Quantification of Viability in Organotypic Multicellular Spheroids of Human Malignant Glioma using Lactate Dehydrogenase Activity : A Rapid and Reliable Automated Assay
Departments of Neurosurgery (PCDWH,SL), Cell Biology and Histology (AJ,CJFVN), and Anatomy and Embryology (JMR), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
Correspondence to: Philip C. De Witt Hamer, MD, Academic Medical Centre, University of Amsterdam, Dept. of Neurosurgery, Room H2-230, PO Box 22660, 1100 DD Amsterdam, The Netherlands. E-mail: P.C.deWittHamer{at}amc.nl
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Summary |
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Key Words: spheroids glioma lactate dehydrogenase enzyme histochemistry metabolic activity toxicity test biological assay drug screening assays image cytometry cryostat sections
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Introduction |
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Screening of cytostatic drugs is commonly carried out with the use of monolayer cell cultures. Apparent advantages of these tumor models are ease of culture and availability of assays that enable quantification of drug response effects. However, new agents with promising effects in these in vitro models repeatedly fail to be efficacious in patients (Wolff et al. 1999). Perhaps the biological behavior of human malignant glioma is not portrayed by monolayer cell cultures, and efforts toward development of a more complex biological model are worthwhile.
The organotypic multicellular spheroid (OMS) model retains the heterogeneity of the original tumor tissue in addition to the presence of extracellular matrix, vascular elements, and cellcell interactions (Sutherland 1988; Hamilton 1998
; Bates et al. 2000
; Oudar 2000
). A technique to culture OMSs has been specified for human malignant glioma (Bjerkvig et al. 1990
). Fragmented surgical specimens are cultured in medium and closely resemble the original malignant glioma tissue histologically and morphologically, including glial fibrillary associated protein (GFAP) positivity, confirming the glial origin (Bjerkvig et al. 1990
; Kaaijk et al. 1995
). Quantification of drug responses in OMSs has been hampered by the heterogeneous character of the OMSs and their three-dimensional structure. To quantify drug response effects in the OMS model, we first introduce a viability score [the ratio of the lactic dehydrogenase (LDH)-active tissue area and total tissue area] and, second, present an image cytometry process that is automated for high-throughput facilitation. The activity of LDH (EC 1.1.1.27) is histochemically determined by reduction of a tetrazolium salt to its formazan, which delineates viable tissue in cryostat sections of OMSs as shown in the present study. Subsequent image cytometry with calibrated absorbance measurements of formazan production enables calculation of a viability index (VI).
This anaerobic glycolytic enzyme was considered as a viability marker for four reasons: (a) stained LDH-active and LDH-inactive tissue areas contrast sharply, allowing accurate discrimination using image cytometry; (b) established techniques also based on reduction of tetrazolium salt are in use for determination of experimental myocardial and hepatic infarction size (Frederiks et al. 1984,1989
) and routine verification of human myocardial infarction at autopsy (Lie et al. 1975
; Fishbein et al. 1981
); (c) the technique has analogy with established cytotoxicity response assays using the same concept for monolayer cell cultures (LDH release and MTT assays) (Korzeniewski and Callewaert 1983
; Decker and Lohmann-Matthes 1988
; Legrand et al. 1992
; Allen et al. 1994
; Sepp et al. 1996
; Hand et al. 1998
); (d) LDH is abundantly present in human malignant glioma tissue (Egami et al. 1983
; Fujii et al. 1984
; Marzatico et al. 1986
; Subhash et al. 1993
; Oudard et al. 1996
).
Here we present the reliability and reproducibility of an automated quantification method using LDH activity as a marker for viability in cryostat sections of OMSs.
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Materials and Methods |
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Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium azide, and dimethylformamide were supplied by Merck (Darmstadt, Germany). Polyvinyl alcohol (average molecular weight 70,000100,000) and nitroblue tetrazolium were supplied by Sigma. Sodium L-lactate and 1-methoxyphenazine methosulfate (mPMS) were supplied by Serva (Heidelberg, Germany). NAD was supplied by Roche Diagnostics (Mannheim, Germany).
Organotypic Multicellular Spheroids in Culture
Tumor specimens obtained at surgery from patients with malignant glioma were fragmented with number 10 and number 15 lancets and transferred to 48-well plates containing minimal essential medium as previously described (Bjerkvig et al. 1990), with slight modifications.
Minimal essential medium consists of Dulbecco's modified Eagle's medium with 10% heat-inactivated normal human serum, 2% L-glutamine, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Each well was coated with 100 µl 50% medium containing agarose to avoid cell adhesion and 300 µl medium overlay was added. Tumor fragments were cultured in a SteriCult 200 tissue culture incubator (CleanAir; Woerden, The Netherlands) at 37C, 100% humidity, 95% air, and 5% CO2. After 1 week, tumor fragments that had evolved to OMSs were manually selected using a phase-contrast microscope. This is the empirically found shortest time in culture for development of fragments to OMSs. The manual selection was based on spherical morphology, cell shedding, and transparency. Figure 1A illustrates a typical fragment meeting these requirements, and Figure 1B shows a typical fragment that disintegrated to a flock of debris that is not suitable for further analysis. The fraction of OMSs that evolved from initial fragments was 6578% [150 from 192 fragments for one tumor from a 74-year-old woman with a GBM (internal reference no. A26); 62 from 96 fragments for another tumor from a 47-year-old man with a GBM (A27)]. OMSs were then cultured for another week after medium overlay replacement with a Pasteur's pipette until harvesting for analysis. The OMSs stabilized in volume and had a mean diameter (SD) of 923 (213) µm.
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Histochemistry of LDH Activity
Tetrazolium salt methods are established precipitation reactions for the localization of the activity of dehydrogenases. Enzyme-catalyzed oxidation of the substrate lactate by LDH releases protons that are picked up by the co-enzyme NAD. The reduced co-enzyme reduces an electron carrier (mPMS), which transfers the electrons directly to the tetrazolium salt as electron acceptor to generate formazan. Formazan is formed as a dark purple water-insoluble precipitate. LDH is a dehydrogenase whose activity can be determined by this histochemical technique (Van Noorden and Frederiks 1992).
The incubation medium consisted of 18% polyvinyl alcohol dissolved in 100 mM phosphate buffer (pH 7.45) containing sodium L-lactate (150 mM), NAD (3 mM), mPMS (0.32 mM), sodium azide (5 mM), and tetra nitroblue tetrazolium (5 mg/ml medium first dissolved in 20 µl ethanol and 20 µl dimethylformamide solution; final dilution of each solvent in the medium 2%). We used tetra nitroblue tetrazolium because it produces the finest formazan precipitate of all tetrazolium salts and thus increases detail (Van Noorden and Frederiks 1992). Slides with unfixed cryostat sections attached were adapted to room temperature and air dried for 10 min. Afterward, incubation was performed using 1 ml of incubation medium per slide. The reaction was aborted using 100 mM phosphate buffer (pH 5.3) at 70C for 15 min to stop the reaction promptly and rinse off all viscous incubation medium (Van Noorden and Frederiks 1992
). Serial sections were stained with HE. Sections were dehydrated using standard ethanol and xylol dilution series and mounted in Pertex (HistoLab Products; Goeteborg, Sweden) to be coverslipped.
Kinetic Measurements of LDH Activity
End point measurements were designed to allow maximal contrast between LDH-active and LDH-inactive tissue compartments. Kinetic measurements of LDH activity were used to determine differences in absorbance values between LDH-active and LDH-inactive tissue compartments over time at RT and 37C [OMS of a 63-year-old man with a GBM with gemistocytary characteristics (A18)]. Control incubations were performed (on serial sections of the same OMSs) omitting the substrate lactate from the incubation medium (Van Noorden and Vogels 1989b).
Digital Image Acquisition
Video microscopy was accomplished with a CCD video camera (high performance CCD, model 4910; Cohu, San Diego, CA) mounted on a light microscope (Vanox AH-2; Olympus, Tokyo, Japan). The image acquisition setup and calibration have previously been described (Jonker et al. 1997). A monochromatic filter of the isobestic wavelength of the half-formazan and formazan (550 nm) was used (Van Noorden and Frederiks 1992
). Images of OMS sections after LDH activity staining were acquired with a x6.3/0.16 microscope magnification objective and frames were grabbed with the CCD video camera in 8-bit images of 786 x 512 pixels. Digital images were calibrated for absorbance measurements with a 10-step absorbance reference strip (Eastman Kodak; Rochester, NY) and pixels were scaled to µm. Acquired images were archived and processed on a Macintosh computer (Cupertino, CA) with a customized macro in the public domain software application Object-Image v2.09 (Norbert O.E. Vischer; University of Amsterdam, Faculty of Science, Amsterdam, The Netherlands; available at http://simon.bio.uva.nl/object-image.html).
Image Cytometry to Determine the Viability Index
Viability of tissue in an OMS section was calculated as ratio of the area (in µm2) expressing an absorbance value compatible with LDH-active tissue and the area (in µm2) expressing an absorbance value compatible with both LDH-active and LDH-inactive tissue. This ratio specifies the VI for a section j of an OMS i:
![]() | (eq.1) |
The VI is a ratio ranging from 0 to 1. A VI of 0.00 represents a minimally viable OMS section, whereas a VI of 1.00 represents a maximally viable OMS section.
Two absorbance thresholds are required to discriminate between absence of tissue, LDH-inactive tissue, and LDH-active tissue. The absorbance thresholds were resolved by correlating contours of LDH-active tissue and LDH-inactive tissue in serial sections of OMSs after conventional HE staining and histochemical localization of LDH activity. Conventional HE staining is generally considered to be the standard for determination of viable tissue in OMS sections. Absorbance thresholds were established on the basis of 40 OMS sections in three separately calibrated acquisition sessions [tumor material from a 47-year-old man with a GBM (A27)]. Average absorbance values (± SD) for the thresholds were determined to be 0.10 (± 0.03) for the value discriminating between absence of tissue and LDH-inactive tissue and 0.40 (± 0.10) for discrimination between LDH-inactive and LDH-active tissue.
Image Procressing Algorithm
OMS sections usually show an outer rim with reduced LDH activity. This phenomenon is considered to be an artifact produced by embedding or cutting. This artifact obviously results in an underestimation of the VI. To exclude the artifactual rim from calculations of the VI, an image processing algorithm was implemented.
The concept of the image processing algorithm is based on the construction of a new OMS section contour to which VI calculations are limited. The new contour is created by shrinking the original OMS section contour to the size of the most frequently occurring distance between the original OMS section rim and LDH-active tissue.
The elementary processing steps are schematically drawn in Figure 2. An example of the algorithm applied to an OMS section is outlined in Figure 3.
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![]() | (eq.2) |
The original total tissue area is the sum of LDH-active and LDH-inactive tissue areas before application of the rim exclusion algorithm, and the new total tissue area is the area remaining after application of the rim exclusion algorithm.
Reliability of the Histochemical VI Analysis
The reliability of demonstration of viability by LDH activity in serial cryostat sections of OMSs was verified in relation to conventional assessment of viable vs nonviable tissue on HE-stained cryostat sections. These were compared with the outlines as determined by the automated cytometry contours after histochemical visualization of LDH activity.
Reproducibility of the VI
For any histochemical technique with application of automated cytometry analysis, perception of its reproducibility is required. Several sources of variation need to be considered, such as cryostat settings, preparation of chemicals, incubation medium, incubation period, image acquisition settings, and cytometric analysis. To test the robustness of all sources of variation after sectioning, the incubation to localize LDH activity, image acquisition and cytometric analysis were repeated on two separate samples of serial OMS sections for 32 OMSs from one tumor [from a 37-year-old woman with a GBM with a significant sarcomatous component (A15)]. Each sample consisted of an average of 5.5 (SD 1.5) sections per OMS. The VI for an OMS i was calculated from K sections as:
![]() | (eq.3) |
The correlation between the two sets of 32 VI estimates was analyzed with a scatter diagram and the Pearson's correlation coefficient.
Detection of Viability Reduction by the VI
The VI for OMSs is expected to detect treatment response in terms of reduction in viability. Therefore, an unequivocal viability-reducing stimulus should induce a substantial reduction in the VI. Sodium azide inhibits the oxidative phosphorylation in mitochondria (Smith et al. 1991; Smith and Wilcox 1994
; Chang and Lamm 2003
). This metabolic uncoupling results in acute cytotoxicity to both normal and malignant cells and provides a means to test the detection of viability reduction. For this experiment, 64 OMSs were produced from two tumors [from a 47-year-old man with a GBM (A27) and a 74-year-old woman with a GBM (A26)]. These OMSs were used as untreated controls and the VIs were determined according to eq. 3. These measurements were compared with the VIs of 8 OMSs produced from another tumor [from a 41-year-old man with an AA dedifferentiated from low-grade astrocytoma (WHO grade 2) who had received temozolomide before surgery (A30)]. OMSs were incubated in the presence of 10 mM sodium azide in culture medium for 1 week before snap-freezing and analysis.
Because of the non-normal distribution, a Kruskal-Wallis test was performed to test differences of the three VI group means.
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Results |
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The Kruskal-Wallis test shows a -square of 22.8, d.f. 2, and p<0.001. This high significance is interpreted as strong statistical evidence for unequality of the three means. This effect is attributed to the significantly reduced VI in the sodium azide group. This finding of reduced VI after sodium azide treatment provides proof of principle for the validity of the VI to detect treatment response in OMSs.
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Discussion |
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This focus on the OMS tumor model to study the behavior of human malignant glioma is our answer to the multitude of treatment strategies that arise rapidly and demand a suitable model for screening of their potential. The ideal tumor model for this purpose would be a system that is rapid in providing results in the short term, valid with respect to the original responsiveness of tumors, efficient, and allows analysis of multiple aspects of tumor biology. On the one hand, it is obvious that assessment of the anticancer potential of an unselected panel of promising novel agents in malignant glioma patients, although highly biologically valid, is slow, inefficient, and ethically unacceptable. On the other hand, initial drug screening in malignant glioma research is generally based on observations with monolayer cell line cultures that allow rapid and efficient high-throughput analysis. However, the discrepancy between results obtained in these basic culture systems and human glioma responses is probably due to shortcomings in the validity of representation of human glioma biology by monolayer cell cultures (Wolff et al. 1999). The more complex three-dimensional OMS tumor model may provide a biological system that more closely represents the human glioma responsiveness because of the native presence of extracellular matrix structures, vascular elements, and cellcell interactions (Sutherland 1988
; Kaaijk et al. 1995
; Hamilton 1998
; Kunz-Schughart et al. 1998
; Bates et al. 2000
; Mueller-Klieser 2000
; Santini et al. 2000
).
However, a test to evaluate drug responsiveness is required for the OMS tumor model that under the best circumstances is rapid, accurate in terms of quantifiability, reproducible, based on transparent principles, takes advantage of the spatial information available from the three-dimensional structure, and allows multiple comparative analysis of different aspects of tumor responses in the same tissue material. Other approaches to quantify responsiveness in OMS models are in use, including growth in diameter (Bjerkvig et al. 1990; Jung et al. 1991
; Chignola et al. 1994
,1995
; Weber et al. 1994
), cell adhesion and migration assays (Bjerkvig et al. 1997
; Narla et al. 1998
; Ohnishi et al. 1998
; Mahesparan et al. 1999
; Santini et al. 2000
; de Ridder et al. 2000
), chemical dissociation to provide cell suspensions that are amenable to conventional monoclonal cell culture cytotoxicity assays (Freyer and Sutherland 1980
; Freyer and Schor 1989
), confocal laser scanning microscopy after fluorescence probing (Wartenberg and Acker 1995
; Wartenberg et al. 1998
; Walenta et al. 2000
), and immunohistochemical detection of proliferation and apoptosis markers (Kaaijk et al. 1996
,1997
; Kolchinsky and Roninson 1997
; Wharton et al. 2000
; Bell et al. 2001
).
Before weighing the merits of these approaches, it is of importance to discern two categories of tumor spheroids. In the first place, there are multicellular aggregated spheroids that evolved from monolayer cell cultures by aggregation and, in the second place, there are OMSs with an architecture native to the surgical specimens from which they are grown. The latter OMSs stabilize in volume over time (Bjerkvig et al. 1990; Kaaijk et al. 1995
) and are not susceptible to chemical dissociation (unpublished data from our laboratory) in contrast to the aggregated spheroids. Although these dissociative characteristics provide an opportunity for quantification of drug responsiveness in aggregated spheroids, the biological behavior of these aggregated spheroids is, again, probably affected by clonal selection and by deficiency of extracellular matrix and vascular elements.
Poor penetration of fluorescence probes in OMSs [up to 60 µm from the margin (unpublished data)] impedes confocal laser scanning microscopy. Hence, immunohistochemical detection of cell proliferation and apoptosis markers is the only approach that provides spatial information and allows the serial analysis of multiple parameters in the same material. However, IHC markers are characterized by the fact that they provide information on one specific protein, which may be expressed only transiently. Therefore, a more general end point was pursued to determine the balance between cell proliferation and (programmed) cell death, which is of primary concern in the malignant glioma patient. One end point meeting this requirement is metabolic activity of cells, also referred to as viability. Here we show that tissue viability can be assessed in OMSs in analogy to various tetrazolium salt reduction assays available for cell cultures, and in essence provides an in situ application of the MTT assay. Quantification of LDH release from dying cells is an established approach to assessment of drug responsiveness in other tumor models based on diffusion of LDH from cells with a leaky plasma membrane, which is an early event in cell death (Frederiks et al. 1983
, 1995
; Korzeniewski and Callewaert 1983
; Decker and Lohmann-Matthes 1988
; Legrand et al. 1992
; Allen et al. 1994
; Sepp et al. 1996
). Many commercially available kits provide colorimetric detection of dehydrogenase activity either indirectly by LDH released in the supernatant from the cytosol of dying cells [i.e., Cytotoxicity Detection Kit (LDH), Roche Applied Science; LDH Cytotoxicity Detection Kit, MoBiTec; TOX7-1KT, Sigma Aldrich] or directly by demonstration of dehydrogenase activity in viable cells [i.e., Cell Proliferation Kit I (MTT), II (XTT), III (WST1), Roche Applied Science; MTT Cell Proliferation Assay, ATCC (Rockville, MD); Cell Proliferation Assay Kit, Chemicon (Temecula, CA)]. Subsequent quantification can be accomplished by colorimetry of the supernatant in an ELISA plate reader for cell culture suspensions.
In the present study, localization of LDH activity in cryostat sections of OMSs is described as a tumor-responsiveness test. This test provides an assay that is rapid, accurate in quantification, reproducible, transparent by being founded on generally established concepts of tissue viability, enables the use of spatial information, and is available for further analysis of other aspects of tumor responsiveness in serial sections from the same tissue material. Furthermore, an example of sodium azide responsiveness has provided proof of principle for the validity of the VI to detect a cytotoxic response in OMSs.
Some issues remain to be resolved, however, before relevant results can be provided with this OMS tumor model and the LDH activity assay. In the first place, LDH-inactive tissue is interpreted as nonviable tissue based on the rationale that viable cells contain LDH and therefore that absence of LDH in tissue is in accordance with absence of viable cells. Nevertheless, the LDH-inactive tissue areas do not entirely consist of necrosis, according to preliminary analysis (unpublished data). Apparently, part of the LDH-inactive tissue stains positive for picrosirius red, indicating collageneous elements of the extracellular matrix. Other parts stain positive for vimentin IX, indicating vascular elements. In the second place, another important issue is whether the OMS model proves to be sufficiently biologically valid. The superior validity of the OMS model compared with the monolayer cell culture has only been theoretically deduced. In the third place, the outliers in VI in untreated and sodium azide-treated samples illustrate the need to compensate for the heterogeneity of OMSs by inclusion of multiple samples in a treatment group. Apparently, eight OMSs sufficed for statistical analysis in the sodium azide experiment. In general, the consequence of heterogeneity of tumor tissue needs to be addressed for a tumor model to be efficient. Crucial to this point are the minimal numbers of OMSs in a treatment group and the minimal numbers of cryostat sections per OMS required (given a predetermined biologically relevant detectable difference in VI, power, and significance level). These issues need to be addressed for this tumor model in succeeding studies.
We conclude that the viability of OMSs can be quantified in a rapid, reliable, and reproducible way using localization of LDH activity in cryostat sections with automated image cytometric analysis. This is an important advance towards relevant drug screening in this human malignant glioma tumor model.
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Acknowledgments |
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We also wish to thank K.S. Bosch and W.M. Frederiks, PhD, from the Department of Cell Biology and Histology, for their contribution to the enzyme histochemical laboratory protocols and facilities.
Support with the software development provided by N.O.E. Vischer, PhD, software engineer at the Faculty of Biology, is kindly appreciated.
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Footnotes |
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