2.1.

Chemicals

A 10 mM stock solution of cisplatin (Sigma-Aldrich, St. Louis, MO) was prepared in distilled water. 2-NBDG was purchased from Invitrogen (Life Technologies, Grand Island, NY). A $5\mathrm{mg}/\mathrm{mL}$ stock solution of 2-NBDG was prepared in distilled water. MTT cell proliferation assay kit was purchased from ATCC (Manassas, VA). Propargyl choline was synthesized using the method described by Jao and others.²³ 4 g propargyl bromide (80% solution in toluene) was added to 3 g of dimethyl-ethanolamine in 10 mL tetrahydrofuran. The mixture was stirred over ice for approximately 30 min. The reaction mixture was then stirred for 24 h under nitrogen. Propargyl choline (white solid) was separated from the solvent by filtration (Millipore filter) followed by repeated washing with tetrahydrofuran. The dried product was stored at $-20°\mathrm{C}$.

2.2.

Cell Culture

Human cervical carcinoma cell line, HeLa, was maintained in a culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Fisher Scientific, Pittsburgh, PA) supplemented with 10% FBS (Fisher Scientific, Pittsburgh, PA) and $100\mathrm{mg}/\mathrm{L}$ penicillin (Sigma, St Luois, MO). HeLa cells ($4\times {10}^4\text{cells}/\mathrm{mL}$) were seeded into culture flasks, grown in a humidified atmosphere of $5\%{\mathrm{CO}}_2–95\%\text{air}$ at 37°C, and subcultured with 0.05% trypsin (Invitrogen). For fluorescence imaging, cells were cultured on 8 well cover slip bottom culture chambers (Fisher).

2.3.

Multicellular Spheroid Formation

The liquid-overlay technique (hanging drop method) was used to form multicellular spheroids.²⁴ Cells from a confluent T-25 flask were detached using trypsin-EDTA and resuspended in a culture medium to a final concentration of ${10}^7\text{cells}/\mathrm{mL}$. To form individual droplets, 20 μL of the concentrated cell suspension was deposited on the lid of a Petri dish. The bottom chamber of a Petri dish was filled with 2 mL of cell culture medium to limit evaporation of moisture from individual droplets. The lid with individual droplets was inverted over a Petri dish to form hanging drops. The Petri dish was placed in an incubator for a period of 5 to 7 days, after which visible cell spheroids were formed in individual hanging drops. To treat individual droplets with cisplatin, an approximately 2 μL of cisplatin stock solution (1 mM) was injected using a micropipette into an individual hanging droplet. Cisplatin concentration in individual droplets was approximately 100 μM. Individual hanging drops were treated with cisplatin for 48 h prior to incubation with propargyl choline or 2-NBDG.

2.4.

Choline Uptake Assay

HeLa cells were treated with cisplatin at selected concentration levels (0 to 100 μM) for a specified incubation time (ranging from 0 to 96 h). After the drug treatment, cells were incubated with propargyl choline (2 mM in regular cell culture medium) for 1 h at 37°C. Propargyl choline labeled cells were rinsed with PBS and fixed using formaldehyde (3.7% in PBS) at 4°C. After 30 min of fixation, cells were rinsed with PBS and incubated with the click chemistry reaction buffer for 30 min in the dark at room temperature. This reaction buffer contains 0.1 M Tris-Buffer ($\mathrm{pH}=8.5$), 0.05% Triton, 10 μM Alexa-488 azide, 1 mM ${\mathrm{CuSO}}_4$, and 50 mM Ascorbic Acid. After the click chemistry reaction, cells were rinsed and imaged using an inverted fluorescence microscope (IX71, Olympus Inc., Center Valley, PA). Excitation and emission filters for fluorescence microscopy were $470/15\mathrm{nm}$ and 520 to 550 nm, respectively. Exposure time of the CCD camera (charge-coupled device, Hamamatsu Photonics, Bridgewater, NJ) was 500 ms for fluorescence imaging of cells. Multicellular spheroids were treated with a fixed concentration of cisplatin for 48 h prior to incubation with propargyl choline using the same procedure as described above for the 2-d cell culture models. Multicellular spheroids were imaged using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc., Thornwood, NY) with 488 nm laser excitation and a band pass emission filter (520 to 550 nm).

2.5.

Glucose Uptake Assay

Changes in intracellular uptake of 2-NBDG following the incubation of cells with cisplatin were measured using similar cell culture and incubation procedures as described in the choline uptake assay. The key differences in the glucose uptake assay as compared to the choline uptake assay were the incubation time (30 min of incubation time for 2-NBDG), use of glucose free DMEM during incubation of cells with 2-NBDG and imaging of cells and spheroids labeled with 2-NBDG without chemical fixation. The incubation time was selected based on the results of our previous study.²¹

2.6.

Quantification of Imaging Data

To quantify imaging data, the mean fluorescence intensity (MFI) of individual cells within the field of view (FOV) was calculated using ImageJ (Public domain, NIH). The MFI of individual cells was corrected by subtracting the background fluorescence signal from a region on a chamber cover slip without any cells. It is important to note that the fluorescence intensity values corresponding to both background fluorescence and cellular autofluorescence were significantly small as compared to the fluorescence signal intensity from propargyl choline or 2-NBDG stained cells. Multiple FOVs (typically 9 to 10 FOVs with approximately 90 to 120 cells in total) were analyzed to calculate the MFI from three independent experiments for each of the experimental conditions. Average MFI and standard deviation were calculated for each experimental condition.

2.7.

Statistical Analysis

Statistical analysis was carried out using Microsoft® Excel 2007 (Microsoft Inc., Bellevue, WA) and SAS® (version 9.1 SAS Inc., Cary NC). Student’s $t$-test or ANOVA was used for evaluating statistical significance between the treatments with a 95% confidence level. Differences between the means were evaluated with Duncan’s $t$-test and differences were considered to be significant if the $p$ values were less than 0.05.

2.8.

MTT Assay

For the MTT cell proliferation, cells cultured in 24-well plates were treated with cisplatin at selected cisplatin concentrations and for specified time (24 to 96 h). After treatment with cisplatin, the MTT assay was conducted according to the instructions of the MTT Cell Proliferation Assay (ATCC, Manassas, VA). Briefly, 10 μL of the MTT reagent was added to individual wells and the plates were incubated for 3 h at 37°C. After 3 h, 100 μL of the detergent reagent (supplied with the kit) was added to individual wells of the plate. The plate was then incubated overnight in the dark at room temperature. Absorbance values at 570 nm were measured from individual wells using a plate reader (Synergy2, Bio Tek Inc., Winooski, VT). The MTT measurements were conducted in triplicate and the experiment was repeated three times for statistical comparison of the data.

2.9.

Apoptosis Assay

For imaging apoptosis, cells were cultured on chamber cover slips and treated with cisplatin (1 and 10 μM) for 24 h. Cisplatin treated cells were washed and incubated with $1\times \text{assay}$ buffer containing Annexin V fluorescein isothiocyanate and propidium iodide (PI) according to the instructions from the vendor [Annexin V apoptosis detection kit (sc-4252 AK), Santa Cruz Biotechnology, Santa Cruz, CA]. In this assay, the binding of fluorescently labeled Annexin V to the cell membrane indicates changes in membrane structure and is a marker for the early stages of apoptosis. Permeation of PI dye (DNA binding dye) across the cell membrane and staining of the nuclei indicate damage to cell membranes induced by cisplatin treatment. PI staining is a marker for the late stages of apoptosis.²⁵ Together, the combination of these agents provides a comprehensive and an established approach to measure apoptosis in cells induced by drug treatment.

3.1.

Effect of Cisplatin Treatment on Propargyl Choline Uptake

Cisplatin induced changes in intracellular uptake of choline were measured using fluorescence imaging. For fluorescence imaging, an analogue of choline (propargyl choline) with a monoalkyne group at the N-terminus of a choline molecule was synthesized as described in the materials and methods section. The click-chemistry reaction between Alexa-488 azide and monoalkyne modified choline (propargyl choline) molecules enabled in-situ detection of propargyl choline in cells.

Fluorescence imaging measurements in Fig. 1(a) show a decrease in the intracellular uptake of propargyl choline with an increase in concentration of cisplatin (0 to 100 μM). In this experiment, HeLa cells were treated with cisplatin (concentration range from 0 to 100 μM) for 24 h prior to incubation with propargyl choline (for 1 h). To quantify the imaging data, the MFI from individual cells was calculated and normalized with respect to the positive control (propargyl choline labeled cells without drug treatment). Figure 1(b) shows changes in the normalized fluorescence intensity as a function of cisplatin concentration (average of three independent measurements). The results show that intracellular uptake of propargyl choline decreased with an increase in concentration of cisplatin. Treatment of cells with 10 μM of cisplatin concentration resulted in approximately 60% decrease in the uptake of propargyl choline in cells after 24 h. At higher concentration levels (50 to 100 μM of cisplatin), the mean fluorescence signal intensity decreased by more than 90%.

Fig. 1

Changes in uptake and intracellular retention of propargyl choline in cells as a function of cisplatin concentration. (a) Fluorescence images of HeLa cells treated with cisplatin at the indicated concentrations for 24 h. (b) Normalized fluorescence signal intensity of HeLa cells labeled with propargyl choline after 24 h of cisplatin treatment. (c) Fluorescence and corresponding brightfield images show high specificity of click chemistry labeling of HeLa cells labeled with Alexa-488 azide without incubation of cells with propargyl choline.

The specificity of the click chemistry reaction was validated using a negative control [Fig. 1(c)]. In this control experiment, cells were labeled with Alexa 488-azide without prior incubation with propargyl choline. The results of the fluorescence imaging measurements show no significant fluorescence staining in the negative control cells [Fig. 1(c)] while significant fluorescence staining was observed in the positive control cells [Fig. 1(a): cells without cisplatin treatment]. This result demonstrates specificity of the click chemistry reaction in cells.

The specificity of intracellular uptake of propargyl choline was also demonstrated using a competition assay (described in the supplementary information section). A fixed concentration of propargyl choline was coincubated with an increasing concentration of unmodified choline for 1 h in a cell culture model. The results of this competition assay show that the uptake of propargyl choline in cells decreased with an increase in concentration of unmodified choline, indicating that propargyl choline was specifically uptaken by cells through choline transporters.

3.2.

Effect of Cisplatin Treatment on 2-NBDG Uptake

Figure 2(a) shows a decrease in the uptake of 2-NBDG with an increase in concentration of cisplatin. This trend is similar to the results in Fig. 1(a). Treatment of cells with 10 μM of cisplatin concentration resulted in approximately a 40% decrease in the uptake of 2-NBDG in cells after 24 h [Fig. 2(b)]. Comparison between the results in Figs. 2(b) and 1(b) demonstrates that the uptake of choline was reduced more than the uptake of 2-NBDG upon treatment with cisplatin.

Fig. 2

Changes in uptake and intracellular retention of 2-NBDG in cells as a function of cisplatin concentration. HeLa cells were treated with cisplatin at the indicated concentrations for 24 h. (a) Fluorescence images show decrease in uptake of 2-NBDG with increasing concentration of cisplatin. (b) Normalized fluorescence signal intensity of HeLa cells labeled with 2-NBDG after 24 h of cisplatin treatment.

3.3.

Effect of Cisplatin Treatment on Cell Viability

Cell viability of HeLa cells treated with cisplatin for 24 and 96 h was measured using the MTT assay. The MTT assay measures the activity of mitochondrial reductase enzymes and is a well-established assay to measure cell viability. The results of the MTT assay are shown in Fig. 3(a). These results show that cell viability was not significantly reduced with exposure to low concentration levels of cisplatin (1 to 10 μM) for both 24 and 96 h incubation time. A significant decrease in cell viability was observed only at higher concentration levels of cisplatin (above 25 μM). These results are in agreement with previously published reports^21,26 and these results highlight that although cells were viable based on the MTT assay, significant changes in the uptake and intracellular retention of both propargyl choline and 2-NBDG were detected. This suggests that changes in the intracellular flux of metabolites are significantly influenced by cisplatin prior to induction of cell death (measured based on the MTT assay).

Fig. 3

Conventional cell viability assays to evaluate the response of HeLa cells to cisplatin treatment. (a) Cell viability assessed by MTT assay after 24 or 96 h of cisplatin treatment. (b) Brightfield/fluorescence overlay images of HeLa cells treated with cisplatin at indicated concentration for 24 h and stained with PI/Annexin V mixture. Annexin V positive cells (green) and PI staining cells (red) indicates the early and late stages of cell apoptosis induced by cisplatin.

To detect apoptosis, HeLa cells were treated with 0, 1, and 10 μM of cisplatin for 24 h and stained using PI/Annexin V [Fig. 3(b)]. The results show no significant increase in Annexin V staining in cisplatin treated cells at 1 μM concentration as compared to the controls (cells not treated with cisplatin). It is also important to note that nonspecific binding of Annexin V to the cell membrane results in significant background fluorescence staining in the control cells. Similarly, no significant PI staining was detected in cells treated with 1 μM of cisplatin. In the case of HeLa cells treated with 10 μM of cisplatin, a significant increase in the number of cells stained with PI and Annexin V was observed as compared to the control cells. These results indicate that PI/Annexin V staining is more sensitive than the MTT assay in detecting the response of cells to cisplatin treatment, but it has limited sensitivity as compared to changes in the uptake and intracellular retention of propargyl choline and 2-NBDG.

3.4.

Early Decrease of Propargyl Choline and 2-NBDG Uptake after Cisplatin Treatment

The next goal was to characterize cisplatin-induced changes in the uptake of propargyl choline and 2-NBDG in cells as a function of time. For these experiments, a sub-lethal dosage of cisplatin (1 μM) was selected to demonstrate sensitivity of the imaging approaches. This sub therapeutic concentration of cisplatin (1 μM) was at least 50 times lower than the IC-50 value determined using the MTT assay. Changes in the uptake of propargyl choline in HeLa cells were measured every 3 h. This time interval of 3 h was selected based on the results of a prior study.²¹ The results of these measurements [Fig. 4(b)] show a significant decrease in the uptake of propargyl choline (approximately 55%, $p=0.032$) during the first 3 h of incubation of cells with cisplatin at 1 μM. These changes in the uptake of propargyl choline precede detection of loss in cell viability by the MTT assay and the PI/Annexin V staining. It is expected that with an increase in concentration of cisplatin, the time required to detect changes in the uptake of propargyl choline can be further reduced.

Fig. 4

Changes in uptake and intracellular retention of propargyl choline in cells as a function of cisplatin incubation time. HeLa cells were treated with cisplatin at the concentration of 1 μM. (a) Fluorescence images show changes in fluorescence intensity as a function of time (over 6 h) of cisplatin treatment. (b) Changes in normalized mean fluorescence signal intensity of HeLa cells treated with cisplatin as a function of time. Cells were labeled with propargyl choline after cisplatin treatment for a defined period of time.

Figure 5 characterizes the changes in the uptake of 2-NBDG in HeLa cells treated with 1 μM cisplatin as a function of time. Figure 5(b) shows the changes in the normalized MFI of 2-NBDG labeled cells as a function of incubation time with cisplatin. The results show that approximately 6 h of drug incubation time (with 1 μM of cisplatin) was required to achieve a significant decrease (approximately 36%, $p=0.043$) in the intracellular uptake of 2-NBDG. These results in Figs. 4 and 5 demonstrate that cisplatin induced changes in the uptake of propargyl choline were rapid and more pronounced as compared to cisplatin induced changes in the uptake of 2-NBDG.

Fig. 5

Changes in uptake and intracellular retention of 2-NBDG in cells as a function of cisplatin incubation time. HeLa cells were treated with cisplatin at the concentration of 1 μM. (a) Fluorescence images show change in fluorescence intensity as a function of time (over 6 h) of cisplatin treatment. (b) Changes in normalized mean fluorescence signal intensity of HeLa cells treated with cisplatin as a function of time. Cells were labeled with 2-NBDG after cisplatin treatment for a defined period of time.

3.5.

Cisplatin Treatment in Multicellular Tumor Spheroid Model

Multicellular tumor spheroids mimicking the structural features of a solid avascular tumor core were formed using the liquid-overlay method as described in the materials and methods section. The results in Fig. 6 characterize the uptake of propargyl choline and 2-NBDG in 3-d tumor spheroids treated with cisplatin (concentration of 100 μM for 48 h) as described in the materials and methods section. The results were compared with control spheroids without cisplatin treatment.

Fig. 6

Confocal fluorescence, brightfield, and brightfield/fluorescence overlay images of multicellular tumor spheroids (MTS) labeled with (a) propargyl choline and (b) 2-NBDG. Control: MTS without cisplatin treatment. Drug treated sample: MTS treated with 100 μM of cisplatin for 48 h.

The optical imaging measurements [Fig. 6(a)] in control spheroids demonstrate uniform uptake of propargyl choline. After treatment with cisplatin, a significant decrease in intracellular uptake of propargyl choline was observed. The results in Fig. 6(b) characterize the uptake of 2-NBDG in 3-d tumor spheroids (control and drug treated samples). These results illustrate uniform delivery of 2-NBDG in control tumor spheroids and a significant decrease in uptake of 2-NBDG in tumor spheroids following treatment with cisplatin. These results demonstrate that the imaging approaches have a significant potential to detect the response of individual cells to cisplatin treatment in a 3-d tumor model.