine (PLL, MW: 30,000-70,000) and N-hydroxysuccinimide (NHS) were achieved from Sigma-Aldrich Chemical Co., Ltd. (USA). Fetal bovine serum (FBS) and DMEM were purchased from Gibco Co. (USA). MCF-7 cells and MDA-MB-231 cells were achieved from Biomedical Translational Research Institute, Jinan University (Guangzhou, China). The isotonic solution (Iso, pH 7.4) included 0.5 mM MgCl2, 70 mM NaCl, 10 mM HEPES, 2 mM CaCl2 and 140 mM D-mannitol. The hypotonic solution (Hypo, pH 7.4) was prepared by omitting the D-mannitol of isotonic solution. The osmolarity of isotonic and hypotonic solutions were respectively adjusted to 300 and 160 mOsM by an osmometer (Osmomat 30, Gonotec, Germany). All reagents were of analytical grade, and ultrapure water (Millipore GmbH, Schwalbach, Germany) was used throughout the whole experiments. Apparatus. 3D cell explorer (Nanolive SA, Switzerland) was used to visualize cell volume changes under isotonic and hypotonic stimulation. Scanning electron microscope (Zeiss Gemini



Ultra-55, Germany) was used to characterize the morphology of the QCM cytosensor. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed to demonstrate the fabrication of QCM cytosensor by an electrochemical working station (CHI 660E, Shanghai Chenhua Instrument Co. Ltd., China). Atonic force microscopy (Bioscope Catalyst Nanoscope-V, Bruker, U.S.A.) was utilized to investigate the membrane stiffness of cells. QCM-Z500 (KSV Instruments Ltd., Finland) was employed to record the changes of frequency (Δf) versus time. The QcmZBrwse software was used to analyze our data. Fabrication of the QCM cytosensor. Gold chips that consisted of 5 MHz AT-cut quartz crystals disks (14 mm diameter, 0.3 mm thickness) and Au electrode (12 mm diameter, 100 nm thickness), were served as the sensing substrate of QCM. The fabrication process of the QCM cytosensor was described as follows. Gold chips were first soaked in a solution containing 25% ammonia, 30% H2O2 and deionized water (volume ratio is 1: 1: 5) at 75 °C for 15 min, and then washed with purified water for three times. After that, these gold chips were incubated with MSA (2 mg·mL-1, 80 μL) at room temperature for 12 h, later added with the mixture of 15 mM NHS and 75 mM EDC and incubated for 1.5 h to activate the carboxylic groups of MSA. At last, PLL (4 mg · mL-1, 80 μL) was coated on the sensor surface at room temperature for 4 h. Then, the as-prepared sensing platform was successfully fabricated. Cell culture and drug treatment. MCF-7 cells and MDA-MB-231 cells were separately grown in DMEM with 1% antibiotics (penicillin/streptomycin) and 10% FBS. Both types of cancer cells were cultured under an appropriate condition of 5% carbon dioxide at 37 °C. During the period of logarithmic phase, the human breast cancer cells were collected through centrifugation at 800 rpm for 5 min. After that, these two kinds of collected cells were resuspended with the isotonic solution for the next stage of experiments. TAM was dissolved with methanol in the concentration of 100 μM, and added into corresponding isotonic and hypotonic solution for drug treatment assays at the final diluted concentrations (5, 10, 20, 30, 40, 60 μM). QCM measurement. The as-prepared gold chips were mounted in QCM flow module carefully. The thermal electric controller was set at 37 °C. Isotonic solution (300 mOsM, pH 7.4) was first injected into the QCM chamber to gain a stable baseline at a velocity of 250 μL·min-1. Then 5×103 cells/mL human breast cancer cells suspension were added into the chamber, followed


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by washing with isotonic solution to remove the unbonded cells. For RVD assays, cells were perfused with hypotonic solution (160 mOsM, pH 7.4) and isotonic solution again in order. For drug treatment assays, isotonic and hypotonic solutions containing different concentration of TAM (0, 5, 10, 20, 40, 60 μM) were injected as the order previously mentioned. The schematic illustration of the proposed QCM cytosensor and measurement principle was exhibited in Scheme 1. Wound-healing assay. Cells were seeded at 1 × 105 cells per well in the standard incubator. After cells grew over each well, a sterile 200 μL pipette tip was used to linearly disrupt the monolayer of cells, simulating a wound-healing model. The scratch was formed vertically in the middle of each well. Then we used PBS to carefully rinse each well in order to remove the detached cells created by the scratch. And the cultured medium containing 0, 5, 10, 20, 40, 60 μM TAM were respectively added into each well which were then incubated for 24 h. Formation of the scratch wound was observed by an inverted light microscopy. Young’s modulus measurement by AFM. Since Young’s modulus could reflect the cellular stiffness, atomic force microscopy (AFM) was utilized to detect the force curves of cell surface. Cell stiffness was measured through the force curves of cells by AFM with the Tap 150Al-G Silicon probe in Force Volume mode. Then, we calculated the Young’s modulus from the force curves via Nanoscope analysis software 8.14 to distinguish the difference in stiffness of MCF-7 and MDA-MB-231 cells. Calculation of the regulatory volume decrease. According to the report,13 the level of cell RVD could be calculated by an equation: RVD (%) = (Vmax – Vmin) / (Vmax – V0)× 100%, in which Vmax was the maximum volume in hypotonic solution, Vmin was the minimum volume in hypotonic solution, V0 was the normal volume in isotonic solution. Based on the proportional relation of cell volume and mass, we can calculate the level of cell RVD by the equation as follows. RVD (%) = (△fmax – △f

min)

/ (△fmax – △f0) ×100%, in which △f0 was the frequency

representing the mass or volume of normal cell in isotonic solution, (△fmax – △f0) was the frequency variation representing the change of cell mass or volume caused by cell swelling in hypotonic solution, and (△fmax – △f min) was the frequency variation representing the change of cell mass or volume caused by cell shrinkage in hypotonic solution. The inhibition rate of RVD by chloride channel blockers TAM was calculated by the equation,13 Inhibition of RVD (%) =



(RVDctrl – RVDi) / RVDctrl × 100%, in which RVDi was RVD in cancer cells inhibited with TAM, and RVDctrl was RVD in control group.

RESULTS AND DISCUSSION Characterization of RVD. Regulatory volume decrease could take place under hypotonic stimulation via the cell osmotic swelling and shrinkage which was accompanied by driving K+, Clions and water out of cells to maintain the normal cell volume. The 3D cell explorer was exploited to characterize the volume changes of human breast cancer cells in isotonic and hypotonic solutions. The 2-dimensional images showed that the average diameter of MCF-7 cells was ~13 μm and displayed a stable state when they were bathed in isotonic solution (Figure 1A). When these cells were exposed to hypotonic solution for 1 min, cell swelling immediately appeared and the diameter was ~16 μm (Figure 1B). After hypotonic stimulation for 5 min, cell volume shrank and the diameter was ~14 μm (Figure 1C). After the cells were placed back in isotonic solution for 5 min, cell volume restored to initial volume (Figure 1D). The 3-dimensional images of cell volume were shown in Figure S1, which were consistent with the 2-dimensional images. The results displayed the RVD response of human breast cancer cells in hypotonic solution. Characterization of QCM cytosensor. SEM was employed to characterize the morphology of QCM sensing interface. As displayed in Figure 2A, the bare gold chip showed a smooth and flat surface. Figure 2B exhibited a finely granular structure and coarse surface layer, indicating that MSA was modified onto the gold chip. Figure 2C showed some tablet-shaped substances appeared on the surface of the coarse structure, which displayed that PLL was assembled on the MSA surface successfully. After the modified gold chip was incubated with human breast cancer cells, some cells were clearly captured on the gold chip (Figure 2D). The stepwise fabrication procedure of the QCM cytosensor was characterized by CV and EIS measurements. Figure S2A showed that a pair of typical redox peaks of ferricyanide ions was obtained by the bare gold electrode (curve a). After MSA was modified on the electrode, peak current significantly decreased, attributing to the electron inert feature of MSA (curve b). And then, peak current increased with the modification of PLL on the electrode that exhibited great conductivity (curve c). By incubating the electrode with human breast cancer cells, peak current decreased further because of the electron inert feature of cells (curve d). In addition, Figure S2B


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showed that the diameter of semicircle of EIS increased after the gold electrode (curve a) was modified with MSA and adhered with human breast cancer cells (curves b and d), but decreased with the modification of PLL (curve c). The results indicated that the QCM cytosensor had been fabricated successfully. Measuring RVD by QCM cytosensor. Cell volume is tightly linked with mass which can be reflected by detecting the frequency variation of QCM. Thus, cell RVD process could be tracked in real time by using QCM to monitor frequency fluctuation, and the graphs of frequency response were obtained for MCF-7 cells and MDA-MB-231 cells in the hypotonic solutions (Figure 3). As cell suspension in isotonic solution flowed into QCM chamber, the resonance frequency decreased and gradually tended to a constant due to the adherence of cells on the chip. With the hypotonic solution injected in QCM chamber, the frequency sharply decreased and peaked in 2-3 min caused by cell swelling with the influx of water (Stage I). Subsequently, the cell shrinkage with the outflux of water led to the increase of frequency (Stage II), which revealed the behavior of cell RVD. And then cells recovered to the normal mass or volume after reinjection of isotonic solution. Therefore, the frequency fluctuation in cell swelling and shrinkage could be used to estimate the variation of cell mass and volume, and hence assess cell RVD. Figure 3 showed the level of RVD of MDA-MB-231 cells with high migratory potential is higher than that of MCF-7 cells with low migratory potential. The results demonstrated that the proposed QCM cytosensor was capable of monitoring the level of cell RVD and identifying the capability of cell migration. Several key experiment parameters were optimized for the measurements including the dosage of MSA and PLL, cell concentration and flow rate. Figures S3A and B showed that the optimal dosage of MSA and PLL were 80 μL, respectively. As presented in Figure S3C, the best concentration of human breast cancer cells in perfusate was about 5 × 103 cells per milliliter. To ensure repeatability of assays ， we stabilized the frequency at a certain value to keep the cell number unchanged on the sensor surface. Besides, stereo microscope was exploited to estimate the cell number after each assay. Figure S3D exhibited the optimal flow rate of the perfusate was 250 μL per minute. Inhibition of RVD by chloride channel blocker. Considerable works10,11,13 reported the outflow of Cl– ions via chloride channels was the crucial mechanism of RVD in live cells, so the level of cell RVD can be regulated through the use of chloride channel blocker tamoxifen (TAM).



Figures 4A and C showed the temporal dynamics of frequency based on RVD in the absence and presence of TAM (10 μM) for two kinds of human breast cancer cells. The statistical analysis was shown in Figures 4B and D, where the level of RVD in TAM-treated cells was markedly inhibited (P < 0.01) compared to that in the control group. Moreover, the RVD in MCF-7 cells and MDA-MB-231 cells without TAM blocking were 32.8±2.9% (n=3) and 49.7±4.2% (n=3), respectively. By treating with TAM (10 μM) on cells, the level of RVD in MCF-7 cells and MDA-MB-231 cells decreased to 23.5±2.4% (n=3) and 34.8±2.6% (n=3). The results exhibited that the inhibition of TAM on cell RVD was more serious in high migratory cells than low migratory cells, which may attribute to their different protein activity of ion channels expressed on the membrane, influencing cell RVD and migration ability as well.6,23 Furthermore, as displayed in Figures 4A and C, the frequency-time curves for cell RVD were obtained by continuous monitoring in three parallel with the coefficient of variation of 2.5%, indicating the excellent stability and reproducibility for the RVD assays. Effect of tamoxifen concentration on the inhibition of RVD. To examine the dosage of TAM on the level of RVD, the QCM cytosensor was applied to monitor the frequency fluctuations in MCF-7 cells and MDA-MB-231 cells induced by increasing TAM dosage from 5 μM to 60 μM (Figures 5A and C). Compared to the control, the level of RVD in 5 μM TAM-treated group of two types of cells was obviously inhibited (P < 0.05). And the results showed the level of RVD in the two kinds of cells reduced with the increasing dosage of TAM from 5 to 60 μM. Specifically, the level of RVD was inhibited from 33.2 % to 14.0 % for MCF-7 cells and from 48.3 % to 21.2 % for MDA-MB-231 cells, respectively (Figures 5B and D). The data provided the further evidences that the inhibition of TAM on the RVD of the breast cancer cells in vitro was concentration-dependent, and revealed a greater inhibitive effect on high migratory cells compared to low migratory cells. Evaluating the capability of cell migration. The wound-healing assay, a convenient and efficient method, was used to investigate the migration differences between MCF-7 cells and MDA-MB-231 cells, and evaluate the inhibition of TAM on cell migration in the two kinds of breast cells. When TAM concentrations increased from 5μM to 60μM, the inhibition ratio of cell migration increased from 32.4% to 88.6% for MCF-7 cells (Figure S4) and from 28.2% to 93.7% for MDA-MB-231 cells (Figure S5). The results indicated that the inhibition of TAM on the


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migration of MCF-7 cells and MDA-MB-231 cells in vitro was concentration-dependent. Because the capability of cell migration was related to cell stiffness, AFM was exploited to characterize the cell stiffness by Young’s modulus maps. As exhibited in Figure S6, the Young’s modulus of MCF-7 cells and MDA-MB-231 cells were 19.5 kPa and 13.7 kPa, which implied that MCF-7 cells were much stiffer than MDA-MB-231 cells and thus the migration ability of the former cells was weaker than the latter ones. Correlation between RVD and cell migration. To confirm the valid relation between cell RVD and cell migration, the inhibition of TAM on cell RVD and migration was investigated through correlation analysis. As shown in Figures 6A and D, the inhibition of TAM on the RVD of MCF-7 cells and MDA-MB-231 cells in vitro was concentration-dependent, so was the inhibition on the cell migration (Figures 6B and E). Both of them exhibited an enhancement of inhibition effect with the increase of TAM concentrations. Therefore, based on this, the calibration plot presented a good linear relationship between the blockage of RVD and the inhibition of cell migration (Figures 6C and F). The linear regression equation, Inhibition of migration (%) = 1.5362 ×Inhibition of RVD (%) +3.877, was obtained with a correlation coefficient R = 0.9892 for MCF-7 cells, and that, Inhibition of migration (%) = 1.7111 × Inhibition of RVD (%) + 0.8784, was obtained with a correlation coefficient R = 0.9921 for MDA-MB-231 cells. The results showed that the blockage of RVD was positively correlated to the inhibition of cell migration. Thus, the QCM cytosensor could be applied to monitor cell RVD to reflect the migration of human breast cancer cells, which could provide a new model for the evaluation of cell migration.

CONCLUSION In summary, a new model based on QCM cytosensor was developed for monitoring real-time cell RVD and evaluating migration of human breast cancer cells. The QCM cytosensor was capable of sensitively tracking frequency fluctuation during the cell swelling and shrinkage in RVD process with hypotonic stimulation. The level of RVD in MCF-7 cells and MDA-MB-231 cells can be distinguished by the QCM cytosensor due to their different capability in cell migration. Furthermore, TAM was used to block chloride channels and then suppress cell RVD, indicating concentration dependence and inhibition difference in both types of cells. The results showed that the blockage of RVD was positively correlated to the inhibition of cell migration.



Therefore, this work established a non-invasive and real-time method for monitoring cell RVD as well as assessing cell migration, which may be further extended to the research of various cell functions in physiology and pathology.

Supporting Information Characterization of cell RVD; Characterization of the QCM cytosensor; Optimization of experimental conditions; Evaluation of cell migration capacity by wound-healing assays; Characterization of cell stiffness by Young’s modulus.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Peihui Yang: 0000-0001-9019-6913 Author Contributions §These

authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 21874057, 21375048) References (1) Christofori, G., New signals from the invasive front. Nature 2006, 441 (7092), 444-450. (2) Huttenlocher, A.; Palecek, S. P.; Lu, Q.; Zhang, W.; Mellgren, R. L.; Lauffenburger, D. A.; Ginsberg, M. H.; Horwitz, A. F., Regulation of cell migration by the calcium-dependent protease calpain. J. Biol. Chem. 1997, 272 (52), 32719-22. (3) Condeelis, J.; Pollard, J. W., Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 2006, 124 (2), 263-266. (4) Mareel, M.; Leroy, A., Clinical, cellular, and molecular aspects of cancer invasion. Physiol. Rev. 2003, 83 (2), 337-376.


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Scheme 1. Schematic illustration of the fabricated QCM cytosensor and measurement principle.



Figure 1. 2-dimensional images of MCF-7 cells under hypotonic stimulation. Cells in isotonic solution (A); Cells swelled in hypotonic solution for 1 min (B); Cells shrank in hypotonic solution for 5 min because of RVD (C); Cells continuously shrank to initial volume with isotonic solution treated again for 5 min (D).


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Figure 2. SEM images of bare gold chip (A), MSA/gold chip (B), PLL/MSA/gold chip (C) and cells/PLL/MSA/gold chip (D).



Figure 3. The QCM frequency shift curves of MCF-7 cells (black) and MDA-MB-231 cells (red) in isotonic (300 mOsM, Iso) and hypotonic (160 mOsM, Hypo) solutions.


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Figure 4. Effect of tamoxifen (10 μM) on the frequency shift of MCF-7 cells (A) and MDA-MB-231 cells (C) in isotonic and hypotonic solutions for three successive parallel assays; Histogram of the level of RVD of MCF-7 cells (B) and MDA-MB-231 cells (D) (n = 3, **P < 0.01).



Figure 5. Effect of different tamoxifen concentrations on the frequency shift of MCF-7 cells (A) and MDA-MB-231 cells (C) in the groups of 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, repectively; Histogram of the level of RVD of MCF-7 cells (B) and MDA-MB-231 cells (D) (n = 3, *P < 0.05).


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Figure 6. Effect of different tamoxifen concentrations on the inhibition of RVD of MCF-7 cells (A) and MDA-MB-231 cells (D); Effect of different tamoxifen concentrations on the inhibition of cell migration in MCF-7 cells (B) and MDA-MB-231 cells (E); Linear relationship between inhibition of RVD and inhibition of cell migration in MCF-7 cells (C) and MDA-MB-231 cells (F).



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Real-time monitoring regulatory volume decrease of cancer cells: A

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