2.1.

Cell Cultures and Treatment

HeLa Kyoto (human cervical cancer) and CT26 (mouse colorectal cancer) cells were used in the study. The cells were cultured in DMEM containing $100\mu \mathrm{g}/\mathrm{mL}$ penicillin, $100\mu \mathrm{g}/\mathrm{mL}$ streptomycin sulfate, and 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% ${\mathrm{CO}}_2$.

To generate tumor spheroids, the HeLa Kyoto cells were seeded into ultra-low attachment 96-well round bottom plates (Corning Inc.), $\sim 100\text{cells}/\text{well}$ in $200\mu \mathrm{L}$ DMEM (Life Technologies) and cultured in standard conditions (37°C, 5% ${\mathrm{CO}}_2$, and 80% humidity). In three days, spheroid formation was verified with light microscopy.

Chemotherapy was performed with cisplatin (Teva, Israel) at dose $2.57\mu \mathrm{M}$ (IC50) for CT26 cells and $1.1\mu \mathrm{M}$ (1/2 IC50), $2.3\mu \mathrm{M}$ (IC50), and $4.5\mu \mathrm{M}$ (2 IC50) for HeLa Kyoto cells grown as monolayer. The IC50 concentrations were determined in our previous experiments using MTT assay.^38,39 HeLa spheroids were treated with $8.6\mu \mathrm{M}$ cisplatin. The tested concentrations of cisplatin were in the range of maximum concentrations of the drug in the blood plasma of patients, 1 to $2\mathrm{mg}/\mathrm{L}$ or 3 to $6\mu \mathrm{M}$ on average.⁴⁰ The cells were incubated with the drug from 10 min to 48 h, spheroids—from 3 to 48 h, and viscosity was measured immediately after the treatment. Untreated cells or spheroids served as a control. Additional experiment was performed to measure viscosity in monolayer cells in 48 h after therapy withdrawal.

The method for establishing the cisplatin-resistant cell subline was adopted from Ref. 41. Briefly, a HeLa cell culture was continuously exposed to gradually increasing concentrations of cisplatin. The start concentration was 1/150 IC50. Each next concentration was increased by 25% from the previous one and added only after clear adaptation of the cells to the drug, i.e., after restarting of cell proliferation without significant cell death in a plate (after 2 to 7 days). In $\sim 4$ months after first exposure, the cells were considered cisplatin-resistant. Measurements of microviscosity in these cells were performed in 48 h after washing out the drug to avoid immediate effects of cisplatin.

2.2.

Cellular Viability Assay

A live/dead double staining kit (Sigma)—calcein and propidium iodide (PI)—was used to stain live and dead HeLa Kyoto cells, respectively, after chemotherapy of cells in monolayer or 3D spheroids, according to the manufacture’s protocol. Fluorescence of calcein was excited by an argon laser at a wavelength of 488 nm and registered in the ranges 500 to 570 nm. PI fluorescence was excited at a wavelength of 543 nm and registered in the range 600 to 700 nm. One-photon fluorescence confocal images were obtained using an LSM 880 (Carl Zeiss, Germany) inverted laser scanning microscope.

2.3.

Fluorescent Molecular Rotor BODIPY 2

BODIPY 2, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Fig. S1 in Supplementary Material), was synthesized according to the previously published procedure²⁸ and was used as a viscosity-sensitive probe. This molecular rotor has already demonstrated the sensitivity of its spectral characteristics to viscosity, with a good dynamic range of fluorescence lifetimes.²⁸ A calibration curve linking fluorescence lifetime of BODIPY 2 to the viscosity of its environment was previously obtained.^25,28

An attractive property of the BODIPY 2 rotor is the selective staining of the plasma membrane, avoiding effective endocytosis. The effectiveness of measuring membrane viscosity was shown not only in cell cultures in cellulo,^4,28–31 but also in vivo on animals with tumors.³⁰ BODIPY rotors also have an excellent dynamic range of fluorescence lifetimes for the determination of viscosity in biologically relevant range, 1 to 5000 cP, which has been demonstrated to be temperature independent.^42,43

For microscopic imaging, the cells were seeded on glass-bottom dishes for confocal microscopy (FluoroDishes, Life Technologies) overnight in complete DMEM media without phenol red (Life Technologies). The membrane viscosity was examined at 10 min, 1, 3, 6, 24, and 48 h after adding cisplatin. Before imaging, the culture media with cisplatin was replaced with ice-cold Hank’s solution without ${\mathrm{Ca}}^{2+}/{\mathrm{Mg}}^{2+}$, and cells were incubated at $+4°\mathrm{C}$ for 5 min. Afterward, Hank’s solution was replaced with ice-cold BODIPY solution ($4.5\mu \mathrm{M}$, 0.1% DMSO).

The spheroids were stained with BODIPY 2 on the day 5 of the growth, when they had a compact heterogeneous structure and the size of $\sim 350\mu \mathrm{M}$. The five to six spheroids were carefully transferred on each glass-bottom dish (FluoroDishes, Life Technologies) in 1 mL DMEM without phenol red and placed in ${\mathrm{CO}}_2$ incubator for 2 h to allow their attachment. The membrane viscosity was examined at 3, 6, 24, and 48 h after adding cisplatin. Before imaging, the culture media with cisplatin was replaced with ice-cold Hank’s solution without ${\mathrm{Ca}}^{2+}/{\mathrm{Mg}}^{2+}$, and cells were incubated at $+4°\mathrm{C}$ for 10 min. Afterward, Hank’s solution was replaced with ice-cold BODIPY 2 solution ($8.9\mu \mathrm{M}$, 0.1% DMSO). Two-photon excited FLIM images were acquired within 5 to 10 min after adding BODIPY 2.

2.4.

Large Unilamellar Vesicles

Lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, and egg yolk sphingomyelin (EYSM) were purchased in powder form from Avanti Polar Lipids^® and resuspended in chloroform (20 mM) before use. A dried lipid film was created by mixing the lipid stock solutions at the appropriate DOPC:EYSM:cholesterol molar ratio before using a rotary evaporator to remove the solvent and to create a lipid film. 0.4 M aqueous sucrose solution was then added to hydrate the film to a final concentration of 1 mM lipid. The mixture was then vortexed and extruded 21 times through a 200-nm polycarbonate filter (Avanti Polar Lipids^®). Lipid vesicles were then doped with an aliquot of BODIPY 2 (3 mM in DMSO), which was externally added to achieve 1:200 dye:lipid ratio. The final concentration of DMSO was 0.5% v/v. Solvents for fluorescence studies were of spectrophotometric grade (Sigma Aldrich^® or VWR). The mixtures were incubated for at least 15 min above the melting temperature of the lipid ($T_m$). After cooling, $200\mu \mathrm{L}$ large unilamellar vesicles (LUVs) were diluted in $1750\mu \mathrm{L}$ $9\mathrm{mg}/\mathrm{mL}$ NaCl solution before measuring.

For cisplatin measurements, $50\mu \mathrm{L}$ of $180\mu \mathrm{M}$ cisplatin solution in $9\mathrm{mg}/\mathrm{mL}$ NaCl was added to the LUV dilution described above and incubated in the dark at room temperature for 1 h before measuring.

All samples were placed in quartz cuvettes (10 mm path length). Time resolved fluorescence decay traces of BODIPY 2 were acquired using a Horiba Jobin Yvon IBH 5000 F time-correlated single-photon counting instrument with detection at $520\pm 6\mathrm{nm}$, after 404 nm pulsed excitation (NanoLED). Acquisition was stopped after peak counts reached 10,000 and the resulting traces were fitted to a biexponential decay using DAS^® software.

2.5.

FLIM

Multiphoton tomograph MPTflex (JenLab, Germany) equipped with a tuneable 80 MHz, 200 fs Ti:Sapphire laser (MaiTai), a single-photon counting module SPC-150 and detector PMC-100-20 (Becker&Hickl, Germany) was used for FLIM. The images were acquired through a $40\times$, 1.3 NA oil immersion objective. The scan head of the MPTflex system was set up in the inverted position for in vitro imaging. The glass-bottom dish with cell monolayer of attached spheroids was placed on the adapter ring connected with a microscope objective. In the case of spheroids, the images were acquired from a depth of $\sim 20\mu \mathrm{m}$.

BODIPY 2 fluorescence was excited at the wavelength of 800 nm and detected in the range 409 to 680 nm using a fixed prefitted emission filter. The average power applied to the sample was $\sim 7\mathrm{mW}$. Image size was $512\times 512\text{pixels}$. The acquisition time was $\sim 7\mathrm{s}$ per image. The collected number of photons per the decay curve was at least 5000. FLIM images were obtained from 10 randomly selected fields of view in each culture dish.

Fluorescence lifetime analysis was performed in the SPCImage software (Becker&Hickl, Germany). Time resolved fluorescence decays at each pixel of the whole image were fitted using a monoexponential model. The goodness of the fit ${\chi }^2\le 1.20$ indicated that the model used provided a reasonable fit. Fluorescence lifetime of BODIPY 2 ($\tau$) was measured in the plasma membranes of cells by manual selecting zones with ${\chi }^2\le 1.20$ as regions of interest.

The viscosity was calculated using the modified Förster–Hoffmann equation in the logarithmic form: $\log {\tau }_f=\alpha \log \eta +\text{const}$, where constant ($\alpha$), log fluorescence lifetime (${\tau }_f$) versus log viscosity ($\eta$). Experimentally measured lifetimes of BODIPY 2 (in ns) were converted to viscosity values (in cP) using the formula $x=y^2/0.0206$, where $x$—viscosity (in cP), $y$—fluorescence lifetime $\tau$ (in ns), on the basis of previously obtained calibration plot.²⁹

2.6.

ToF-SIMS

$5\times {10}^5\text{cells}$ were seeded on glass-bottom dishes (FluoroDishes, Life Technologies) containing clean and dry poly-L-lysine-coated cover glass and were incubated in an incubator at 37°C and an atmosphere of 5% ${\mathrm{CO}}_2$ for 24 h. The next day, cisplatin ($2.6\mu \mathrm{M}$) was added to the culture medium. After 20 min or 24 h incubation with cisplatin, cells were washed three times with phosphate-buffered saline (PBS) and then cells were incubated with 4% paraformaldehyde for 60 min at room temperature for chemical fixation. Afterward, cells were washed three times with PBS. In total, three samples were prepared—control sample without cisplatin and cells incubated with cisplatin for 1 and 24 h. Fixed cells were then stored in PBS for $\sim 5$ to 6 h due to shipping to ToF-SIMS laboratory. Since ToF-SIMS analysis involves studies in vacuum, a cell dehydration procedure was applied. Cells were washed with mQ water to remove excess of salts. Drying was carried out under gentle stream of argon at room temperature.

Positive and negative mass-spectra were acquired with a ToF-SIMS 5 (ION-TOF Gmbh, Germany) equipped with a 30-keV ${{\mathrm{Bi}}_3}^{+}$ liquid metal ion source. Twelve mass spectra were recorded for each sample in both, positive and negative, ion mode, with an analysis area of $300\times 300{\mu \mathrm{m}}^2$, and raster was $64\times 64\text{pixels}$. The primary ion dose density was $4\times {10}^{11}\text{ions}/{\mathrm{cm}}^2$ to maintain static SIMS conditions. A low-energy electron flood gun was used for charge compensation in all experiments. Ion yields were calculated as an intensity of the corresponding peak of interest normalized to the total ion count amount.

2.7.

Statistics

The mean values ($M$) and standard deviations (SD) were calculated for the long and short components of fluorescence lifetimes of BODIPY 2. Student’s $t$-test was used to compare the data ($p\le 0.05$ was considered statistically significant). The number of cells for mean value calculations was 20 to 30 in 7 to 10 fields of view.

3.1.

Viscosity in Monolayer Cells upon Cisplatin Treatment

Using FLIM with fluorescent molecular rotor BODIPY 2, plasma membrane viscosity in cancer cells was measured during chemotherapy with cisplatin that was applied to the cell monolayers in the clinically relevant concentrations. It is important to mention that membrane viscosity was measured only in viable cells, as in dead (PI positive) cells the rotor did not stain the plasma membranes, but accumulated inside the cells, where its fluorescence decayed biexponentially, indicative of rotor aggregation⁴⁴ (Fig. S2 in the Supplemental Materials).

In untreated colorectal cancer CT26 cells, time-resolved fluorescence decays collected from plasma membrane locations could be fitted with monoexponential function, and fluorescence lifetime of BODIPY 2 was measured to be $\sim 2.55\pm 0.07\mathrm{ns}$, which corresponded to the viscosity value $322\pm 21\mathrm{cP}$. In the time period from 10 min to 3 h of incubation with $2.57\mu \mathrm{M}$ cisplatin, the membrane microviscosity decreased compared to the control and amounted to $\sim 290$ to 300 cP (differences with control statistically not significant). At further incubation with cisplatin the viscosity increased and at 24 h time point became greater than the control value, $397\pm 17\mathrm{cP}$ ($p\le 0.005$) [Figs. 1(a) and 1(b) and Fig. S3 in the Supplemental Materials].

Fig. 1

Plasma membrane microviscosity in monolayer CT26 and HeLa cells before (control) and during 24-h-exposure to cisplatin. (a) Viscosity images of cells acquired with FLIM. Quantification of viscosity of (b) CT26 and (c) HeLa cells during chemotherapy. $\text{Mean}\pm \mathrm{SD}$, $n=20$ cells. Bar, $50\mu \mathrm{m}$. *, $p\le 0.005$ compare with control.

To find out whether cisplatin induces similar changes of viscosity in a different cell line and whether these effects depend on a drug dose, we have tested three doses of cisplatin (1.1, 2.3, and $4.5\mu \mathrm{m}$) on cervical carcinoma HeLa cells [Fig. 1(a)]. Interestingly, no dose-dependent effects on viscosity were detected at the range of the doses we used, which likely indicates that the observed alterations in membrane viscosity are not associated with drug–lipids interactions that may occur as a result of a passive diffusion process but rather reflect the effect of a biophysical process that is identical in all treated cells at these treatment doses. Upon treatment of HeLa cells with cisplatin, a decrease in membrane viscosity was observed at early time points (10 to 60 min), when the viscosity was reduced from $326\pm 22\mathrm{cP}$ to $\sim 300\mathrm{cP}$ (not significant). Starting from 6 h, the value of the membrane viscosity increased. At 24 h, the membrane viscosity was $427\pm 26\mathrm{cP}$ [Fig. 1(c) and Fig. S3 in the Supplemental Materials].

We have further confirmed that cisplatin at $4.5\mu \mathrm{M}$ concentration (the highest concentration used in cells) does not have a direct effect on the lipid bilayer viscosity, by performing fluorescence lifetime measurements using BODIPY 2 in aqueous suspension of LUVs produced from DOPC, DOPC + cholesterol (50/50), and DOPC/Sphingomyelin/Cholesterol (25/25/50). The lifetime corresponding to the rotor embedded in the membrane increased in these series, from 1.8 to 2.6 ns and to 3.0 ns, as expected,⁴⁵ due to an increased packing of lipids in the presence of cholesterol and sphingomyelin and the corresponding phase change from liquid disordered to liquid ordered lipid bilayer. However, the addition of cisplatin did not alter viscosity in any of these compositions (Fig. S4 in the Supplemental Materials). We note that the lifetime value (and viscosity) of DOPC/cholesterol (50/50) is very close to the lifetime value seen in colorectal cancer CT26 and in HeLa cells. The lack of viscosity change in LUVs upon incubation with a high concentration of cisplatin confirms that the effect of cisplatin on the plasma membrane viscosity is a consequence of its biological action on cellular function and not simply an effect of passive incorporation or association of this drug with the bilayer.

It is seen from PI- and DAPI-staining and bright-field microscopy (Fig. S2 in the Supplemental Materials) that most cisplatin-treated cells remained viable after 24 h, though their morphology was altered. After 24 h of incubation with cisplatin, the number of dead (PI-positive) HeLa cells increased to 7%, for CT26 cells the number of dead (PI- and DAPI-labeled) cells increased to 10%. As we previously showed, inhibition of cell proliferation, but not a cell death, is the main effect of cisplatin on HeLa cells upon the use of IC50 concentration over 24 h.³⁸

To check how stable and persistent the observed viscosity changes are, we removed cisplatin from the culture medium after 24-h incubation and measured viscosity following additional 48 h of culturing. During this time, the cells did not recover their initial viscosity, but preserved the elevated viscosity of their plasma membranes (Fig. S5 in the Supplemental Materials).

3.2.

Viscosity in Tumor Spheroids upon Cisplatin Treatment

Next, we assessed the plasma membrane viscosity of living cells within tumor spheroids during therapy with cisplatin. Spheroids are often used as an in vitro multicellular model of a solid tumor that mimics its basic structural and functional features and drug resistance profile. Previously, we have shown that microviscosity of plasma membranes in spheroid cells did not differ for spheroids of different sizes and between the inner (quiescent) and outer (proliferative) cellular layers of a spheroid.²⁹ These data indicate that microviscosity of membrane is not affected by the metabolic nor proliferative activity of cells and by the heterogeneity of the cellular microenvironment in conventional conditions.

Membrane viscosity of HeLa cells in the tumor spheroids was identical to cells in a monolayer, $325\pm 29\mathrm{cP}$. Treatment with cisplatin resulted in a dramatic increase of the viscosity up to $425\pm 25\mathrm{cP}$ after 24 to 48 h of incubation ($p=0.0005$) [Fig. 2(b)]. As expected, the treatment was accompanied by the increase in the number of dead cells, but the viscosity of viable cells did not change between 24 and 48 h. Moreover, we evaluated the changes in the morphology of spheroids upon incubation with cisplatin. For imaging, multicellular compact tumor spheroids of 5 days of growth, $\sim 350\mu \mathrm{m}$ in diameter, were taken, in which the outer layer of proliferating cells and the inner, more dense nucleus could be distinguished. After 48 h of incubation with cisplatin, the spheroid size increased to $\sim 450\mu \mathrm{m}$ (Fig. S6 in the Supplemental Materials). Although cisplatin treatment did not affect the size of the spheroids, an increase in the number of dead cells (PI-positive) and structural disorganization of the spheroids can be detected after 24- and 48-h exposure [Fig. 2(a)].

Fig. 2

Plasma membrane microviscosity in tumor spheroids HeLa before (control) and during 48-h-exposure to cisplatin. (a) Bright-field microscopy, viscosity images of spheroids acquired with FLIM, and live (calcein)/dead (PI) cells assay. (b) Quantification of viscosity during chemotherapy. $\text{Mean}\pm \mathrm{SD}$, $n=4$ spheroids, 20 cells in each. Bar, $80\mu \mathrm{m}$. * Statistically significant difference with control, $p\le 0.005$.

These experiments in cancer cell monolayers and in spheroids clearly showed that a pronounced increase in viscosity, observed at prolonged drug exposures ($\ge 24\mathrm{h}$) was not directly related to cell death because the viscosity changes observed were similar in all cells in a cell population, in those that died and in those that survived the treatment. It was also not a transient response to the treatment, because the increased viscosity persisted for at least 2 days after the drug withdrawal. This allows us to assume that increase in the membrane viscosity could be an early adaptive response to stress conditions (e.g., DNA damage), a part of cell defense mechanisms.

3.3.

Viscosity in Cisplatin-Adapted Cells

In line with the idea that increased membrane viscosity can be a consequence of physiological adaptation of cells to the drug, we analyzed viscosity in HeLa cell subline that had been adapted to cisplatin by prolonged ($\sim 4$ months) exposure to the drug. Drug-adapted cancer cell lines are commonly used as a preclinical model of acquired drug resistance.^46,47 Cisplatin-adapted cells had the same morphology and proliferation rate as nonadapted counterpart, whereas the IC50 value increased from 2.3 to $6.26\mu \mathrm{M}$, indicating that sensitivity to the drug decreased by 2.7 times. Using FLIM of BODIPY 2, we found that the viscosity of the plasma membrane in cisplatin-adapted cells was higher than in control cells, $407\pm 38\mathrm{cP}$ versus $301\pm 27\mathrm{cP}$, $p=0.0005$ (Fig. 3 and Fig. S7 in the Supplemental Materials).

Fig. 3

Plasma membrane microviscosity in cisplatin-adapted (resistant) HeLa cells. (a) Bright-field images and viscosity images of cells acquired with FLIM of fluorescent molecular rotor BODIPY 2. (b) Quantification of viscosity in control and resistant cells. $\text{Mean}\pm \mathrm{SD}$, $n=20\text{cells}$. Bar, $50\mu \mathrm{m}$.

Noteworthy, the increase of membrane viscosity detected in both cisplatin-treated cells at late time-points and the cells adapted to the drug suggests that these changes in the membrane properties can favor the cell survival and contribute to the acquisition of drug resistance.

3.4.

Lipid Composition of Plasma Membranes

To determine whether the changes in plasma membrane viscosity during chemotherapy are caused by the alterations in the lipid composition, we obtained lipid profiles for CT26 cells upon cisplatin treatment using ToF-SIMS. Figure 4 shows typical mass spectra of CT26 cells plasma membranes. Labels show characteristic fragments and molecular ions. Since ToF-SIMS analyze only topmost layers of a sample (i.e., plasma membranes), fatty acids peaks [Fig. 4(b)] are likely originated as fragments of phospholipids. Phosphatidylcholine and sphingomyelin have similar chemical structure and therefore generate common peaks. Nevertheless, these species could be distinguished by $m/z$ 224.1 and $m/z$ 264.3 peaks that were utilized for analysis here. Lipid composition was analyzed at 1 and 24 h exposures to cisplatin, when the decrease and increase in viscosity were detected, respectively, and compared with untreated control.

Fig. 4

Representative mass spectra of CT26 plasma membranes: (a) positive ions and (b) negative ions. Fatty acids are designated as $CX:Y$, where $X$ is the chain length and $Y$ is a number of double bonds. PC, phosphatidylcholine and SM, sphingomyelin.

Figure 5 reveals ion yield variation of phosphatidylcholine ($m/z$ 224.1), sphingomyelin ($m/z$ 264.3), and cholesterol ($m/z$ 369.3) ions for cisplatin exposed cells compared to a control sample. Sphingomyelin signal increases for 24 h administration compared with control and 1 h samples, which is in good accordance with detected increase in microviscosity. Interestingly, phosphatidylcholine intensity drops by more than 20% for 1 h of exposure and increases by more than 20% for 24 h of exposure to cisplatin. In addition, we recorded an increase in cholesterol peak intensity in the membranes of the treated cells during 24 h of incubation with cisplatin. Cholesterol is known as a regulator of membrane stiffness, therefore, the obtained data correlate well with the increase in viscosity that we registered using FLIM of BODIPY 2.

Fig. 5

Lipid composition of CT26 plasma membranes obtained by ToF-SIMS: (a) positive ions. PC, phosphatidylcholine and SM, sphingomyelin. Data normalized to the control sample. (b) Fatty acids chains analysis in negative ions. Data normalized to saturated fatty acids. * Statistically significant difference with control, $p\le 0.001$.

Figure 5(b) represents sum intensity of main peaks of saturated, unsaturated, and polyunsaturated fatty acids observed on mass spectra obtained in negative ion mode. For better presentation, data are normalized to signal of saturated fatty acids.

Unsaturated to saturated fatty acids ratio showed a tendency to decrease after cisplatin exposure. Although the effect is more pronounced after 24 h, it remained within statistical error. The ratio changes were mainly due to oleic acid signal variation. Polyunsaturated fatty acids also showed similar dynamic decrease, which is more pronounced for the 24-h sample.