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Molecular Cancer Therapeutics 6, 644-654, February 1, 2007. doi: 10.1158/1535-7163.MCT-06-0358
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells

Laszlo Markasz1,3, Gyorgy Stuber1, Bruno Vanherberghen1, Emilie Flaberg1, Eva Olah3, Ennio Carbone4, Staffan Eksborg2, Eva Klein1, Henriette Skribek1 and Laszlo Szekely1

1 Department of Microbiology, Tumor and Cell Biology (MTC) and Center for Integrative Recognition in the Immune System, Karolinska Institute; 2 Karolinska Pharmacy and Department of Woman and Child Health, Childhood Cancer Research Unit, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; 3 Department of Pediatrics, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; and 4 Department of Experimental and Clinical Medicine "G Salvatore," University "Magna Graecia," Catanzaro, Italy

Requests for reprints: Laszlo Szekely, Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Box 280, SE-171 77 Stockholm, Sweden. Phone: 46-8-524-86760; Fax: 46-8-330-498. E-mail: lassze{at}ki.se

Abstract

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell–based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells. We found that treatment of NK cells with the drugs vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited NK cell–mediated killing without affecting the viability of NK cells. On the other hand, the following drugs permitted efficient NK cell–mediated killing even at concentrations comparable with or higher than the maximally achieved therapeutic concentration in vivo in humans: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine. [Mol Cancer Ther 2007;6(2):644–54]

Introduction

The goal of immune-based adjuvant therapeutic strategies is to stimulate immune effector cells to kill malignant cells, which may result in a better clinical outcome. Infiltration of solid tumors with inflammatory cells is a well-known phenomenon (1–3). Several studies showed that the infiltration of natural killer (NK) cells in malignant tumors was associated with a better clinical outcome (4, 5).

NK cells are large granular lymphocytes that are part of the innate immune system (6). Mature NK cells are mostly circulating in the peripheral blood, where they represent ~10% to 15% of all blood lymphocytes. Phenotypically, NK cells express surface antigen CD56, which has unknown function on NK cells, and lack the typical T-cell antigen CD3 (7). Physiologically, NK cells have direct cytotoxic activity against tumor targets and virus-infected cells (8). NK cells can regulate adaptive immune response by IFN-{gamma} production and can mediate antibody-dependent cellular toxicity through membrane Fc{gamma}RIII (CD16; ref. 6). These cells have the ability to directly kill target cells and produce immunoregulatory cytokines in transfused cancer patients (9–12).

The outcome of NK cell-target cell interactions is regulated by a fine integrative balance between inhibitory and activating receptors; the first elicited by the recognition of MHC class I molecules by NK inhibitory receptors (killer immunoglobulin–like receptors, CD94/NKG2A, and immunoglobulin-like transcript) and the latter by NK cell–activating (NKG2D and natural cytotoxicity receptor) receptors that can sense stress inducible glycoprotein (MHC class I chain–related proteins and UL16 binding proteins) expression on tumor- and virus-infected target cells (13). Because NK cells are able to spontaneously lyse tumor cells, considerable effort has been focused on investigating the possible use of NK cells in the treatment of human cancer. Recent understanding of NK cell development and function has permitted the investigation of novel NK cell–based immunotherapeutic modalities. A promising approach is to expand allogeneic NK cell clones in vitro, with typed killer immunoglobulin–like receptor expression, and to transfuse these cells into leukemic patients (14–16).

Basse et al. (17) showed that the level of accumulation of NK cells in murine tumor metastases in vivo is dependent on the dose and frequency of interleukin 2 (IL-2) administration. NK cells have also been used in human therapeutic regimens that involved the administration of high or low doses of IL-2, alone or in combination with IL-2–activated blood lymphocytes or purified populations of NK cells, for primary lung and renal cell carcinomas (18, 19). In other studies, extended low-dose IL-2 was used to activate NK cells in patients with solid tumors who received intensive chemotherapy or in patients with acute myeloid leukemia (20, 21). Harker et al. (22) suggested that IL-2–induced lymphokine-activated killer cells may be useful in the treatment of chemotherapy-resistant cancers.

The chemotherapeutic regimens for malignancies restrict the effectivity of the immune system and significantly decrease the level of NK cells (23). The activity of NK cells is also affected by some cytostatic drugs (24). Utsugi et al. (25) observed enhanced NK cell–mediated cytotoxicity following treatment of the target cells with topoisomerase inhibitors. Killing by NK cells requires microtubule integrity, which may be influenced by antimicrotubule agents (26). It is important to consider the drug-induced effects when chemotherapy and NK cell–mediated immunotherapy are planned to be used in parallel.

The aim of this study was to systematically investigate the effect of the most frequently used cytostatic drugs on NK cell–mediated cytotoxicity. We carried out more than 4,000 cell survival assays on single-cell levels using automated laser confocal microscopy to establish the pattern of in vitro effect of 29 different cytotoxic drugs on the efficiency of NK cell–mediated killing.

Materials and Methods

Isolation and Culturing of Human Polyclonal Peripheral Blood NK Cell Cultures
Polyclonal NK cells were acquired from lymphocyte-enriched buffy coat derived from healthy donor blood (Blodbank, Karolinska Sjukhuset, Stockholm). Peripheral blood mononuclear cells were isolated by centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). NK cells were isolated from peripheral blood mononuclear cells by negative magnetic bead selection (Stemcell Technologies, Vancouver, Canada) according to the manufacturer's instructions. Purified NK cells were >95% CD3–CD56+. NK cells were cultured either overnight or for several weeks in IMDM supplemented with 10% human serum (AB, Blodbank), 2 mmol/L L-glutamine, 1x nonessential amino acids, 1 mmol/L sodium pyruvate, 50 units/mL penicillin-streptomycin, 50 µmol/L 2-mercaptoethanol (media and supplements bought from Life Technologies, Inc., Carlsbad, CA), and 100 units/mL IL-2 (Peprotech, Inc., London, United Kingdom).

Eight primary NK cell isolates were tested in the present study. Five NK cell isolates were restimulated every 3 to 4 days with fresh IL-2–containing media for 1 week and were periodically monitored for the CD3–CD56+ phenotype. Three NK cell isolates were stimulated with IL-2 only for 24 h.

Cell Lines and Culture Conditions
Cytotoxic NK tumor cell lines Nishi (27), NKL (28), NK92 (29), and KHYG-1 (30) were cultured in IMDM supplemented with different concentrations of IL-2 (Nishi and NK92, 100 units/mL; NKL, 200 units/mL; KHYG-1, 450 units/mL). K562 cell line was used as target in the in vitro killing assay. These cells were cultured in IMDM (Sigma, Dorset, United Kingdom) supplemented with 10% FCS (Sigma), 100 mmol/L L-glutamine (Sigma), and 80 µg/mL gentamicin (Sigma). Cell suspensions were grown in a humidified incubator at 37°C in an atmosphere containing 5% CO2. Cell counts were adjusted to 1 x 106/mL and the cells were fed twice a week. The absence of Mycoplasma contamination was ensured by regular monitoring with Hoechst 33258 staining.

In vitro Killing Assay (Standard Chromium-51 Release Assay)
Target cells were labeled for 1 h at 37°C, 7.5% CO2 with 100 µCi of Na251CrO4 (Amersham Biosciences, Uppsala, Sweden) added directly to the cell pellet. Radioactively labeled target cells were washed thrice with RPMI, and 15 x 103 cells were plated per well in flat-bottomed 96-well microtiter plates to replicate cell densities in the flat-bottomed microtiter plates. NK cells were subsequently added at 5:1 effector-to-target ratio per well (total volume of 200 µL/well) and incubated for 5 h at 37°C, 5% CO2. Spontaneous release was determined by incubation of labeled target cells with media alone whereas maximal release was determined by lysis of target cells in 10% SDS. Spontaneous release was <15% of the maximal release. One hundred microliters of supernatant were collected for scintillation counting on a {gamma} irradiation counter. Each measurement was done in triplicate and the average of three wells was used for calculation of specific lysis. Specific lysis (killing effectiveness) was calculated as follows: killing effectiveness (%) = [(cpm experimental well – cpm spontaneous release) / (cpm maximal release – cpm spontaneous release)] x 100.

In vitro Killing Assay (Novel Fluorescent Labeled Killing Assay)
The effects of drugs on in vitro killing efficacy of different NK cell lines were assessed using microtiter plates. Twenty-nine drugs were tested, each at four different concentrations in triplicates on flat-bottomed 384-well plates. Each well was loaded with 20-µL cell suspension of NK cells containing 15,000 cells and was incubated for 20 h at 37°C in 5% of CO2. Target (K562) cells were stained with CellTracker green CMFDA (Invitrogen, Sweden). A time-lapse image sequence of NK cell killing of target cells is illustrated in Fig. 1 . As a next step, 20 µL of target cells (K562) containing 3,000 cells were seeded on the same plate and were incubated with the NK cells for 5 h (effector-to-target ratio, 5:1). Dead cells were differentially stained red with ethidium bromide. The precise numbers of green target cells and red dead cells were determined by fluorescent detection using a custom-designed automated laser confocal fluorescent microscope (a modified Perkin-Elmer UltraView LCI) at the Karolinska Institute visualization core facility. The images were captured using the computer program QuantCapture 4.0, and the living and dead target cells were identified and individually counted using the computer program CytotoxCount 3.0. The target cells were identified as dead if they were stained red and green at the same time. Both programs were developed at Karolinska Institute visualization core facility using OpenLab Automator programming environment (Improvision). On each plate, eight wells were set up as controls to determine the baseline killing activity. These wells contained NK and K562 cells in culture medium only (without drugs). As additional control, effector NK cells alone were incubated under the same conditions. To monitor the possible short-term effect on the target cells, K562 cells were incubated alone and with all the individual drugs for 5 h. Killing effectiveness of NK cells was calculated for every well using the following formula: [number of dead target cells (both red and green signal in the same cell) / number of all (green) target cells] x 100.


Figure 1
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  		Figure 1. Time-lapse image sequence of NK cells killing a K562 target cell. Target cells were labeled with CellTracker green (CMFDA; Invitrogen). NK cells were labeled with CellTracker blue (CMAC; Invitrogen). Dead cells were labeled with ethidium bromide (red). Images were taken with an automated laser confocal microscope (a modified Perkin-Elmer UltraView LCI). Arrows, green target cells as they get killed by the surrounding NK cells and turn red. Time scale is given in seconds.

 
To normalize the killing effect values of different NK populations, the following formula was used: (killing effectiveness in the wells with drugs / mean killing effectiveness values of the control wells) x 100%. Mean killing efficacy was determined from the average killing of all NK populations. The red-stained cells that were not green at the same time were considered as dead NK cells. The cytotoxic effect (%) of the drugs on NK cells was calculated with the following formula: (number of dead NK cells in the wells with drugs / number of dead NK cells in the control wells) x 100%.

Drugs
For the in vitro killing test, 29 drugs were used (summarized in Table 1 ). Twenty-eight of these drugs are regularly used in the treatment of human patients. All the drugs were dissolved in 50% DMSO and printed on 384-well flat-bottomed plates using a high-density metal pin array (50-nL replica volumes) in Biomek 2000 fluid dispenser robot (Beckman, Fullerton, CA). The same robot was used to generate the drug masterplates containing the triplicates of four different drug dilutions using a single-tip automatic dispenser head.


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  		Table 1. AUC values of the used drugs and calculated RAUC values for NK cells

 
The highest drug concentration was selected as the maximum drug concentration physiochemically achievable at 600x dilution (50 nL of drug in a 30-µL assay volume). These concentrations are in a comparable range with the in vivo plasma concentrations in patients (31–52).5 The starting concentrations of the dilution series (1x, 5x, 25x, 125x) for the individual drugs were initially determined based on the solubility of the different agents. The fifth to sixth rows of Table 1 represent the drug concentrations that were presented in the wells with NK cells alone and after increasing the volumes with 20-µL cell suspension of target cells.

Testing the drug plates on a large number of in vitro tumor cell lines and primary tumors, it was possible to find sensitive cell lines for each individual drugs, showing that all the drugs on the plate were active (data not shown).

A way to calculate a relationship between the in vitro drug concentrations and the in vivo ones is to use the area under the curve (AUC) values of the individual drugs. For this comparison, ratio of area under the curve (RAUC) values were determined by the following formula: in vitro used concentration (µg/mL) x 20 h/ AUC (µg h/mL) in vivo. A RAUC value >1 indicates that the in vitro used dose is higher than the one used in clinical practice. If this value is 1, it means that our in vitro dose corresponds to the clinical dose. In vivo AUC levels, clinical doses, and calculated RAUC values of the individual drugs from the literature are summarized in Table 1. RAUC values are also shown in Fig. 2 .


Figure 2
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  		Figure 2. RAUC values for NK cells (data are also represented in Table 1). To calculate relationship between the in vitro used drug concentrations and the in vivo used values, RAUC values were determined by the following formula: in vitro used concentration (µg/mL) x 20 h / AUC (µg h/mL) in vivo. RAUC > 1, the in vitro dose is higher than the clinical dose. RAUC < 1, the in vitro dose is lower than the clinical dose. RAUC = 1, the in vitro dose is corresponding to the clinical dose.

 
Results

Comparison of the Novel In vitro Killing Assay to the Standard Chromium-51 Release Assay
The two methods were compared with each other in the absence of drugs under the same conditions (incubation time, effector-to-target ratio, flat-bottomed plates instead of V-bottomed plates). The results of our single-cell fluorescence method correlate closely to the standard chromium-51 release assay on different NK cell lines. Figure 3 shows the in vitro killing capacity of a NK tumor cell line (NKL) and two NK cell clones tested with both methods.


Figure 3
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  		Figure 3. Comparing of the fluorescent in vitro killing assay to the standard chromium-51 release assay. NKL cell line and two IL-2–activated primary NK cell polyclonal preparations were incubated together with K562 target cells for 5 h. Effector-to-target ratio was always 5:1. KE, killing effectiveness.

 
Effect of Drugs on the Killing Capacity of Different NK Cells
The eight different primary NK cell polyclonal preparations from healthy donors and four NK tumor cell lines that were tested with the novel fluorescent method in the present study represent a variety of cells with different origin and in vitro history. The investigation included separated NK cells from healthy donors with 1 week of in vitro culturing together with IL-2 along with freshly separated NK cells cultured with IL-2 only for 24 h. NK tumor cell lines showed relatively low killing efficiency (≤10%) in our novel experimental model system. Microscopy evaluation of killing activity requires the use of flat-bottomed plate instead of conventional V-bottomed plates. Many of the NK tumor lines showed low spontaneous mobility and required high-level cellular crowding to be able to kill effectively. Control killing effectiveness values for all investigated NK cell lines can be seen in Table 2 .


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  		Table 2. Killing effectiveness of different NK cell lines

 
The NK cells of different origins showed a very similar killing efficacy pattern in the presence of most of the drugs. This phenomenon is illustrated in Fig. 4 , summarizing the killing effectiveness of all NK cell populations in the presence of 29 drugs at four different concentrations. Each individual thin curve represents the average of three independent measurements. The thick broken line is the mean of all curves, the mean killing efficacy. The curves labeled by black spots are NK cell isolates, treated with IL-2 only for 1 day. The gray shaded area marks the SD. Figure 5 illustrates the cytotoxic effect (%) of all of the drugs on NK cells, as shown in a similar way in Fig. 4. All the mean killing efficacy values are represented in Table 3 . Importantly, none of the drugs influenced the viability of the K562 target cells during the 5-h incubation (data not shown). The number of dead NK cells in the drug-treated cultures that received IL-2 only for 1 day was higher than those in the drug-treated cultures that received IL-2 for 1 week. None of the NK populations showed dose-dependent increase in the number of dead cells. Consequently, the dose-dependent decrease in the killing effect of NK cells in case of certain drugs cannot be simply explained by drug-induced NK cell death (Fig. 5).


Figure 4
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  		Figure 4. Killing effectiveness of NK cells tested with the fluorescent killing assay. The figure summarizes the killing effectiveness of all NK cell populations in the presence of 29 drugs at four different concentrations. Thin curves, average of three independent measurements. Thick broken line, mean of all curves, the mean killing efficacy (MKE). Curves with black spots, NK cell isolates that were treated with IL-2 only for 1 d. Gray shaded area, SD. A, effective drugs (lowest mean killing efficacy, <60%). B, marginally effective drugs (lowest mean killing efficacy, 60–70%). C, noneffective drugs (lowest mean killing efficacy, >70%). X axis, increasing concentrations of the drug. Y axis, killing effectiveness of NK cells 0% to 120%.

 

Figure 5
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  		Figure 5. Cytotoxic effect on NK cells tested with the fluorescent killing assay. Thin curves, average of three independent measurements. Curves with black spots, NK cell isolates treated with IL-2 only for 1 d. The cytotoxic effect (%) of the drugs on NK cells was calculated using the following formula: (number of dead NK cells in the wells with drugs / number of dead NK cells in the control wells) x 100%. [Cytotoxic effect (%) of >100% means more dead NK cells.] A, effective drugs. B, marginally effective drugs. C, noneffective drugs. X axis, increasing concentrations of the drug. Y axis, cytotoxic effect of the drug compared with the control 0% to 500% (control means NK cell survival without the drug, which is 100%).

 

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  		Table 3. Mean killing effectiveness of NK cells in the presence of different drugs

 
Identification of Highly Effective and Noneffective Drugs
We found that the most effective drugs that inhibited NK cell–mediated killing were chlorambucil, MG-132, docetaxel, cladribine, paclitaxel, bortezomib, gemcitabine, and vinblastine (Fig. 4). For MG-132, no RAUC value could be defined because this drug has not been clinically used. Oxaliplatin, dactinomycin, cytarabine, daunorubicin, vincristine, and topotecan were only marginally effective.

Most NK cell lines were not affected by bevacizumab, bleomycin, doxorubicin, epirubicin, vinorelbine, carboplatin, methotrexate, ifosphamide, etoposide, hydroxyurea, asparaginase, 5-fluorouracil, 6-mercaptopurine, streptozocin, and cyclophosphamide.

Discussion

In our experimental model, the killing effectiveness of NK cells was tested in the presence of different cytostatic drugs. Previous studies investigated NK cell–mediated cytotoxicity following treatment of target cells with certain cytotoxic drugs (25) or treatment of NK cells with a few chemotherapeutic agents before the NK cell killing assays (24, 26). No systematic study has been carried out to investigate the effect of a large number of different drugs at different concentrations.

In our experimental setup, the NK cells were preincubated alone for 20 h with the drugs to test the drug effect only on the NK cells. This was followed by coincubation with the target cells for 5 h. No drug-induced cytotoxicity was detectable on target cells during this incubation, although other effects on target cells, which influence NK cell killing, cannot be excluded.

The presented data suggest that IL-2–activated NK cell lines share a common profile of cytotoxic drug sensitivity. This profile does not change with different lengths of in vitro culturing with IL-2.

Topotecan, 6-mercaptopurine, and bleomycin did not inhibit NK cell killing effectively even at very high AUC levels. Almost no effect could be seen in case of carboplatin, methotrexate, and dactinomycin, although this might be explained by the relatively low range of RAUC levels achieved in the microcultures.

Although chlorambucil efficiently inhibited the killing effect of NK cells, other alkylating agents like cyclophosphamide and ifosphamide did not affect NK cell killing. This might be explained by the fact that both of the latter compounds are prodrugs that have to be converted into active metabolites by the liver in vivo.

The effectiveness of chlorambucil may also be explained by the high in vitro RAUC levels (Fig. 2; Table 1). The in vitro doses (in vitro AUC levels) of the other effective drugs were in close vicinity to the clinical doses.

Unlike Reiter et al. (53) who found no inhibition of NK cell killing ability when effector cells (NK cells) alone were treated with cladribine, this agent was the only drug among the base analogues that significantly decreased the killing effect of IL-2–activated NK cells in our experimental setup.

Our data also showed that NK cells are particularly sensitive to antimicrotubule drugs and proteosome inhibitors. The dependence of NK cell–mediated killing on microtubule integrity is well known (24, 26). Here we show that different microtubule inhibitors have a variable effect on NK cell–mediated killing. Vinorelbine and vincristine had much less effect on NK cell–mediated killing than vinblastine, docetaxel, or paclitaxel.

Several studies showed that the chymotryptic activity of proteasomes in NK cells might play a role in their cell-mediated cytotoxicity (54–56). Kitson et al. (54) reported that synthetic proteasome inhibitors like CEP-1508, CEP-1612, and CEP-3117 can inhibit the rat NK proteasome in a dose-dependent manner and found a 50% inhibition of IL-2–activated NK cell–mediated cytotoxicity. Lu et al. (57) pointed out that MG-132 could reduce NK cell–mediated cytotoxicity of rat NK cells, but this was due to the apoptosis-inducing property of the drug. Here, we show that proteosome inhibitors severely compromise the killing efficiency of human NK cells even in the absence of apoptosis induction.

Bortezomib is a clinically used reversible inhibitor of the chymotrypsin-like activity of the 26S proteasome. A study showed that bortezomib can sensitize tumor cells to the lytic effects of dendritic cell–activated immune effector cells like NK cells or CD8+ lymphocytes (58), but no data have been published on the direct inhibition effect of bortezomib on NK cell–mediated cytotoxicity.

Our results suggest that chemotherapy protocols that include proteosome inhibitors or antimicrotubule drugs may interfere with NK cell–based immunotherapy, if applied simultaneously.

Based on the almost complete absence of inhibitory effect on NK cell–mediated killing even at high RAUC levels (concentrations that are directly comparable with maximally achieved in vivo therapeutic doses), we suggest that the following drugs may be effectively combined with adjuvant NK cell–based immunotherapy: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine.

Several anticancer drugs have been reported to induce the expression of ligands for activating NK cell receptors on the tumor cell membrane potentially increasing NK cell–mediated cytotoxicity against tumor cells. This information, in combination with the data presented here, could potentially be used in the future to design new NK cell–based adjuvant immunotherapy protocols.

Footnotes

Grant support: The Swedish Cancer Society, the Swedish Research Council, and the Integrative Recognition in the Immune System Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: L. Markasz and G. Stuber contributed equally to this work.

5 GlaxoSmithKline Research Triangle Park N. Prescribing Information Leukeran. Available from: http://us.gsk.com/products/assets/us_leukeran.pdf. Back

Received 6/21/06; revised 11/14/06; accepted 12/22/06.

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S. Armeanu, M. Krusch, K. M. Baltz, T. S. Weiss, I. Smirnow, A. Steinle, U. M. Lauer, M. Bitzer, and H. R. Salih
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