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QUANTITATIVE EVALUATION OF CISPLATIN UPTAKE IN SENSITIVE AND RESISTANT INDIVIDUAL CELLS BY SINGLE-CELL ICP-MS (SC-ICP-MS) Mario Corte Rodríguez, Roberto Álvarez-Fernández García, Elisa Blanco Gonzalez, Jorg Bettmer, and Maria Montes-Bayon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02746 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Analytical Chemistry

QUANTITATIVE EVALUATION OF CISPLATIN UPTAKE IN SENSITIVE AND RESISTANT INDIVIDUAL CELLS BY SINGLE-CELL ICP-MS (SC-ICP-MS). M. Corte Rodríguez+, R. Álvarez-Fernández García+, E. Blanco, J. Bettmer* and M. Montes-Bayón* Department of Physical and Analytical Chemistry. Faculty of Chemistry. University of Oviedo. C/ Julián Clavería 8, 33006 Oviedo. Spain. *[email protected] *[email protected]

+

These two students have contributed equally to this work.

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ABSTRACT One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated to a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and/or alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in solution). Quantitative uptake studies of a non-toxic Tbcontaining compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest. Key-words: cisplatin, single cell, ICP-MS, resistance


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INTRODUCTION Since its approval by the Food and Drug Administration in 1978, cisplatin is one of the most commonly used chemotherapeutics in the treatment of different cancers, such as ovarian, testicular or lung cancer.1,2 The main mechanism of action of cisplatin begins with the uptake of cisplatin by the cells, followed by its transport through the cytosol to the cell nucleus. In this process, cisplatin is activated by aquation of its Pt-Cl bonds, followed by its binding to DNA, preferentially to the nitrogen-7 of the guanine residues. 3,4 This ability to crosslink with the purine bases on the DNA generating the so-called Pt-DNA adducts causes DNA damage, which is the ultimate event in its cytotoxic activity that eventually engenders cell death.5 However, cisplatin acquired resistance is one of the major drawbacks of this drug therapy and the paths that lead to it are unclear and multifactorial.6 Resistance to cisplatin can occur due to a combination of events such as an altered cellular accumulation of the drug, its cytosolic inactivation and altered DNA repair of the formed cisplatin-DNA adducts.7 A decreased accumulation of cisplatin in cancer cells seems to be an important source of resistance that has been frequently observed in cisplatin-resistant cells.7 Such event could be due to either a decreased cellular uptake or an increased cellular export of the drug.8 Incorporation of cisplatin into cells was thought to occur through passive diffusion although more recent reports reveal the important role of specific membrane transporters, such as the copper transporters CTR1 and CTR2 for the uptake9 or P-type ATPase or ATP-binding cassette transporters for the export.10 In any case, the determination of intracellular Pt concentration seems to be an important parameter to consider for detecting cellular resistance.11



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In this vein, several attempts have been made to quantify platinum incorporation in cisplatin-treated cellular cultures and animal models. However, most of these experiments are bulk studies, in which Pt concentration obtained after cell lysis is averaged with respect to the number or mass of cells or is made relative to the cell protein concentration.12,13 This strategy provides the average Pt content in the cells but does not consider the variation between individual cells once exposed to a given cisplatin dose. In this regard, it has been long known that biological populations are heterogeneous and, thus, that it is more interesting to obtain information about individual cells rather than settling for average information. For detection of metals like Pt in cells, colorimetric methods have been classically applied.14 They rely on the fact that many inorganic complexes are distinguished by their intense visible absorption spectrum and many histochemical methods are based on such changes. Although these methods are useful for studying metal distribution in tissues in a qualitative manner, they do not show sufficient sensitivity for single-cell studies. Regarding mass spectrometric based methods, metal cellular distribution has been accomplished by using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and secondary ion mass spectrometry (SIMS).15,16 The latter one with spot sizes around 50 nm permits, in addition, the imaging of subcellular structures. For the monitoring of elements in single cells it is mandatory to introduce individual cells into the ionization source. This requires an appropriate dilution of the sample accompanied with short integration times. Li et al. introduced the first approach to investigate the behaviour of bacteria introduced into an ICP-MS using fast


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data acquisition for U+ detection (4 ms)17. Further work involved the use of mass cytometry, which is a variation of flow cytometry, in which antibodies were labeled with heavy metal ion tags rather than fluorochromes for multiplex detection of different cell biomarkers using a similar cell introduction principle.18 In this approach, antibodies against specific biomarkers present on the cell surface have been designed and then labelled with different lanthanides. The individual cellular introduction and the (quasi)-simultaneous time of flight (TOF) selection of the m/z of the different lanthanides permits multiplex biomarker determination. Recently, such individual cellular introduction into the ICP-MS has been exploited to conduct initial studies on individual cisplatin uptake into cells.19 Finally, single cell techniques employing special sample introduction systems involving microfluidic devices like microdroplet generators (MDG) 20,21,22 or different nebulizers have been successfully applied to the determination of constituting elements or specific metallodrugs in single cells with ICPMS detection (single cell ICP-MS, SC-ICP-MS).23,24 Recently, the combination of a concentric micronebulizer (PFA-ST) and a heated single-pass spray chamber was applied to the study on the variation of element masses and distribution patterns in individual cells25. A novel coupling of a droplet chip for single cell encapsulation to a microflow nebulizer was applied to the analysis of single cells after incubation with ZnO nanoparticles26. Finally, Zheng et al. studied the Gd@C82(OH)22 and cisplatin uptake of Hela and 16HBE cells and reported Pt contents of up to approximately 2 pg per cell27. Thus, in this work, the evaluation of single-cell analysis with ICP-MS detection using a self-developed liquid introduction system based on a total consumption microflow nebulizer for introduction of cells into the ICP-MS is conducted. The system has 5 ACS Paragon Plus Environment


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been evaluated with three different models: Fe as a constitutive element; a Tbcomplex (non-essential and non-toxic) and finally, the cisplatin (non-essential and toxic to cells). The combination of flow cytometry data with ICP-MS measurements is shown to address, accurately, the number of cells in the suspension and thus, the efficiency of the sample introduction system. This set-up is explored to address cisplatin uptake in ovarian cancer cell lines that are sensitive (A2780) and resistant (A2780cis) to the drug in order to obtain clinically relevant data in cancer chemotherapy.

EXPERIMENTAL Materials and methods Reagents.- All chemicals were of analytical reagent grade or better. Ultrapure water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA) and was used throughout this work. HPLC-grade methanol was obtained from Sigma Aldrich. Cisplatin (cis-diamminedichloroplatinum(II)) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved daily to obtain a 1 mg mL-1 stock solution in water. For cell culture, Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS) and trypsin were from LabClinics (Barcelona, Spain), sodium chloride (ACS reagent,, ≥ 99.0%, Sigma-Aldrich), fetal bovine serum was from Invitrogen (Fisher Scientific, Madrid, Spain) and plasmocin was from InvivoGen (San Diego, USA). CellstripperTM was purchased from Thermo Fisher Scientific (Madrid, Spain). A Tb complex of diethylenetriaminepentaacetic acid (Tb-DTPA) was used to evaluate cell uptake. It was synthesized using DTPA and TbCl3·6H2O, both from Sigma


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Aldrich. They were mixed and incubated at 37 ºC for 2 h to obtain a solution of the complex in a concentration of 20 mmol L-1. Instrumentation.- All the experiments were conducted in the ICP-MS Agilent 7700 (Agilent Technologies, Japan) fitted with an EnyaMist nebulizer (Burgener, Ontario, Canada) and a self-developed single pass spray chamber of an internal volume of about 10 mL. A similar system is described elsewhere28, however, within this work no additional gas connection was needed. The samples and rinsing solutions were pumped at a flow rate of 10 µL·min-1 into the nebulizer using the syringe pump SP101i (Florida, USA) fitted with a 1 mL Hamilton syringe (Nevada, USA). The dwell time was set to 10 ms in order to be able to detect single-cell events. The data were recorded in time-resolved analysis mode during 3 min per analysis. Under these conditions, only a single isotope could be measured in one run due to the sequential nature of the measurements in a quadrupole system. A Flow Cytometer FC 500 series from Beckman Coulter (California, USA) was used. For accurate cell counting, BD Trucount™ Tubes (BD Biosciences, New Jersey, USA) were used. These contain a determined number of beads, which are mixed with a known volume of cells and then vortexed thoroughly (at low speed) to re-suspend the beads and reduce cell aggregation before running the mixture in the flow cytometer. The instrument separates the population of beads from the cells and calculates the number of cells that pass through the laser beam related to the known number of beads also counted. The flow cytometry increases the accuracy of cell counting compared to haemocytometers.



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Cell culture conditions.- A2780 human ovarian carcinoma cell line was a kind gift of Dr. J.M. Pérez Freije (Department of Biochemistry and Molecular Biology, University of Oviedo). The in vitro-derived cisplatin-resistant A2780cis cell line was purchased from the European Collection of cell cultures through Sigma-Aldrich. The cells were cultured at 37ºC in a 5% CO2 incubator using DMEM medium supplemented with 10% fetal bovine serum and 5 mg L-1 of plasmocin. The cell cultures were exposed to cisplatin at 10 and 20 µmol L-1 and to Tb-DTPA at 5, 10 and 20 µmol L-1, respectively, during 24 h at 37ºC. In order to obtain a homogeneous contact between the drug and the cells, solutions containing cisplatin or Tb-DTPA in the cell growing medium were freshly prepared. The initially used cell culture medium was then replaced by metallo-complex containing medium. Non-treated cells (control) were also processed alongside the drug-treated cells. Afterwards, the cells were washed three times with PBS and collected by trypsinization. At this point, cells were always counted by means of two different systems: a haemocytometer and a flow cytometer using the BD Trucount™ Tubes. As alternative to PBS washing, the cells were three times washed with 0.9 % solution of NaCl. Finally, CellstripperTM was tested as gentle, non-enzymatic reagent to dissociate adherent cells in cell cultures29. For single cell analysis, the collected cells were diluted with 10% methanol to the required concentration. As recommended by the manufacturer, the addition of small amounts of an organic modifier is necessary for the undisturbed operation of the EnyaMist nebulizer. For the bulk analysis of Tb in the cell suspension, cells were digested with concentrated HNO3 at 70ºC for 1 h and 30% H2O2 for 3 h at the same temperature. Then, the digested solution was diluted with ultrapure water and the metal content determined by ICP-MS as previously optimized.12 8 ACS Paragon Plus Environment

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Data treatment.- For the data treatment of single cell suspension measurements, an established iterative procedure was followed30,31, based on averaging the entire data set and collecting all data points that are three standard deviations (3σ) above the mean. The resulting data set after removing the selected events is rearranged and the procedure is repeated until no new data points are above the 3σ threshold. After the selection of the single cell signals, those higher than 3σ above their mean were discarded. RESULTS AND DISCUSSION Optimization of liquid sample introduction system and time-resolved analysis for single cell analysis.- First experiments were conducted using the A2780 cell line incubated with the complex Tb-DTPA to address the possibility of using the total consumption sample introduction system to detect events corresponding to single cells in the ICP-MS. These cells of ovarian carcinoma represent eukaryotic cells with an average diameter of 5.6 µm and were introduced into the nebulizer by continuous pumping at 10 µL min-1. Most of the tested cells maintained an intact cellular morphology and stay in a monodisperse state during nebulization. Only a low percentage of cells were disrupted during the transport and nebulization process as can be seen in Figure S1. In such way, each ICP-MS spike signal corresponds to one individual cell event. However, one of the limitations of such simple introduction system might be the possibility of having more than one cell simultaneously passing through the nebulizer at a time, generating overlapping events that cannot be distinguished from each other. The common strategy to prevent such situation is to prepare highly diluted suspensions of particles (or cells, in this case), in which mostly



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individual events correspond to single cells. For this aim, the cell number density was optimized in order to obtain a statistically relevant number of cells in a time period of 3 minutes and minimizing double- and multiple-cell events. The cell number concentration has to be low enough, so that most droplets do not contain any cell and the probability of having two or more cells can be neglected. This optimization was done by measuring different cell densities of Tb-treated cells. The complex Tb-DTPA penetrates into cells without showing significant toxicity and can be easily detected by ICP-MS (low blank levels from cells).32 Figure 1 shows the events corresponding to an approximate number of cells per mL of 2.5 x 104, 5 x 104, 1 x 105 and 3 x 105, respectively. As can be observed, an increase in the number of cells seems to be directly related with the number of detected events in the ICP-MS, as reported in the case of single particle analysis. In addition, even in the case of low number of cells, the events can be clearly distinguished from the background noise. The mean net signals varied between 4,000 to 10,000 cps for the lower concentrated cell populations and increased for the higher ones due to the enhanced introduction of more than one cell per integration time. Each individual cell event detected in the ICPMS using 10 ms integration time provides an ion cloud from an individual cell that is large enough to produce a detectable signal . It has to be taken into account that even when the typical lifetime of a droplet or particle in the ICP has been reported to be about 0.2 - 0.5 ms, 10 ms is within the range of optimum reading efficiency obtained in previous experiments conducted by other authors with a similar sample introduction system.23 As mentioned above, with increasing cell concentrations the number of very intense signals (> 500,000 cps) ascended. This indicated the introduction of two and more cells within the integration time of 10 ms. 10 ACS Paragon Plus Environment

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Efficiency for single cell introduction: the combination with flow cytometry.According to Degueldre et al.33, and adapting the terms from single-particle to singlecell analysis, the frequency of the cell events (f) (number of pulses per millisecond) can be related to the cell number concentration, NC, the sample flow rate, qliq (mL ms-1) and the transport efficiency (ƞn) by Equation [1]: ݂ = ܰ஼ · ‫ݍ‬௟௜௤ · ߟ௡

[1]

Thus, transport efficiency, ƞn, is proportional to the ratio f/NC. While increasing NC, f increases proportionally while double-cell events are negligible. Above a certain cell concentration, this cannot be assumed anymore and, due to these double-cell events, f does not increase proportionally to NC, thus decreasing the ratio f/NC. The maximum transport efficiency and the maximum cell concentration that can be used with the presented set-up will then be obtained just before double-cell events are too frequent, which can be obtained from the maximum obtained when plotting ƞn vs NC. In order to obtain this plot, a more accurate evaluation of the number of introduced cells (than the conventional haemocytometer) was conducted by using flow cytometer which permits accurate cell counting (to obtain NC) by using the BD Trucount beads following the manufacturer’s instructions. Such beads are pre-mixed with a known volume of cells and then vortexed thoroughly to re-suspend beads and reduce cell aggregation before running the mixture on the flow cytometer which counts the sorted populations of either beads or cells. After counting a certain number of beads, the flow cytometer calculates the number of cells of an aliquot considering the number of events detected and the initial and detected number of beads (see



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Figure 2A). This system permits an accurate determination of the number of cells in suspension. We compared the number of events obtained for 56Fe (measured in untreated cells) and

159

Tb in Tb-DTPA-treated cells during 180 s at 10 µL·min-1. The cell number

density of the suspension was determined by flow cytometry. With these parameters it was possible to calculate the efficiency of the system for cell introduction and the results can be observed in Figure 2B. The total number of detected cell events within 180 s increased with rising cell number concentration from approximately 110 to 680 for Tb and 95 to 456 for Fe. Under optimum conditions, the cell introduction efficiency is about 25% for the two tested elements using 5 x 104 cells mL-1. The corresponding histograms are shown in Figure S2. Higher cell number density can cause overlapping of cell signals, with a final decrease in system efficiency, as observed at 3 x 105 cells mL1

in Figure 2B. All these results are summarized in Table 1.. Since the aim of the work was to quantitatively address cellular uptake of

metallodrugs, additional experiments were conducted in order to address the changes in the height of the events with regard to the Tb-DTPA uptake by the cells. Therefore, the events corresponding to the solutions containing the same number of cells (about 6x105 cells) of different cell cultures exposed to 5, 10 and 20 µmol L-1 of Tb-DTPA, respectively, were analysed. The variation on the height of the events with the TbDTPA concentration revealed to be related to the Tb-doses employed for cell exposure. This relation was not linear which is not surprising since different uptake mechanisms can be triggered at specific doses. The next step was to address if we can use the single cell method to calculate the individual Tb content in each cell. For this aim, a


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calibration curve of Tb was constructed using inorganic Tb standards that were measured under the same conditions. The mass of Tb in each of these standards was represented versus the count rate for each of the calibration standards. Similarly, the mass of Tb in the single cells exposed to 10 µmol L-1 Tb-DTPA was calculated using the equation [2]:

݉஼ =

ఎ∙ி∙௧∙ூ ௕

[2]

Where mc is the mass of the element in the cell, η is the transport efficiency of the inorganic standard solutions (calculated using NIST certified gold nanoparticles RM 8012 in the single particle mode using the particle number method suggested by Pace et al.30); F is the sample flow into the system, t is the integration time, I is the count rate of Tb in the single cell and b is the slope of the Tb calibration curve previously obtained. The calculated transport efficiency using the total consumption nebulizer used in this work turned out to be about 48%. The value was much higher than the transport efficiency determined for the cells (approximately between 5 and 25 %, Figure 2B). This observation could be explained with the dimensions of cells in the µm3 range and its surface structure. The surface might have a relatively enhanced affinity to the transport and nebulizer capillaries, so that adsorption could be expected resulting in a certain loss of cells. Furthermore, the cell sizes are in the order of magnitude of the size of droplets produced during nebulization and a part of them might be captured at the walls of the nebulization chamber. In any case, using these calculations the average mass of Tb found in the cells turned out to be 2.9 ± 0.8 fg Tb per cell (n = 81 events). The methodology was validated by conducting the bulk 13 ACS Paragon Plus Environment


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analysis of the pooled cells using a previously established strategy12 and a calibration curve of Tb using flow injection analysis with ICP-MS detection (FIA-ICP-MS). The results provided an overall Tb mass of 2.6 ± 0.1 fg Tb (n=3 independent cultures) per cell, which is in good agreement with the results obtained for individual cells and that validates the proposed quantitative strategy. Quantitative cellular uptake of cisplatin in sensitive and resistant cell models.- The next step was to evaluate the suitability of the system to address changes in the uptake of Pt-drugs from cell cultures. For this aim, different cell cultures of A2780 and A2780cis from ovarian cancer (sensitive and resistant to cisplatin, respectively) were treated with concentrations of 10 and 20 µmol L-1 cisplatin. Initial experiments using PBS as washing medium resulted in increased background level in the exposed cells that ranged from 50,000 cps (in the cells exposed to 10 µmol L-1 cisplatin) to almost 100,000 cps (in those exposed to 20 µmol L-1 cisplatin). Although several washing steps were included, the Pt background contribution was always observed. This is probably due to a leaching effect of the cell trypsination solutions on the cisplatin that is adsorbed on the surface of the cell growing surface together with the Pt that could be weakly attached to the cell surface. Previous studies12 have revealed through mass balance experiments the unspecific loss of cisplatin when exposed to cell cultures in plastic culture flasks. As alternative NaCl was tested, but did not improve the high Pt background levels. Moreover, as demonstrated in Figure S1C, a low percentage of cells did not maintain integrity during the transport and nebulization process. This surely contributed to the elevated background signal.


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Apart from the latter observation, better results were obtained by using CellstripperTM to collect cells from the adhering surface29. Figure 3 shows a significantly improved background signal between approximately 10,000 and 30,000 cps and the change on the event´s height upon increasing cisplatin concentration for the A2780 (Figure 3C and D) and the absence of events in the case of the control cells or the background (Figure 3A and B). Corresponding measurements of A2780cis cells are represented in Figure S3 (see electronic supplementary material). The Pt concentration in the single cell events was calculated, as mentioned for Tb, using the equation given in [2]. The obtained results revealed an average Pt concentration in the cells exposed to 10 µmol L-1 cisplatin of 1.26 ± 0.63 fg Pt (n=202 events) for the A2780 (sensitive) and 0.82 ± 0.42 fg Pt (n=260 events) for the A2780cis (resistant). The same lines exposed to 20 µmol L-1 showed values of 3.58 ± 1.05 fg Pt (n=134 events) for the A2780 and 1.33 ± 0.36 fg Pt (n=368 events) for the A2780cis. These results are collected in the box plot of Figure 4. This represents about 4 x 106 Pt atoms and 3 x 106 Pt atoms per cell, respectively, in the sensitive and resistant lines exposed to 10 µmol L-1 cisplatin which is in the order of previous studies in other cell models.19 As can be observed, the fg of Pt per cell obtained when duplicating the cisplatin exposure concentration is more than double in the sensitive line but not in the resistant one, as observed in the bulk analysis.12 Thus, the methodology here developed permits to directly discriminate among cell populations (sensitive or resistant to the drug) with no need to digest the sample and with relatively few cells (5x105). In addition, the data dispersion (rejecting events above mean + 3σ) turns out to be higher in the sensitive cells while the resistant type shows more homogeneous behaviour supporting the possibility of a resistance mechanism associated to an 15 ACS Paragon Plus Environment


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inhibition on the drug influx. These observations and the application of the developed methodology might be, for instance, very useful for pre-clinical studies of novel metallodrugs. Such possibility can be further explored with this type of instrumental set-up. CONCLUSIONS The developed system based on the use of the EnyaMist nebulizer and a total consumption spray chamber turned out to be a useful approach for individual cell monitoring with ICP-MS detection. The optimized system yields cell introduction efficiencies of about 25%, which is higher than most of the set-ups published until now. Such data can be accurately obtained by the combined application of flow cytometry using fluorescent beads for cell counting. In addition, the optimization of the system with the complex of Tb-DTPA allows the quantitative estimation of the Tbuptake in individual cells that can be directly compared to the results obtained in the bulk experiments. Furthermore, the proposed strategy permits to discriminate between cisplatin-sensitive and resistant cell lines obtaining also information on the differences among individual cells. Thus, this analytical tool seems to be a promising approach for many studies in individual cells.

ACKNOWLEDGMENTS The authors would like to acknowledge the grant of M. Corte referenced BP13114, the regional funding through the project GRUPIN14-010 and the funding from the Spanish


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MICINN (Spanish ministry for science and innovation) through the project MINECO-16CTQ2015-69583-R.



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TABLES AND FIGURES

Table 1

Comparison of the cell transport data for 56Fe (constitutive element) and 159 Tb (as cell marker)

Figure 1 Time-resolved scan of 159Tb obtained for different cell concentrations treated with 20 μmol L-1 of Tb-DTPA: A) 2.5 x 104 mL-1, B) 5.0 x 104 mL-1, C) 1.0 x 105 mL-1, and D) 3.0 x 105 mL-1

Figure 2 A) Flow cytometric analysis of the beads and cells (FL1 Log represents, in logarithmic scale, the forward scatter signal intensity, which is proportional to cell size and SS Lin means the side scatter signal intensity, which is proportional to cell granularity) and B) determined transport efficiency versus cell number concentration of 56Fe and 159Tb (N = 3)

Figure 3 Time-resolved scans of 195Pt obtained for the A2780 cells treated under different conditions. A) Obtained background signal without introducing any cell suspension, B) without cisplatin incubation, C) after treatment with 10 μmol L-1 cisplatin; D) after treatment with 20 μmol L-1 cisplatin

Figure 4 Box plots showing Pt content observed in A2780 and A2780cis cells treated with 10 and 20 μmol L-1 cisplatin, respectively


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Table 1

Comparison of the cell transport data for 56Fe (constitutional element) and 159

Tb (as cell marker)

56

Fe as constitutive element

Cell number concentration

Event frequency

Number of events Transport efficiency

[mL-1]

[Hz]

in 180 s

[%]

25,000

0.53

95

12.7

50,000

2.08

375

25.0

100,000

2.10

378

12.6

300,000

2.53

456

5.1

159

Tb-DTPA as cell marker

25,000

0.65

117

15.6

50,000

2.06

371

24.7

100,000

2.87

517

17.2

300,000

3.73

672

7.5



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Figure 1 Time-resolved scan of 159Tb obtained for different cell concentrations treated with 20 μmol L-1 of Tb-DTPA: A) 2.5 x 104 mL-1, B) 5.0 x 104 mL-1, C) 1.0 x 105 mL-1, and D) 3.0 x 105 mL-1


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Figure 2 A) Flow cytometric analysis of the beads (A) and cells (B) (FL1 Log represents, in logarithmic scale, the forward scatter signal intensity, which is proportional to cell size and SS Lin means the side scatter signal intensity, which is proportional to cell granularity) and B) determined transport efficiency versus cell number concentration for 56Fe and 159Tb (N = 3)


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Figure 3 Time-resolved scans of 195Pt obtained for the A2780 cells treated under different conditions. A) Obtained background signal without introducing any cell suspension, B) without cisplatin incubation, C) after treatment with 10 μmol L-1 cisplatin; D) after treatment with 20 μmol L-1 cisplatin



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Figure 4 Box plots showing Pt content observed in A2780 and A2780cis cells treated with 10 and 20 μmol L-1 cisplatin, respectively



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