Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs
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Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients’ own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine.
KeywordsBreast cancer Apoptosis Viability Cytostatic drugs Lab-on-a-Chip
Breast cancer is the leading cause of cancer among non-smoking women in the world, although variation in incidence, prevalence and mortality exists between countries (Bray et al. 2004).
Treatment modalities used in breast cancer include surgical removal of the tumor, followed by radiotherapy, chemotherapy and/or hormone therapy. The various protocols that exist for chemotherapy and hormone therapy have different and limited rates of success (EBCTG 2005). Currently, selection of chemotherapy and/or hormone therapy is based on a broad scale of factors, including a patients’ age and tumor characteristics, such as nodal status, the presence of estrogen receptors and the Her-2/neu status (Carney et al. 2007). One potential approach to improve the therapeutic efficacy is to use ex vivo approaches to evaluate the sensitivity to specific hormone therapies and chemotherapeutic agents using patients’ tumor cells. This requires the harvesting of tumor cells via biopsy, conducting chemo- and hormone sensitivity tests and extrapolating the results to the clinical setting.
Microfluidic devices are very helpful in the design of such chemo- and hormone sensitivity tests for multiple reasons. Microfluidic devices offer the possibility of using lower quantities of cells, parallel processing (high-throughput analysis) and detailed cellular analysis (Andersson and van den Berg 2003, 2004). Because of the lower quantities of cells (typically <100), cells can even be obtained via fine-needle aspiration. Furthermore, the obtained cells can be subdivided into numerous samples to test various concentrations and combinations of different chemotherapeutics and hormones (high-throughput drug screening). Furthermore, individually tested samples in parallel promotes statistical analysis.
The balance between programmed cell death (apoptosis) and cell proliferation determines the tumor growth rate, and any alteration between these two factors may be the key element for the uncontrolled expansion of malignant tumors. Chemotherapeutics and/or hormones are prescribed to tackle this homeostatic imbalance. Conventional apoptosis and proliferation assays were performed to evaluate the hormone sensitivity of different breast cancer cell lines in vitro (Franke and Vermes 2002, 2003; Franke et al. 2003; Werner et al. 2005). Attempts of testing chemosensitivity of tumor cells ex vivo were already made in the eighties, but have not developed into a standard chemosensitivity test so far (Selby and Raghaven 1981). Recently, research groups have established a link between clinical responses to chemotherapeutics and ex vivo chemosensitivity analysis (Nakada et al. 2005; Yamashita et al. 2005; Trojan et al. 2005). Moreover DNA microarrays (Gianni et al. 2005) and multiparametric microsensor chips (Otto et al. 2003) have been developed for chemosensitivity testing. The assays described above provide valuable leads for novel therapeutic modalities and improve cancer treatment, however there are limitations. For example, the in vitro hormone sensitivity assays need cell culture to obtain millions of cells for analysis and the techniques are very time-consuming and extremely laborious. Cell handling is easier with DNA microarrays, however, the link between genetics and the ultimate goal, cell death in response to chemotherapeutics, yields uncertainty and adds to statistical difficulties.
The focus of this article is to develop a microfluidic chip which enables the analysis of the chemosensitivity of first breast cancer cell lines, and in a later stage a patients’ own breast cancer cells in the presence of various chemotherapeutics. As the goal of chemotherapy is to induce cell death, the degree of apoptosis was assessed using fluorescent dyes. It is not until recently that research groups have become interested in developing chips convenient for detecting apoptosis (Wolbers et al. 2004a, b; Qin et al. 2005). So far, microfluidic chips have been developed to characterize the various hallmarks in the apoptotic cascade in real-time at a single-cell level using fluorescent dyes (Valero et al. 2005; Munoz-Pinedo et al. 2005; Chan et al. 2003), measuring the release of biological substances (Tamaki et al. 2002; Kurita et al. 2003), and the fragmentation of the DNA (Kleparnik and Horky 2003). Apoptosis assays place greater demands on cell handling, especially when cell culture of adherent cells is required. Therefore it has to be ensured that the microfluidic device and experimental set-up do not negatively influence the cellular behavior prior to adding chemotherapeutics (Wolbers et al. 2006). Besides the fact that the microfluidic device and experimental set-up must be compatible with cell survival, a flow system is needed to deliver medium and chemotherapeutics. The applied flow rate and the accompanied shear stress have to ensure cell survival. For the chip material, the silicone elastomer poly(dimethylsiloxane) (PDMS) together with glass, for sealing, is chosen. PDMS is biocompatible, gas permeable, and a cheap material in which structures on a micrometer scale can be easily made (McDonald et al. 2000; Sia and Whitesides 2003), and Pyrex glass is a good supporting material for cell adhesion and promotes optical analysis. Overall, the microfluidic device and flow system consist of disposables, which is a great advantage when performing biological experiments.
This paper describes the cellular studies performed on the estrogen receptor positive human invasive lobular carcinoma cell line MCF-7 in two different microfluidic PDMS chips. Firstly, a low volume microfluidic device with cell trap was used to evaluate cell culture. Secondly, a chip with a higher volume without cell trap was used to apply a controlled flow of medium and a chemotherapeutic to study the effect of this drug on the apoptotic pathway. In the experiments presented here, apoptosis is induced by the protein kinase C (PKC) inhibitor staurosporine (SSP; Koivunen et al. 2006) to optimize the microfluidic device towards a potential clinically relevant apoptosis assay. Staurosporine is a frequently used agent for the induction of apoptosis, and known to induce apoptosis in MCF-7 cells (Mooney et al. 2002; Xue et al. 2003; Sayeed et al. 2007). In a later stage, specific chemotherapeutic agents for breast cancer, such as cyclophosphamide or doxorubicin (anthracyclin) will be used. Cell viability was assessed using two fluorescent dyes, i.e., the life-stain Calcein-AM (CAAM; Bratosin et al. 2005) and the death dye propidium iodide (PI; Zamai et al. 2001). This work provides us with valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells and improve cancer treatment.
2 Material and methods
2.1 Chip design and fabrication
2.1.1 Chip 1
After curing, holes were punctured in the PDMS, creating an in- and outlet. The PDMS chip was sealed onto a slide of Pyrex glass (1 × 2 cm) using oxygen plasma. Therefore, the PDMS and Pyrex glass were cleaned with acetone and ethanol. The surfaces were placed in the plasma oxygenator (Harrick PDC001) for 4 min at an RF of 29.6 W and a pressure of 400 mTorr (high). After sealing, phosphate buffer saline (PBS) was introduced into the chip to preserve the hydrophilicity of the PDMS and facilitate cell loading.
2.1.2 Chip 2
2.2 Cell culture
The estrogen receptor positive human invasive lobular carcinoma cell line MCF-7 was purchased from DSMZ (Braunschweig, Germany). Cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine and 0.4 μg/ml fungizone (RPMI+ medium). Media and supplements were all obtained from Invitrogen (Grand Island, NY, USA). Cell cultures were sustained in a 5% CO2 humidified atmosphere at 37°C. The medium was refreshed every 3–4 days and cultures were split weekly at a ratio of 1:3–1:6 after treatment with versene in distilled water. Versene consists of distilled water with 137 mM NaCl, 1.47 mM KH2PO4, 2.68 mM KCl, 7.37 mM Na2HPO4·2H2O and 0.54 mM NA2EDTA dissolved. In experiments, MCF-7 cells which had satiated the conventional culture flasks (>80% confluence) were used for cell loading.
2.3 Experimental set-up
2.3.1 Chip 1
Cell loading was accomplished by pipetting cells in the inlet. Hydrostatic forces moved the cells into the channels and cells were trapped at the trapping site. The chip was put in a Petri dish and covered with RPMI+ medium to prevent further flow. The chip was placed back in the incubator to analyze long-term cell culture. For this, cells were cultured for 1, 2, 3, 5 and 7 days, without changing the medium. For every time point a separate chip was used. Viability was assessed by staining the cells in the chip with 2 μg/ml Calcein-AM (CAAM; Molecular Probes Invitrogen) and 10 μg/ml Propidium Iodide (PI; Sigma, St. Louis, MO, USA). Cell viability was obtained optically, checking cell morphology with light microscopy and fluorescence with a mercury lamp.
2.3.2 Chip 2
Cells were introduced by pipetting 20 μl of 4 × 106 cells/ml in the inlet. Cells dispersed evenly throughout the chip by hydrostatic forces. The chip was put in a Petri dish, covered with RPMI+ medium and placed in the incubator for 2 days. The medium in the chip and in the Petri dish were refreshed after 1 day. After 2 days, flow was applied. In the main inlet, a pipette tip (Finntip 200Ext 5–200 μl; Thermo Electron Corporation, Vantaa, Finland) was punctured, in which a PA-6 (inner Ø 0.25 mm and outer Ø 0.75 mm; Liquid scan) tube was put to connect it to a BD plastic syringe (Becton Dickinson, Franklin Lanes, NJ, USA) via a BD Microlance needle (30 G × 1/2 in.; 0.3 × 13 mm, yellow). Flow was applied by a KdScientific 101 syringe pump at a flow rate of 0.5 μl/min. The outlet and secondary outlets were left open. Flow was applied for 30 min. Apoptosis was induced by 50 μM staurosporine (SSP; Sigma). RPMI+ medium served as a control. After 30 min of flow, the chip was disconnected from the flow system and put in a Petri dish fully covered with RPMI+ medium and placed in the incubator for 24 h.
After 24 h, the chip was connected to the flow system to flow a solution of 2 μg/ml CAAM and 10 μg/ml PI through the chip at a flow rate of 0.5 μl/min for 30 min. Cell viability was obtained optically, checking cell morphology with light microscopy and fluorescence with a mercury lamp.
2.4 Microscope system
The microfluidic chip was mounted on to an X–Y–Z translation stage of an inverted wide fluorescence microscope (Leica DM IRM, Leica Microsystems, Wetzlar, GmbH, Germany). The microscope is equipped with a mercury lamp as an excitation source for fluorescence measurements, 5×, 10×, 20×, 50×, 63× objectives, and different fluorescent cubes, of which the BGR filter cube was used in these experiments (excitation BP 420/30 and emission BP 465/20 [blue]; excitation BP 495/15 and emission BP 530/30 [green]; excitation BP 570/20 and emission BP 640/40 [red]). In addition, a computer-controlled CCD camera (Leica DFC300 FX) is mounted on to the microscope for image recording, using the accompanied Leica Application Suite Software (version 2.3.4 R2).
3.1 Chip 1
For the culture of adherent cells in a microfluidic device, cells must be able to adhere to the bottom of the chip. PDMS is non-toxic and MCF-7 cells adhered optically. However when the PDMS was rinsed the cells flushed away. Coating with gelatin 1% or BSA 3% did not improve this. Possibly other coating agents would improve adherence. Nonetheless Pyrex glass was used for the cells to adhere (bottom of the chip), since MCF-7 cells adhere to Pyrex glass without the need of coating. Structures (upper part of the chip) were made in PDMS because of its fabrication properties and oxygen permeability.
Chip 1 consists of a cell trap, which promotes the entrapment of MCF-7 cells in the middle of the channel (Fig. 1). In this way, the amount of cells which could adhere to the Pyrex glass, increased. Thus, the immediate flow of cells from the inlet directly to the outlet was prevented. By loading cells into the vertical inlet, it was expected, due to the hydrophilicity of the chip, that medium including cells would easily flow through the channel because of hydrostatic forces. However, only part of the loaded cells flew into the channel, others sank to the bottom of the inlet. This could be caused by the relatively small size of the channel compared to the size of the inlet, the used cell concentration or the stickiness of the cells. The used cell concentration is convenient for the chip dimensions of chip 1. MCF-7 cells are adherent epithelial-like cells which grow in colonies, forming aggregates, as it is the nature of these tumor cells. However, MCF-7 cells need to be in suspension to transfer these cells into the chip. This might account for the stickiness, and together with the narrow channel, complicate the entrance into the channel. In this way, a natural selection could occur due to differences in stickiness. If stickiness is related to cell viability, this could confound the viability analysis. In our case, MCF-7 cells, cultured in a conventional culture flask, were detached with versene, and immediately loaded in the chip, to minimize the effect on the cell viability. Resuspending did not yield extra cells in the channel and possibly damages the cells mechanically.
To conduct apoptosis experiments, a controlled flow was required to (continuously) introduce the apoptotic inducer in the microfluidic chip. Therefore, pipette tips were chosen because of the simplicity and the disposability. Unfortunately, it was not possible to use pipette tips to apply a controlled flow in chip 1, because puncturing a pipette tip in the inlet caused a sudden, strong flow (which detached the cells) as a result of the large volume of the pipette tip in comparison with the channel volume. To reduce this sudden strong flow, a new chip was developed (chip 2), with dimensions which enabled the connection to the flow system with use of pipette tips.
3.2 Chip 2
3.4 Goals and obstacles
Matching cancer patients to the appropriate chemotherapeutics would increase the effectiveness of these pharmaceuticals. Microfluidic devices have the potential to aid in this process. For microfluidic devices to fulfill their potential multiple obstacles must be taken. For example, cells obtained via biopsy have to survive in the microfluidic device and the obtained results have to be extrapolated to the clinical situation. This paper described the first steps towards a microfluidic device which enables chemosensitivity testing of breast cancer cells to improve treatment. The requirements for such assays were analyzed in two different PDMS chips. The first design was used to explore the cell culture properties of MCF-7 cells in the microfluidic device and the second design to test the chemosensitivity in response to the apoptotic agent staurosporine. However, it should be stated that the microfluidic device and experimental set-up as described in this paper is not specific for breast cancer, but applicable for other (cancer) cell lines and any other drug of interest to fine-tune multiple therapies and the treatment of diseases, in which specifically the process of apoptosis is suppressed or enhanced.
3.5 Chip design and cell culture
In apoptosis assays it is essential that the chip design and materials used create a microfluidic environment that meets cellular needs and sustains cellular viability (Wolbers et al. 2006). MCF-7 cells have been cultured in microfluidic devices, either in a 3D-cone structure (Torisawa et al. 2005) or a channel device to which continuous flow was applied (Neville et al. 2007). In our microfluidic PDMS-glass device type 1, cell viability was maintained for up to 7 days in a static cell culture fashion in the incubator. It is described that the thickness of the PDMS has an influence on the permeation of gasses such as O2 and CO2 (Leclerc et al. 2003), however, in our experiments PDMS with a thickness range in between 100 μm to several mm showed to have no influence on the cell viability (data not shown). Cell survival is also dependent on the quality of the available medium. A higher concentration of cells will exhaust the medium faster, however, cell–cell contacts are necessary for survival (Yu et al. 2005). During long-term cell culture no flow was applied in the microchannel. This could have had an adverse effect on cell survival and proliferation because of acidification of the medium and depletion of nutrients (Zhu et al. 2004). However, Fig. 4 shows that cells managed to survive and proliferated in the medium present in the chip for up to 7 days. Although observed in a different cell line and in a microchannel with dimensions in the mm range, the relationship between the microchannel geometry and cell proliferation was explored (Yu et al. 2005). It was described that cells cultured in microchannels in the absence of flow proliferated significantly more slowly than cells cultured in conventional culture flasks, and entered a quiescent state. However, this inhibited cell proliferation disappeared when cells returned to a conventional culture flask. A possible explanation for the inhibited proliferation is that (cell-secreted) functional molecules surround the cells, enclosing the cell in a special microenvironment. Figure 5 of this paper also showed that cells grew better under a ceiling of only medium (in the punctured part) than under the ceiling of medium and PDMS. This suggests that the culture conditions change and even possibly deteriorate rapidly when under a PDMS ceiling, because of the lack of diffusion, causing the cells not to die, but entering a quiescent state.
Applying a continuous flow is a possible solution to provide the cells with the appropriate amount of nutrients and oxygen, and to remove waste products (Hung et al. 2005). However, the applied flow rate will expose the cells to mechanical forces, such as the shear stress. The maximum amount of shear stress that can be endured by cells depends on the cell type, since certain cells (e.g., endothelial cells) require shear stress for their development, whereas other cell types are negatively affected by shear stress (such as chondrocytes; Li et al. 2005; Healy et al. 2005; Kim et al. 2007). The optimal flow rate is therefore different for every cell type and chip design, depending on the chip dimensions. MCF-7 cells are from an epithelial origin, hence they do not require a (high) shear stress for development. In this case, the optimal flow rate needs to provide a good balance between on the one hand favorable medium conditions and on the other hand a low as possible shear stress. As stated in the results section, a flow of 0.5 μl/min was applied in chip 2, resulting in a shear stress of 0.17 dyn/cm2 in the inlet and outlet channel. The shear stress in the cell culture chamber is even lower, due to broadening of the channel and the presence of micropillars. This flow rate did not affect the cellular viability, though it could be observed that after 60 min of flow, the characteristic colony-forming growth pattern diminished and individual cells could be identified (data not shown). Applying a continuous flow could introduce a confounding variable in the apoptosis assays. To prevent this, the shear stress needs to be reduced by further decreasing the flow rate, changing the chip dimensions, or applying flow in intervals. In our experiments flow was applied for only 30 min, which preserved the cellular viability (Fig. 6).
3.6 Apoptosis induction
To analyze the chemosensitivity of MCF-cells apoptosis assays were performed. Staurosporine induced apoptosis in MCF-7 cells, exhibiting a diminished aggregate-like phenotype and PI positivity (Fig. 6). Morphology and fluorescence analysis are widely used in apoptosis assays. For an apoptosis assay to be useful the assay has to be controllable and apoptosis rates must be quantifiable and repeatable. Quantification of apoptosis in adherent cells that grow in colonies and maybe even in layers is only indicative when solely relying on optical methods, and therefore it is better to implement the measurement of electrophysiological parameters, such as pH, pO2 and impedance measurements (Otto et al. 2003). Furthermore, this aggregate-like phenotype complicates the interaction of the drug with all cells separately, hence probably not every cell was exposed to the same drug concentration during the 30 min of flow and the subsequent 24 h incubation. Moreover, the cells that in the final stage of the apoptotic process have detached from the surface and flow towards the outlet have to be counted in. In the experiments presented here, these detached cells were not included, so the amount of apoptosis observed might be an underestimation, for not only the untreated cells, but especially the SSP-treated cells. The chemosensitivity results obtained in the microfluidic chip are difficult to compare with standard conventional assays, such as flow cytometry, due to differences in experimental settings. The chosen staurosporine concentration used in our experiments was substantial higher than described in literature, in order to assure quick apoptotic effects in our caspase-3 negative MCF-7 cells (Mooney et al. 2002; Xue et al. 2003; Sayeed et al. 2007). The implementation of a dose-gradient generator in future chip designs will enable dose–response analysis (Thompson et al. 2004).
3.7 External validity and future plans
If eventually the chip as described here is optimized to serve as a solid apoptosis assay to analyze drug responses, it will still be doubtful if in vivo cancer sensitivity to a certain chemotherapeutic agent can be predicted by in vitro testing. It is impossible to copy in vivo circumstances to the in vitro situation. However the quality of the model depends on predicting chemosensitivity, not on the resemblance between the in vitro and the in vivo environment. For testing chemosensitivity, apoptosis assays were chosen since apoptosis is the objective and most downstream effect of chemotherapy. DNA microarrays have less requirements regarding cell handling, nevertheless are more upstream. The uncertain link between genetics and apoptosis in response to certain chemotherapeutics introduces more variability. Breast tumors are heterogeneous and the cells of a single tumor could be heterogeneous because of tissue conditions (ischemia and necrosis) (Stingl and Caldas 2007). Therefore, it is essential to use a patients’ own (tumor) cells obtained via biopsy and perform experiments in an ex vivo setting. For high-throughput analysis, the obtained cells can be subdivided into numerous samples to test various concentrations (dose-gradient generator) and combinations of different chemotherapeutics. The objective of such a high-throughput analysis would be to rank different available chemotherapeutics based on their effectiveness for the specific tumor of a specific patient. In this way patient survival can be increased and costs decreased. Specific for breast cancer patients, such an assay could be used for example to decide between a cyclophosphamide-methotrexate-5-fluorouracil (CMF) treatment or an anthracyclin containing agent. For this, a detailed validation of the chip in a clinical setting is greatly required.
Our future plans are first focused on optimizing the experimental set-up and be able to quantify apoptosis percentages, which is possible in conventional flow cytometry analyses (Wolbers et al. 2004a, b) and in multiparametric tests on chip (Gouaze et al. 2002; Mooney et al. 2002; Otto et al. 2003). Quantifying apoptosis makes it easier to correlate the obtained values with results published in literature, and undertake steps towards the clinic. This will increase the chemotherapeutic effectiveness and improve personal heath care. Although there is an increasing knowledge about the different chemotherapeutics and intracellular pathways, in our opinion one of the reasons for lack of chemotherapeutic effectiveness arise from the fact that there are hardly any predictive tests to match patients to chemotherapeutics.
In this paper the development towards a microfluidic chip which enables the cultivation of cells and the induction of apoptosis using a flow system is described. We found that MCF-7 cells could be cultivated statically for up to 7 days in a microfluidic PDMS-Pyrex chip. Upon induction of apoptosis with SSP, the characteristic aggregate-like phenotype disappeared, and MCF-7 cells were predominantly present in small PI positive clusters. Compared to untreated MCF-7 cells, SSP diminished the ratio of the fluorescence of the life-stain CAAM vs. the fluorescence of the death dye PI with almost a factor 2, indicating that apoptosis is induced. Next steps are focused on validating this device using clinically relevant hormone and chemotherapeutic agents in combination with patients’ tumor cells, to prove the importance of such a device for the clinic.
Financial support from the Dutch technology association STW (TMM.6016 NanoSCAN project, matching project of NanoNed project TMM.7128 Flow sensing and control in nanochannels) and technical assistance of J.W. van Nieuwkasteele, S. Le Gac and P.M. ter Braak are gratefully acknowledged.
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