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Keywords:

  • flow cytometry;
  • UV fluorescence;
  • chronic myeloid leukemia;
  • imatinib mesylate;
  • intracellular imatinib

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Imatinib (IM) was the first specifically targeted drug to be developed and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). To evaluate cellular uptake of IM, we developed a method based on the chemical structure of the molecule and using the natural UV fluorescence that we quantified by flow cytometry. In two CML cell lines, we obtained a satisfactory relationship between intracellular IM (ICIM) levels and media concentrations, and we found a strong correlation between ICIM at 1 h and IM efficacy at 24 h, demonstrating that ICIM at 1 h might be a relevant predictive parameter of cell sensitivity. Our method was more sensitive than the standard physicochemical method. We applied our method to primary cells and found cell morphology-dependant IM accumulation. Moreover, in CML cells from patients at diagnosis, IM accumulation was heterogeneous. In all cases, ICIM at the single-cell level was much higher than in culture media arguing in favor of a predominantly active uptake process. We developed a simple method directly applicable to primary cells that has shown two major advantages: only a small number of cells are required, and cell subsets can be identified according to morphological criteria and/or the presence of particular antigenic sites. This method provides a new tool to assess CML cell sensitivity to IM, and ICIM levels in native CML cells could be used to monitor therapeutic response. © 2012 International Society for Advancement of Cytometry

One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Chronic myeloid leukemia (CML) is a clonal, multistep, multilineage myeloproliferative disorder that has become a model of targeted therapy. CML is characterized by the presence of the Philadelphia chromosome (Ph) generated by a reciprocal translocation of the long arms of chromosomes 9 and 22 (1). The resulting BCR-ABL fusion gene encodes a chimeric oncoprotein (p210BCR-ABL) that displays constitutively elevated tyrosine kinase activity and drives the pathogenesis of the disease (2). Imatinib mesylate (IM; Gleevec®, Novartis), originally designated as Signal Transduction Inhibitor 571 (STI571), is a 2-phenylaminopyrimidine derivate. This molecule is the first targeted therapy through selective BCR-ABL kinase inhibitor activity and is now the front-line therapy for chronic phase CML, producing durable response and prolonging survival (3). However, both initial and acquired resistances to IM have emerged and, in a minority of cases, are explained by mutations in the BCR-ABL kinase domain (4). Some studies using cell-line models have identified other events leading to IM resistance, such as BCR-ABL gene amplification (5), high expression of efflux transporter of the drug (6, 7), clonal evolution with acquisition of additional chromosomal abnormalities, or persistence of quiescent stem cells (8).

Another obvious resistance mechanism concerns IM pharmacokinetics, because it is essential that sufficient concentrations of the drug reach the cell target to be fully effective. IM bioavailability is subject to considerable interindividual variability, due in part to digestive absorption, plasma protein binding (9), interaction with others drugs, and CYP3 A4 activity (10). A residual plasma threshold value (1,002 ng/ml) with a predictive value for achieving complete cytogenetic response (CCyR) and major molecular response has been identified (11). However, plasma IM levels show considerable interindividual fluctuations as well as intraindividual variability. Some patients, despite achieving the required plasma IM threshold value, do not develop CCyR and vice versa, indicating that clinical response does not always correlate with plasma IM levels (12). Such variability may be multifactorial and can be partly explained by some membrane pump activity, like the organic cation transporter 1 (OCT-1) protein, which is responsible for the active uptake of IM (13), and which is predictive of CML response (14). Despite these considerations, some unsatisfactory responses remain unexplained.

Intracellular IM (ICIM) level is likely to result from all these pharmacodynamic variables and could therefore be a relevant parameter for anticipating the CML response. The simplest technique for evaluating the amount of drug in the cells is to destroy a high number of cells and measure the drug in the cell lysate using a standard technique (HPLC/mass spectrometry). Apart from these standard physicochemical methods for measuring ICIM, only IM uptake experiments using [14C]-labeled drug have been performed (13, 15, 16). However, these methods require a large number of cells and do not allow for identification of target malignant cells; furthermore, radiolabeled drug cannot be used routinely in vivo for patients' blood and bone marrow cells.

In our opinion, given the potential interest of intracellular IM concentrations for evaluating treatment response in leukemia patients and the possibility of identifying target cells, or even the CML stem cells (17) persisting after treatment (18), it was relevant to evaluate IM in the CML model in order to develop a new procedure for detecting IM. In this context, we developed a patented method (CNRS/CHU patent PCT PCT/FR2010/050474) for evaluating the ICIM level in target cells without modifying the molecule or altering its binding to BCR-ABL but using its natural UV fluorescence (19) by flow cytometry. After having validated our process using CML cell lines and established the correlation between our method and the standard assay method using a physicochemical technique, HPLC, we then analyzed the relationship between ICIM level and IM cytotoxicity. Our results suggest that the ICIM level is a relevant parameter that could be used to improve understanding of interpatient variability with regard to clinical response. Here, we present a simple method that is directly applicable to primary cells and which could prove to be a new tool for assessing the sensitivity of CML cells to IM.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Cell Lines and Patients

K562 and KCL22 are BCR-ABL-positive cell lines derived from patients with CML in blast crisis. K562 was purchased from ATCC, and KCL22 was obtained thanks to our partnership with Dr Maguer Satta (Lyon). K562 cells were grown in Iscove's modified Dulbecco's medium (Lonza, Verviers, Belgium) and KCL22 in RPMI 1640 (Lonza). For the two cell lines, culture media were supplemented with 10% fetal calf serum (Biowest, Nuaillé, France), 1% L-glutamine (Lonza), and 1% ciprofloxacine (Merck). All the cells were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO2. Every 3–4 days, cells were counted and seeded at 5 × 105 cells/ml with fresh media. To standardize our experiments, the input-seeding cell-line density was 5 × 105 cells/ml in all experiments (log phase of growth, data not shown).

Blood samples from CML patients in chronic phase (n = 22) before exposure to IM were collected in lithium heparinate tubes. Written informed consent was obtained for all the patients, and the study was approved by the local Ethics Committee. Peripheral blood samples from normal adults (n = 5) were obtained from the remaining blood collected during routine analysis. These samples could be used for research, because patients had been informed and did not verbally express any disagreement, as stipulated by French law. All the experiments were carried out with fresh cells, within 24 h of sampling. Nucleated cells were isolated by collecting the buffy coat, and erythrocytes were lysed using ammonium chloride (Stemcell Technologies, Vancouver, Canada). The cells were counted and plated at 1 × 106 cells per milliliter in minimal essential medium (Lonza) supplemented with 4% fetal calf serum.

Imatinib Solubilization—Determination of IM Absorption and Emission Spectrum

Imatinib mesylate (Sequoia Research Product, Pangbourne, UK) was dissolved in sterile distilled water. Stock solutions were prepared at 10 mM and kept at 4°C.

IM absorption and emission spectrum were measured using a Kontron Instrument (Montigny Le Bretonneux, France) model SFM 25 spectrofluorimeter in scan mode. The results showed maximal absorption and emission at 258 and 412 nm, respectively.

Flow Cytometry Analysis of ICIM Level

Intracellular imatinib (ICIM) level was measured by flow cytometry using a Coulter Epics Elite™ flow cytometer (Beckman Coulter, Roissy Charles de Gaulle, France) equipped with an Innova 90C-4 UV laser (Coherent, Orsay, France), used at a power supply of 100 W UV and argon laser (488 nm), are spatially separated, allowing them to be used simultaneously. Argon laser allowed detection of the fluorescence from PI and Annexin V-FITC. The time delay between the two lasers was 40 μs and was verified before each use. UV fluorescence was detected using a 408 long pass filter.

We applied the principle that in a controlled system, the additional UV fluorescence detected by flow cytometry between control and treated cells must be directly correlated with the amount of added naturally fluorescent molecule. Numerous cellular components may have intrinsic fluorescence, and each cell has spontaneous weak natural fluorescence in UV light. It is therefore essential to predetermine the amount of light naturally emitted by each cell population. We chose flow cytometry because of its sensitivity and the potentially interesting possibility of evaluating UV fluorescence at cellular level and identifying cell subsets. In our study, IM is a molecule naturally fluorescent in UV light, making it possible to trace its intracellular accumulation using this principle. Thus, the difference in fluorescence units between treated and control cells will be proportional to the amount of IM that has penetrated the cell. Consequently, for each experiment, the ICIM level was defined as the different fluorescence of control and treated samples, presuming that the cell system and the cytometer were stable. For the last point, we used calibrated beads (Supporting Information Fig. S1) to ensure that the cytometer did not vary during analysis. They were used immediately before and at the end of each series of analyses. Only a few commercial beads are available as UV beads, and so we also tested calibrated beads for FITC able to fluoresce in UV (Supporting Information Table S1), which, according to the manufacturer, would most likely obtain reliable results. In our experiments, the best were Flow-Check Fluorosphere (Beckman Coulter), which were able to fluoresce at low intensity with a fine peak in linear scale (Supporting Information Fig. S1F).

In all cases, we added propidium iodide (PI; 2 μl per 5 × 105 cells) to analyze only viable (PI negative) cells. In our first experiments, we used two CML cell lines (K562 and KCL22) to implement the protocol with a cell population that was as homogeneous as possible. This removed the variability related to cell morphology and cell lineages (see Results section). Lymphocytes, monocytes, and polymorphonuclear cell subsets were identified with forward and low-side light-scattering characteristics. Acquisition and analysis of at least 50,000 target events were performed.

Assessment of the In Vitro Kinetics of IM Uptake Using Flow Cytometry

In our culture conditions, we carried out a first series of experiments to assess IM uptake kinetics, by measuring UV fluorescence at 5, 15, 30, 60, 120, and 240 min of incubation at two different doses (5 and 50 μM) in the usual growth cell-culture media. At each time point, we stopped IM uptake by subjecting cells to cold temperature (tubes on ice) (13), and cells were then analyzed according to the procedure described earlier. The preliminary data showed that IM uptake reached a plateau from 1 h of incubation, and we chose this time as the endpoint for subsequent experiments.

Correlation Between ICIM Levels Measured by Flow Cytometry and Physicochemical Assay

To evaluate the correlation between flow cytometry and HPLC (a standard analytical method), we quantified the IM-related UV fluorescence from each cell, and, in parallel, the amount of IM released after lysis of a known number of cells. A defined number (5 × 106) of K562 cells were incubated for 1 h with different concentrations of IM (1, 5, 10, 15, 20, 25, 50, and 100 μM) at 37°C in a saturated humidified atmosphere of 5% CO2. After 1 h, we stopped IM uptake by immediate dilution of the cell suspension with cold medium; the cell suspension was then washed twice and kept at cold temperature (tubes on ice, cold centrifugation). After the last wash, 150 μl of cell suspension was removed for measuring intracellular imatinib (ICIM) level by flow cytometry. The cells were counted with a hemocytometer, and viability was evaluated by trypan blue exclusion to calculate precisely the total number of cells available. Preliminary data showed viability > 95% at 1 h. As much supernatant as possible was removed, and the cell pellet was cryopreserved at −80°C until analysis by the standard method (Dr MC Gagnieu' Laboratory, Lyon, France) in order to determine the total amount of IM released from lysed cells. After liquid/liquid extraction, IM was assayed using HPLC with a UV diode array detector. The amount of IM was calculated using three wavelengths (264, 240, and 290 nm), and a spectral analysis was performed to ensure the purity of chromatographic peaks (Supporting Information Fig. S2). The first wavelength (264 nm) enabled us to analyze the UV spectrum corresponding to the IM peak, and the two other wavelengths used (240 and 290 nm) enabled us to check that there was no contamination under the chromatographic peak.

We could thus calculate the average intracellular quantity of IM (pg/cell). From this value, we could then compare intracellular and extracellular amounts of IM, because we knew that an extracellular dose of 5 μM corresponded to 2.9 ng of IM per microliter, which in turn corresponded to 2.9 × 10−6 pg of IM per fl (10−15 l). For example, we estimated that the average size of CML cell line cells was around 4,000 fl, containing around 1 pg corresponding to 2.5 × 10−4 pg IM/fl. We applied this reasoning to other peripheral blood leukocyte subsets from healthy donors and CML patients.

In Vitro Functional Test

With the aim of evaluating the relationship between ICIM levels and cell sensitivity to IM, cells were incubated in the presence of 1, 5, 10, 25, and 50 μM of the drug, and the ICIM levels were evaluated at 1 h. Then, a cell viability assay was done at 24 h by using trypan blue exclusion. The samples were diluted to half in trypan blue solution, and viable and died cells were counted using a hemocytometer. The results were confirmed with Annexin V/PI staining (20) (Annexin V—FITC kit, Beckman Coulter) according to the manufacturer's instructions. Briefly, the cell samples were washed with ice-cold PBS, and the cell pellets were resuspended in 100 μl of 1× binding buffer. The cells were then incubated for 15 min with 1 μl of Annexin V-FITC solution and 5 μl PI. The cell preparations were immediately analyzed by flow cytometry and the percentage of viable cells (PI/Annexin V) evaluated (Supporting Information Fig. S3).

Statistical Analyses

Because of the sample size, the correlation between data obtained with the two methods was found by the calculation of the nonparametric Spearman correlation coefficient. This analysis was completed by linear regression analysis, and straight line equation established the correspondence between additional fluorescence and intracellular IM concentration. To calculate the statistical significance of the difference between two dependent correlation coefficients, the test proposed by Steiger (21) was performed. Differences between paired series were analyzed using the Student's t-test (significance: P < 0.05). The results were expressed as mean ± SEM.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Measurement of ICIM Levels Using UV Fluorescence Detected by Flow Cytometry

With the aim of evaluating intracellular imatinib (ICIM) levels at single-cell level, we used a quantitative flow cytometry technique that was able to analyze each cell separately. In this case, the amount of IM in one cell is low, and, consequently, the UV-fluorescence emitted from IM molecules is limited, requiring linear scale analysis. Thus the intrinsic variability of the cytometer could significantly influence results, and so we used calibrated beads to standardize fluorescence intensity measurements. After testing different commercially available beads, we chose the Flow-Check Fluorosphere for which UV fluorescence was relatively low (detectable at the intensity at which we detected ICIM), but homogenous, with a narrow intensity peak (Supporting Information Fig. S1). These beads were used systematically just before and after each sample series to ensure UV laser stability.

In a first series of experiments, we used CML-derived cell lines to validate our process. After incubating K562 cells in the presence of IM (5 and 50 μM), we quantified UV-fluorescence through the usual FS/SS and PI negative gating (Figs. 1A and 1B). We observed a significant (P < 0.001, n = 6) shift in UV fluorescence intensity between control and treated cells (Fig. 1C), called additional UV fluorescence intensity, which corresponded to the IM amounts that had penetrated the cells. Our first observations showed that ICIM levels were dependant on FS/SS characteristics, with less accumulation in smaller cells (Figs. 1A and 1D). These differences were visible from an extracellular concentration of 5 μM and became significant at 50 μM (P < 0.001 between the smallest and largest cells). The study of IM accumulation kinetics showed additional IM dose-dependant UV fluorescence intensity that was proportional to extracellular IM concentration and could be ascribed to IM stored in these intact cells, thus validating the possibility of detecting UV-fluorescence emitted from IM (Fig. 1E). The kinetics showed that IM rapidly penetrated into cells as the drug was detectable from 5 min of incubation, with 10 and 30 U of additional fluorescence for extracellular concentrations at 5 and 50 μM, respectively. From 1 h, and irrespective of extracellular concentration, ICIM levels stabilized at a maximum (43 U of additional fluorescence for extracellular IM at 50 μM) and became stable over time. From these data, we chose 1 h of incubation for further experiments.

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Figure 1. Measurement of IM uptake in K562 cells using single cell UV fluorescence detected by flow cytometry. A UV-laser equipped-cytometer was able to detect UV fluorescence emitted from IM in K562 cells gated on FS/SS dot plot (A) and PI negative alive cells (B). A demonstrative example (C) shows a shift of the fluorescence peak from natural UV fluorescence. We used the difference in mean intensity of fluorescence in linear scale between treated and control cells to evaluate the amount of ICIM. We noted that UV fluorescence depended on morphological characteristics, with significantly lower IM accumulation in smaller cells (n = 14) (D). Evaluating IM uptake kinetics (5, 15, 30, 60, 120, and 240 minutes of incubation) and dose response (5 and 50 μM IM) in whole-cell population (ab gating) by applying this method (n = 6) showed rapid dose-related IM uptake then a plateau (E). The correlation between flow cytometry and quantification of IM after lysis of a known number of cells confirmed that in these conditions, UV fluorescence was directly related to the amount of ICIM (n = 57) (F). *P < 0.05, **P < 0.01, ***P < 0.001.

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Correlation Between Additional Fluorescence and ICIM Quantified Using the Standard Method

To relate the additional fluorescence evaluated by flow cytometry to the quantity of IM expressed in picogram per cell, we determined the relationship between additional UV fluorescence and IM content. In this series of experiments (n = 57), K562 cells were incubated with increasing IM concentrations (1, 5, 10, 15, 20, 25, 50, and 100 μM) for 1 h, and the intracellular IM amount was measured in the same sample by flow cytometry at single-cell level and also in parallel with a standard physicochemical technique after lysis of a known number of cells. We obtained a significant correlation between additional fluorescence measured with flow cytometry and intracellular imatinib (ICIM) quantified using the standard method (r2 = 0.73, P < 0.001; Fig. 1F). Indeed, under our test conditions, ICIM levels (pg/cell) were equivalent to 0.0599-fold of the additional fluorescence detected by flow cytometry, and we applied this calculation to all experiments. This was how we obtained a value in the order of picogram per cell.

Furthermore, we noted that (i) flow cytometry was more sensitive than the standard technique, because it detected IM at lower concentrations while samples were negative after cell rupture (n = 7) and (ii) we were able to analyze fewer cells (1.5 × 105 vs. 5 × 106), an interesting advantage for subsequent cell subset analysis.

Correlation Between Extracellular IM, ICIM, and Cell Sensitivity to the Drug

To investigate whether intracellular imatinib (ICIM) levels could prove a potential parameter for predicting IM efficiency, as speculated, we incubated cell lines in media supplemented with 0, 1, 5, 10, 25, or 50 μM IM and determined the correlation between the ICIM levels measured at 1 h and IM efficiency evaluated by the proportion of dead/apoptotic cells at 24 h. We tested two cell lines: K562 and KCL22.

We found a correlation between IM uptake at 1 h and IM concentration in the culture media. We observed significant differences in IM accumulation between the two cell lines, particularly for lower extracellular concentrations (1–5 μM) corresponding to therapeutic doses (11), with ICIM levels being higher in KCL22 cells than in K562 cells (0.7 vs. 0.2 pg/cell, respectively, for 1 μM in culture media, P < 0.01, Fig. 2A). These differences were not related to membrane pump (OCT-1, ABCG2, ABCB1, and ABCC1) expression (Supporting Information Fig. S4). For example, KCL22, which had a higher ICIM level, had lower expression of OCT-1 transcript than K562 cells and no lower expression of efflux membrane pumps. Moreover, in this model, we observed a much higher IM concentration in cells than in culture medium. For example, for 5 μM of IM in extracellular media, the ICIM amount per femtoliter was 86 and 43-fold higher in KCL22 and K562 cells, respectively, when compared with the same volume of culture medium. Finally, the curve inflected for IM extracellular concentration higher than 25 μM, which is consistent with the previously observed regulated IM uptake (Fig. 1E).

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Figure 2. In K562 and KCL22 cells in vitro, amounts of intracellular IM at 1 h are predictive of apoptosis at 24 h. We used the two CML-derived cell lines as an in vitro model to study the relationship between extracellular IM (1, 5, 10, 25, and 50 μM) and ICIM at 1 h, then IM efficiency evaluated at 24 h by quantifying the proportion of dead cells. In the first series of experiments (n = 5), we assessed the relationship between extra- and intracellular IM content and observed a significantly different ICIM uptake between the two cell lines depending on the medium (A), KCL22 cells showing a greater capacity to store IM at low-extracellular dose. Second, the relationship between extracellular IM concentration (μM) or ICIM at 1 h and the percentage of cell death evaluated by Annexin V-PI staining at 24 h showed a comparable correlation with K562 cells (B, C) but a stronger correlation of ICIM with IM sensitivity in KCL22 cells (D, E). Results are expressed as mean ± SEM; n = 5. **P < 0.01, ***P < 0.001.

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We then compared the relationship between cell sensitivity, expressed by the proportion of dead cells after 24 h of treatment and either extracellular or intracellular IM concentration. We observed an equivalent strong correlation between the two IM parameters and K562 cell sensitivity (r2 = 0.93 vs. 0.96; Figs. 2B and 2C). With KCL22 cells, the correlation between cell sensitivity and extracellular IM dose was lower (r2 = 0.66, Fig. 2D), but the correlation with intracellular IM at 1 h was significantly stronger (r2 = 0.92 vs. 0.66, P < 0.05, Fig. 2E), suggesting the better predictive value of ICIM levels with regard to efficiency. These data are consistent with IM uptake at low-extracellular dose in this cell line (Fig. 2A).

Quantification of ICIM in Primary Normal and CML Blood Cells

We then applied our procedure to normal leukocytes and native primary CML blood cells before any treatment with tyrosine kinase inhibitor (TKI). We compared IM storage in the presence of increasing concentrations of IM at 1 h in normal and CML cells (Fig. 3A).

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Figure 3. Intracellular IM uptake in primary healthy donor leukocytes and CML leukocytes at diagnosis. We used flow cytometry to analyze primary healthy donor leukocytes and CML leukocytes. The different type of blood leukocytes was identified according to morphological criteria (FS/SS graph) (A). The cells were incubated in the presence of 0.2, 1, 5, 25, and 50 μM IM, and the ICIM level was measured at 1 h in lymphocytes (L), monocytes (Mo), and polymorphonuclear cells (PMN) from healthy donors (n = 5) and CML patients at diagnosis (n = 22) (B). PMN systematically stored more IM than did L and Mo. For CML leukocytes, ICIM interpatient heterogeneity was far greater in PMN (C). Results are expressed as mean ± SEM.

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Given the preliminary result obtained with K562 cells showing that intracellular imatinib (ICIM) levels were dependant on cell morphology, it was necessary to compare malignant cells with their normal counterparts. Our approach using flow cytometry, unlike the standard physicochemical method, makes this possible. In all categories of blood cells, we observed a roughly similar dose-response curve as with Philadelphia chromosome positive cell lines: IM penetrated lymphocytes (L), monocytes (Mo), and polymorphonuclear cells (PMN) at the lowest IM medium concentration and then increased in line with available extracellular IM. However, as anticipated, we observed different ICIM levels depending on blood cell subsets (L < Mo < PMN) with minor differences between CML and their normal counterparts. Moreover, and whatever the cell morphology, we noted a relatively homogeneous accumulation in normal blood cells.

To evaluate the predictive value of this new parameter with regard to CML therapeutic response, we included the cells of 22 CML patients in a pilot trial. As shown in Figure 3B, IM accumulation in granular CML cells was highly heterogeneous, ranging from 1 to 13 pg/cell for an extracellular concentration of 50 μM. We have started patient follow-up, which should show whether the different profiles obtained in vitro before treatment can predict clinical response after 18 months of IM therapy.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

For targeted therapy such as IM, the amount of drug penetrating the targeted cells is likely to be a major efficiency parameter, because it is essential that the therapeutic molecule be as close as possible to the target molecule. Studies have been conducted on IM metabolism (22, 23), but the subject of intracellular accumulation in the malignant clone has been rarely addressed; indeed, the synthetic molecule is too small to be immunogenic; also, the reference technique for tracing its intracellular penetration is 14C-labeled IM (15), which is difficult to manipulate and cannot be used to follow IM accumulation in patient cells.

In this study, using K562 and KCL22 cell lines, we validated a standardized method for measuring ICIM by flow cytometry without modifying the chemical structure of IM or altering its binding to the BCR-ABL protein. The good correlation between our data and those obtained with the standard physicochemical assay after lysis of a known number of cells allowed us to convert the results of additional fluorescence into amounts of ICIM. Moreover, the kinetics for IM uptake in K562 cells was similar to those already described using 14C-labeled IM (16, 24).

It is generally accepted that OCT-1 mediates influx of IM by an “active” mechanism (13). Furthermore, inhibition of OCT-1 results only in a partial decrease in IM uptake (15), which suggests that penetration is not related exclusively to OCT-1. In our in vitro model, we noted that the ICIM level varied between cell lines with no correlation with membrane pump expression, which suggests another intracellular accumulation mechanism. However, the evaluation of the mean quantity of drug in each cell showed that the intracellular concentration was markedly higher than the extracellular concentration, indicating a predominantly active mechanism (13, 25). Although this process is not fully understood, it probably involves a saturable-type transporter (26).

Because our method was more sensitive than the physicochemical assay, we could observe the differences in IM accumulation between the K562 and KCL22 cell lines for low dose of IM. The correlation between the amount of ICIM and cell death at 24 h may be more relevant than that observed for extracellular IM as demonstrated with KCL22 cells, suggesting the possible predictive value of ICIM toward the sensitivity of malignant cells. The study of IM penetration into primary CML cells showed marked interpatient variability; the significance of these variations could only be evaluated by studying the correlation between the ability of the native clone to store IM and CML response. This study is ongoing. The ICIM level is probably a determining parameter of treatment efficiency and may explain, at least to a certain degree, why some patients with low-plasma levels of IM respond well whilst others with high-plasma levels of IM are poor responders. A recent study (27) did not find any correlation between the amount of IMIC and CML response, but the assay was performed 30 days after the start of treatment. However, we observed, in a few patients (n = 3), that the analysis of malignant cells can only be done in the first week after the start of treatment, because analysis at D30 reveals a majority of normal cells (data not shown).

The main advantage of our method is to identify a target subpopulation accurately, which is not possible even with a nonradioactive assay (28). After adapting the method to primary blood leukocytes, we demonstrated the influence of cell morphological parameters (ICIM L < Mo < PMN) and confirmed that the ability of cells to store IM varied amongst cell types (29). This cell-dependant uptake is poorly understood, but the accumulation of IM in the PMN of CML patients appears to be relatively heterogeneous, suggesting that cell morphology is only one of the parameters influencing IM uptake. For example, the expression levels of plasma membrane pumps (particularly OCT-1), which could be proportional to the membrane surface, appear to be higher in PMN than in MNC (30). Moreover, membrane pump activity may be a determining factor, which could be modulated quite extensively by certain polymorphisms (31, 32), but the intrinsic characteristics of the clone responsible for IM accumulation as a result of genetic, epigenetic, and environmental factors (33) remain unknown. Overall, this observation demonstrates that it is necessary to compare cell subpopulations that are cytologically equivalent, which is possible with our method without cell immunoselection, as, for example, the CD34+ subset.

However, the UV fluorescence of a molecule is due to the presence of conjugated binding in its chemical structure and is thus a physicochemical phenomenon shared by several molecules. Consequently, our method is not specific to IM, and we must make sure that the differences in fluorescence measured in the processed samples and controls are due exclusively to IM, which was the case in our controlled, in vitro experiment. In vivo measurement of ICIM levels during treatment would require knowing about other potentially fluorescent drugs that might interfere with IM emission, and results must be interpreted with great care. We detected no UV fluorescence for Hydrea (hydroxycarbamide) or interferon α and these molecules, potentially used in CML treatment, did not interfere with IM penetration (data not shown). However, in vivo, the main metabolite synthesized is N-desmethyl IM, which represents 10–15% of administrated IM. We checked that our method was able to detect N-desmethyl IM (Supporting Information Fig. S5), but we could not differentiate it from IM. In our in vitro model, the analysis of chromatographic peaks obtained at the time of IM assay confirmed no generation/production of N-desmethyl IM (data not shown), a significant difference with in vivo metabolism. We choose to use IM rather than its metabolite to be able to relate UV fluorescence with pharmacologically determined amounts of IM and to compare our data with published studies.

To conclude, we developed a simple, rapid method for evaluating accurately, and with great sensitivity, the amount of IM accumulated in a living cell. Its limited specificity requires careful laboratory work and knowledge of all the molecules likely to interfere with IM fluorescence. The use of flow cytometry has two main advantages in comparison with standard techniques used in the literature: only a few cells are needed, and cell subsets can be differentiated based on morphological criteria or the use of membrane antigens. We observed differences in accumulation of IM in different categories of blood leukocytes; this opens new horizons for research into CML stem cells. Our method also established a strong correlation between the amount of ICIM and the sensitivity of cells to TKI, suggesting that ICIM could be a relevant biomarker for assessing the sensitivity of the CLM clone. This method makes it possible to envisage, for the first time, a prospective study of CML cells at diagnosis with in vitro tests to predict the ability of the malignant clone to accumulate TKI and help monitor therapeutic response.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

The authors thank Dr Véronique Maguer-Satta for providing the KCL22 cell line and Dominique Chadeyron for manuscript preparation.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22118_sm_SuppTab1.tif5288KTable S1: UV fluorescent calibrated beads tested to standardize ICIM measurement. Several commercialized beads were tested to ensure flow cytometer stability during analysis. Only a few beads are designed to have a specific UV application, and they did not correspond to our experimental conditions. We also tested FITC fluorescent beads and chose the Flow Check Fluorosphere whose fluorescence in linear scale was homogeneous and located in the area of IM fluorescence.
CYTO_22118_sm_SuppFig1.tif4563KFigure S1: UV fluorescence of marketed calibrated beads. Different calibrated beads were analyzed through UV laser excitation. In most cases, the signal obtained was neither homogeneous nor accurate enough (large peak), or not in the space corresponding to fluorescence intensity emitted from one cell containing IM requiring projection on linear scale. Demonstrative examples in (A) ImmunobriteTM, (B) Cyto-CalTM, (C) SHEROTM Rainbow, (D) SpheroTM UV Carboxyl Particle. Only Flow Check FluorospshereTM (E, F) met our criteria to calibrate the cytometer before each experiment; their UV fluorescence intensity was uniform and stable over time and within the space of IM fluorescence. For each tested beads, the CV is indicated next to the peak.
CYTO_22118_sm_SuppFig2.tif4881KFigure S2: UV spectrum of IM (A) and demonstrative example of sample chromatogram obtained with a solution used in the set of standards and containing 0.150omg/L of imatinib and its metabolite N-desmethyl imatinib at a wavelength of 264onm (B).
CYTO_22118_sm_SuppFig3.tif4917KFigure S3: The ICIM level in cell lines is not related to membrane pump expression. We evaluated, in parallel, membrane pump expression and the ability of K562 and KCL22 cells to store IM. Total RNA was isolated from cells using the Nucleospin RNA II kit (Macherey Nagel); cDNA was synthetized using the cDNA High Capacity Reverse Transcription kit, according to the manufacturer's instructions. A TaqMan quantitative real time PCR was then performed on a RotorGene 6000 real time thermal cycler (Corbett Biosciences) using TaqMan Gene Expression Assays (Hs00427552_m1 for OCT-1, Hs01053790_m1 for ABCG2, Hs01067802_m1 for ABCB1, Hs00219905_m1 for ABCC1, Hs00984230_m1 for β2-microglobuline; Applied Biosystems). The amount of target transcript was analyzed using the 2-ΔΔ Ct method and was normalized to the endogenous reference gene (β2-microglobuline) and K562 cells as the calibrator (n=3) The relative expression of the membrane pump in KCL22 cells are 0.32±0.1 for OCT-1, 0.31±0.12 for ABCG2, 1.36±0.4 for ABCC1 and 0.05±0.006 for ABCB1, according to the expression in K562 cells used as calibrator (arbitrary value: 1) (A); hOCT-1 expression was also analyzed at the protein level using western blot. Briefly, K562 and KCL22 cells were lyzed in an extraction protein buffer supplemented with protease inhibitors (Roche Diagnostics), and 30µg of protein were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were incubated with diluted antibody (anti-OCT1, Aviva Systems Biology, 2.5µg/ml, and anti-tubulin, Ozyme) overnight at 4oC with gentle shaking. After incubation with appropriate secondary antibody for 1 hr at room temperature, immunoblots were visualized with an ECL system (ECL prime Western Blot Detection Reagent Kit, VWR). (B): ICIM level in K562 and KCL22 cell lines after 1 hr of incubation with 1 µM IM (n=5) (C).
CYTO_22118_sm_SuppFig4.tif4888KFigure S4: Demonstrative example of flow cytometric analysis of apoptotic K562 cells. K562 control cells (A) and K562 treated by 25µM of IM for 24 hrs (B) were stained with PI and Annexin-V. Viable cells are visualized in B3 and A3 quadrant (PI-/AnnexinV-).
CYTO_22118_sm_SuppFig5.tif4767KFigure S5: The active metabolite N-Desmethyl IM remains detectable by flow cytometry. N-Desmethyl IM, the main active metabolite of IM, could be detected by flow cytometry using UV-laser equipped-cytometer. K562 and KCL22 cell lines were incubated for 1 hr with 0.5/1/5/10/25 or 50 µM. We then applied the protocol used to detect IM to follow N-Desmethyl IM uptake in cells. We observed a similar dose-response curve as with IM; at lower extra-cellular concentrations (0.5 and 1 µM) of N-Desmethyl IM, the uptake was significantly higher in KCL 22 cells (**p<0.01). Results are expressed as mean ± SEM, n=5.
MIFlowCyt-Item-Location.doc46KSupporting Information: MIFlowCyt

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