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Quantification of Viability in Organotypic Multicellular Spheroids of Human Malignant Glioma using Lactate Dehydrogenase Activity: A Rapid and Reliable Automated Assay
Witt Hamer Philip C. De, Ard Jonker, Sieger Leenstra, Jan M. Ruijter and Cornelis J.F. Van Noorden J Histochem Cytochem 2005 53: 23 DOI: 10.1177/002215540505300104 The online version of this article can be found at: http://jhc.sagepub.com/content/53/1/23

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© The Histochemical Society. 2002.jhc. 2002. Lactate dehydrogenase (LDH) activity was demonstrated in cryostat sections of spheroids using the tetrazolium salt method. Fragmented 23 Downloaded from jhc.30 Hanash et al. 0022-1554/05/$3. vascular elements. Room H2-230. Inc. 2001. Oudar 2000). despite standard treatment consisting of gross total surgical resection and radiotherapy (Gupta and Sarin 2002. Reddy and Kaelin 2002. Ard Jonker. Correspondence to: Philip C. E-mail: P.CJFVN). and efforts toward development of a more complex biological model are worthwhile. PO Box 22660. (J Histochem Cytochem 53:23–34. A technique to culture OMSs has been specified for human malignant glioma (Bjerkvig et al. 2004. Ruijter. De Witt Hamer.org ARTICLE Quantification of Viability in Organotypic Multicellular Spheroids of Human Malignant Glioma using Lactate Dehydrogenase Activity: A Rapid and Reliable Automated Assay Philip C. respectively. The viability index (VI) was calculated as ratio of LDH-active areas and total spheroid tissue areas. angiogenesis inhibitors. Accurate and reproducible quantification of response effect has been lacking to determine drug responses in this three-dimensional tumor model. Cell Biology and Histology (AJ. MD.sagepub.F. Fully automated image cytometry reliably demarcates LDH-active and LDH-inactive tissue areas by thresholding at specific absorbance values.deWittHamer@amc. However.Volume 53(1): 23–34. 2012 . TremontLukats and Gilbert 2003). of Neurosurgery. 1100 DD Amsterdam. Academic Medical Centre. The organotypic multicellular spheroid (OMS) model retains the heterogeneity of the original tumor tissue in addition to the presence of extracellular matrix. Apparent advantages of these tumor models are ease of culture and availability of assays that enable quantification of drug response effects. accepted August 10. Bates et al. Jan M. WHO grade 3 (AA) or glioblastoma multiforme. and endotoxins (Brandes et al. Amsterdam. 1990). in addition to conventional cytostatics. Therefore. The Netherlands. The Netherlands SUMMARY Organotypic spheroids from malignant glioma resemble the biological complexity of the original tumor and are therefore appealing to study anticancer drug responses. 2004 [DOI: 10. We conclude that quantification of viability in cryostat sections of organotypic multicellular spheroids from malignant glioma can be performed reliably and reproducibly with this approach. Signal transduction modifiers have recently extended the list of potential agents. Dept. Effective treatment strategies are notoriously lacking. 2005 Journal of Histochemistry & Cytochemistry http://www. De Witt Hamer.2005]. Sodium azide incubation of spheroids induced reduction in VI to almost zero. Hamilton 1998. University of Amsterdam. Laws et al. Academic Medical Centre.C.SL). Duplicate staining and processing on the same tissue showed good correlation and therefore reproducibility. University of Amsterdam. 2003).nl Received for publication February 27. immunotherapy.com by guest on May 4. Sieger Leenstra. WHO grade 4 (GBM)] is a devastating primary CNS tumor with a median patient survival of 10 and 18 months. and Anatomy and Embryology (JMR).1369/jhc. screening of novel anticancer agents is of primary importance. Screening of cytostatic drugs is commonly carried out with the use of monolayer cell cultures. and cell–cell interactions (Sutherland 1988. Perhaps the biological behavior of human malignant glioma is not portrayed by monolayer cell cultures.4A6301. 2005) KEY WORDS spheroids glioma lactate dehydrogenase enzyme histochemistry metabolic activity toxicity test biological assay drug screening assays image cytometry cryostat sections Malignant glioma [anaplastic astrocytoma. Calibrated digital image acquisition of the stained cryostat sections enables quantification of LDH activity. Van Noorden The Journal of Histochemistry & Cytochemistry Departments of Neurosurgery (PCDWH. Basso et al. 1999). new agents with promising effects in these in vitro models repeatedly fail to be efficacious in patients (Wolff et al. 2000. and Cornelis J.

second. Multiple OMSs were embedded in one gelatin unit to incorporate OMSs from a 48-well plate all together. IL) using disposable knives at a cabinet temperature of 22C to cut sections of 8. 100% humidity. 1992. MO). and transparency. Allen et al. 95% air. penicillin (100 IU/ml). penicillin. The reduced co-enzyme reduces an electron carrier (mPMS). 1993.sagepub. 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. with slight modifications.45). Van Noorden (Heidelberg.000–100. allowing accurate discrimination using image cytometry. 1984.1989) and routine verification of human myocardial infarction at autopsy (Lie et al. and 5% CO2. The Journal of Histochemistry & Cytochemistry Gelatin Embedding and Cryosectioning After harvesting. The fraction of OMSs that evolved from initial fragments was 65–78% [150 from 192 fragments for one tumor from a 74-year-old woman with a GBM (internal reference no. The activity of LDH (EC 1. 62 from 96 fragments for another tumor from a 47-year-old man with a GBM (A27)]. Sectioning was carried out on a Jung Frigocut 2800-E cryostat (Leica Microsystems. Formazan is formed as a dark purple water-insoluble precipitate. and Figure 1B shows a typical fragment that disintegrated to a flock of debris that is not suitable for further analysis. Potassium dihydrogen phosphate. Oudard et al. The unit was then snap-frozen in liquid nitrogen and stored at 80C until cryostat sectioning. Leenstra. 1990). After 1 week. Subhash et al. Bannockburn. The Netherlands) at 37C. The Netherlands). 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. Fishbein et al. Agarose and gelatin were supplied by Sigma (St Louis. 2012 . The Netherlands) supplied the Costar 48-well culture plates. LDH is a dehydrogenase whose activity can be determined by this histochemical technique (Van Noorden and Frederiks 1992). MD). Quantification of drug responses in OMSs has been hampered by the heterogeneous character of the OMSs and their three-dimensional structure.24 surgical specimens are cultured in medium and closely resemble the original malignant glioma tissue histologically and morphologically. Minimal essential medium consists of Dulbecco’s modified Eagle’s medium with 10% heat-inactivated normal human serum. disodium hydrogen phosphate. Each well was coated with 100 l 50% medium containing agarose to avoid cell adhesion and 300 l medium overlay was added. Ruijter. Woerden. A26). 1994. (d) LDH is abundantly present in human malignant glioma tissue (Egami et al. This is the empirically found shortest time in culture for development of fragments to OMSs. Kaaijk et al.27) is histochemically determined by reduction of a tetrazolium salt to its formazan. Marzatico et al. sodium azide. 1983. Germany). Figure 1A illustrates a typical fragment meeting these requirements. Legrand et al. OH) and normal human serum by BioWhittaker (Walkersville. cell shedding.1. Materials and Methods Suppliers of Resources Dulbecco’s modified Eagle’s medium was supplied by ICN Biochemicals (Aurora. confirming the glial origin (Bjerkvig et al. including glial fibrillary associated protein (GFAP) positivity. 1986. tumor fragments that had evolved to OMSs were manually selected using a phase-contrast microscope. 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) m. De Witt Hamer. Germany). Corning Life Sciences (Schiphol. Hand et al. Germany). To quantify drug response effects in the OMS model. This anaerobic glycolytic enzyme was considered as a viability marker for four reasons: (a) stained LDH-active and LDH-inactive tissue areas contrast sharply. we first introduce a viability score [the ratio of the lactic dehydrogenase (LDH)active tissue area and total tissue area] and.000) and nitroblue tetrazolium were supplied by Sigma. Enzyme-catalyzed oxidation of the substrate lactate by LDH releases protons that are picked up by the co-enzyme NAD. 2% l-glutamine. which transfers the electrons directly to the tetrazolium salt as electron acceptor to generate formazan. (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. 1984. 1975. 1998). (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. Gelatin embedding resulted in a better quality of the sections compared with various other embedding agents tested (data not shown). Fujii et al. OMSs were embedded in plastic containers (20 30 mm) containing a liquefied solution of 8% gelatin at 37C in 100 mM phosphate buffer (pH 7. NAD was supplied by Roche Diagnostics (Mannheim. 1981). 1990. Downloaded from jhc.1. and streptomycin were supplied by Gibco Invitrogen (Breda. 1995). and dimethylformamide were supplied by Merck (Darmstadt.m thickness. Sodium l-lactate and 1-methoxyphenazine methosulfate (mPMS) were supplied by Serva Histochemistry of LDH Activity Tetrazolium salt methods are established precipitation reactions for the localization of the activity of dehydrogenases. and streptomycin (100 g/ml). The manual selection was based on spherical morphology. Tumor fragments were cultured in a SteriCult 200 tissue culture incubator (CleanAir. Polyvinyl alcohol (average molecular weight 70. l-Glutamine. Sepp et al. Jonker. Subsequent image cytometry with calibrated absorbance measurements of formazan production enables calculation of a viability index (VI). Decker and Lohmann-Matthes 1988. 1996).com by guest on May 4. which delineates viable tissue in cryostat sections of OMSs as shown in the present study. 1996. present an image cytometry process that is automated for high-throughput facilitation.

Vischer.09 (Norbert O. Sweden) to be coverslipped.(eq. 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).nl/object-image.uva. Kinetic Measurements of LDH Activity End point measurements were designed to allow maximal contrast between LDH-active and LDH-inactive tissue compartments.bio.html). Afterward. 2012 .3) at 70C for 15 min to stop the reaction promptly and rinse off all viscous incubation medium (Van Noorden and Frederiks 1992). Rochester. Goeteborg. Acquired images were archived and processed on a Macintosh computer (Cupertino. Tokyo. San Diego. model 4910. Faculty of Science. Digital images were calibrated for absorbance measurements with a 10-step absorbance reference strip (Eastman Kodak. Serial sections were stained with HE. This ratio specifies the VI for a section ‘j’ of an OMS ‘i’: The incubation medium consisted of 18% polyvinyl alcohol dissolved in 100 mM phosphate buffer (pH 7. Amsterdam. Images of OMS sections after LDH activity staining were acquired with a 6.Quantification of Viability in Glioma Spheroids 25 in Pertex (HistoLab Products.E. Figure 1 Phase-contrast micrograph of (A) a glioma spheroid that has evolved from a fragment of a surgical specimen after 1 week in culture. and tetra nitroblue tetrazolium (5 mg/ml medium first dissolved in 20 l ethanol and 20 l dimethylformamide solution. A VI of 0. The Netherlands.16 microscope magnification objective and frames were grabbed with the CCD video camera in 8-bit images of 786 512 pixels. 1). NAD (3 mM). mPMS (0. University of Amsterdam. and LDHactive 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. CA) mounted on a light microscope (Vanox AH-2. Two absorbance thresholds are required to discriminate between absence of tissue. 1997). Downloaded from jhc. whereas a VI of 1. area LDH-active tissue + ∑ area LDH-inactive tissue ∑ The VI is a ratio ranging from 0 to 1. CA) with a customized macro in the public domain software application Object-Image v2.com by guest on May 4. sodium azide (5 mM). The image acquisition setup and calibration have previously been described (Jonker et al. LDH-inactive tissue.3/0. NY) and pixels were scaled to m. Bar 500 m.45) containing sodium l-lactate (150 mM). incubation was performed using 1 ml of incubation medium per slide. Kinetic measurements of LDH activity were used to determine differences in absorbance values between LDHactive 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)]. and (B) a fragment of a surgical specimen that does not meet the requirements of a spheroid as specified in Materials and Methods. available at http:// simon. Japan). Slides with unfixed cryostat sections attached were adapted to room temperature and air dried for 10 min. A monochromatic filter of the isobestic wavelength of the half-formazan and formazan (550 nm) was used (Van Noorden and Frederiks 1992). Cohu.32 mM). Sections were dehydrated using standard ethanol and xylol dilution series and mounted ∑ areaLDH-active tissue VI ij = ----------------------------------------------------------------------------------------------------------------. The Journal of Histochemistry & Cytochemistry Digital Image Acquisition Video microscopy was accomplished with a CCD video camera (high performance CCD. Conventional HE staining is generally considered to be the standard for determination of viable tissue in OMS sections. Image Cytometry to Determine the Viability Index Viability of tissue in an OMS section was calculated as ratio of the area (in m2) expressing an absorbance value compatible with LDH-active tissue and the area (in m2) expressing an absorbance value compatible with both LDH-active and LDH-inactive tissue.sagepub.00 represents a maximally viable OMS section.00 represents a minimally viable OMS section. The reaction was aborted using 100 mM phosphate buffer (pH 5. Control incubations were performed (on serial sections of the same OMSs) omitting the substrate lactate from the incubation medium (Van Noorden and Vogels 1989b). final dilution of each solvent in the medium 2%). Olympus.

(E) The most frequently observed distance is determined (to preserve LDH-inactive tissue area bordering the artifactual rim that is to be included in VI calculations) and applied to determine the adjusted OMS section contour as shown in red. This artifact obviously results in an underestimation of the VI. (eq.03) for the value discriminating between absence of tissue and LDH-inactive tissue and 0.26 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)]. The artifactual rim that is to be excluded surrounds the LDH-active and LDH-inactive tissue areas. Reproducibility of the VI For any histochemical technique with application of automated cytometry analysis. preparation of chemicals. To test the robustness of all sources of variation after sectioning.40 ( 0. (B) The lower absorbance threshold value determines the entire OMS section contour as shown in yellow. 2).5 (SD 1. incubation period. 2012 . These were compared with the outlines as determined by the automated cytometry contours after histochemical visualization of LDH activity. perception of its reproducibility is required.10 ( 0. an algorithm impact (AI) on total tissue area was calculated for each OMS section (n 4136) of 64 OMSs from two tumors [from a 47year-old man with a GBM (A27) and a 74-year-old woman with a GBM (A26)] as: The Journal of Histochemistry & Cytochemistry AI = 100% original total tissue area [ µm ] – new total tissue area [ µm ] --------------------------------------------------------------------------------------------------------------------------------------------------------2 original total tissue area [ µm ] 2 2 Figure 2 Schematic illustration of the fully automated image processing algorithm to determine the viability index (VI) in organotypic multicellular glioma spheroids (OMS) on the basis of LDH activity. incubation medium. Van Noorden Image Procressing Algorithm OMS sections usually show an outer rim with reduced LDH activity. Several sources of variation need to be considered. and the new total tissue area is the area remaining after application of the rim exclusion algorithm. The original total tissue area is the sum of LDH-active and LDH-inactive tissue areas before application of the rim exclusion algorithm. De Witt Hamer. image acquisition settings. (C) The upper absorbance threshold value determines the LDH-active tissue contours as shown in green. and (3) an LDH-inactive tissue area bordering the artifactual rim. An example of the algorithm applied to an OMS section is outlined in Figure 3.com by guest on May 4. (G) The numerator in the VI calculation is provided by the surface measurement after application of the upper absorbance threshold. Leenstra. such as cryostat settings. and cytometric analysis. (2) an LDH-inactive tissue area surrounded by LDH-active tissue. (H) The denominator in the VI calculation is provided by the surface measurement after application of the lower absorbance threshold. (D) The closest distance to LDH-active tissue is determined for every point of the rim of the entire OMS section. the incubation to localize LDH activity. The VI for an OMS ‘i’ was calculated from K sections as: 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. 3). This phenomenon is considered to be an artifact produced by embedding or cutting. (A) OMS section with a dark gray LDH-active tissue area and a light gray LDH-inactive tissue area.5) sections per OMS. To exclude the artifactual rim from calculations of the VI. The adjusted contour in F is used as an overlay for the original image A. 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. (F) The adjusted contour in E is combined with the LDH-active tissue contour in C to include the incorrectly excluded LDH-active tissue area not surrounded by the artifactual rim. an image processing algorithm was implemented. Three elements (1–3) illustrate algorithm handling of contour finding problems: (1) an LDH-active tissue area not surrounded by the artifactual rim. Jonker.10) for discrimination between LDH-inactive and LDH-active tissue. Average absorbance values ( SD) for the thresholds were determined to be 0. 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 elementary processing steps are schematically drawn in Figure 2. VI i = -------------------------------------------------------------------------------------------------------------------------------K j=1 j=1 ∑ ( ∑ areaLDH-active tissue ) K ∑ ( ∑ areaLDH-active tissue + ∑ areaLDH-inactive tissue ) (eq.sagepub. Ruijter. To demonstrate the impact of this rim exclusion algorithm on the total tissue area measurement. Each sample consisted of an average of 5. Downloaded from jhc. 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)].

68. The correlation between the two sets of 32 VI estimates was analyzed with a scatter diagram and the Pearson’s correlation coefficient. an unequivocal viability-reducing stimulus should induce a substantial reduction in the VI. (F) The result image of Figure 3E combined with Figure 3D as mask.com by guest on May 4. Bar 250 m. (J) Combined image of Figure 3B and 3I showing that LDH-active tissue containing information required for the VI calculations is located outside the new contour. Smith and Wilcox 1994. This metabolic uncoupling results in acute cytotoxicity to both normal and malignant cells and provides a means to test the detection of viability reduction. (H) The most frequently occurring distance value 11 is located in red on a distance-transformed image map of Figure 3C. Chang and Lamm 2003).Quantification of Viability in Glioma Spheroids 27 The Journal of Histochemistry & Cytochemistry Figure 3 Rim exclusion algorithm to determine the viability index (VI) in glioma OMSs on the basis of LDH activity. 1991. (C) Binary image after thresholding for LDH-active and LDH-inactive tissue with low absorbance threshold. (K) Result of application of the contour of Figure 3J as mask for the original image in Figure 3A with LDH activity information. 2012 . Consequently. Sodium azide inhibits the oxidative phosphor- ylation in mitochondria (Smith et al. new outer contour (red). 3. For this experiment. (L) Outlines of original outer contour (yellow). These measurements were compared with the VIs of 8 OMSs produced from an- Downloaded from jhc. (B) Binary image after thresholding for LDH-active tissue with high absorbance threshold. The VI calculated on the basis of the rim-exclusion algorithm was 0. Therefore. (I) A threshold of the modal distance applied to the distance-transformed map inside the entire OMS section constructs a new outer contour. (D) One-pixel-wide contour of Figure 3C after filling in the interior holes. Detection of Viability Reduction by the VI The VI for OMSs is expected to detect treatment response in terms of reduction in viability. These OMSs were used as untreated controls and the VIs were determined according to eq. (G) Histogram of Figure 3F showing distribution of distances of outer rim pixels to LDH-active area. 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)]. and LDH-active area (green) plotted on the original image in Figure 3A. (A) Original 8-bit 400 400 pixel image acquired by a CCD video camera coupled to a light microscope with a monochromatic filter. For illustration purposes. the LDH-active tissue area (Figure 3B) is added to the new contour (Figure 3I). (E) The inverted binary image of Figure 3B is distance-transformed to an image map with the 8-bit gray scale value specifying the closest distance in pixels to LDH-active tissue for each pixel.sagepub. showing the shortest distance of outer rim pixels to LDH-active tissue. the contour Figure 3D is also shown. note that the most frequently occurring distance is 11 pixels (red bar).

28 other 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)]. this incubation period was applied for end-point measurements to refrain from absorbance values above 0. i. Frederiks et al. it is evident that LDH-active and LDH-inactive tissue areas contrast sharply after LDH activity staining. object glass background ( ) and respective control reactions without lactate. ( ) LDH-inactive tissue. Maximal difference between the absorbance values is observed 30 min after incubation. such as the center of the OMS section in Figure 5A (located with *). Reliability of the Histochemical VI Analysis The comparison between tissue appearance using conventional HE-stained sections of OMSs. The LDH-inactive tissue reaction curve is linear with time.8 to avoid the out-of-range error (Van Noorden and Butcher 1986). or are predominantly LDH-inactive (Figures 5J–5L). Because of the non-normal distribution. 1989). On the basis of these kinetic measurements. Reaction curves at 37C (data not shown) showed a rapid increase in absorbance over time. Although the tissue was sectioned in a cryostat. tions appeared difficult to determine on the basis of morphological aspects.e. OMS sections stained for LDH activity.. and digital images with contours for LDH-active and LDH-inactive tissue areas provided by automated image cytometry is illustrated in Figure 5. and this was subtracted from all test reactions (Figure 4B). OMSs shown are either predominantly LDH-active (Figures 5A–5C) or harbor complex alternations of LDH-active and LDH-inactive tissue areas (Figures 5D–5I). This area shows marked LDH activity. showed a complete lack of formazan formation.. Jonker. the morphology of the tissue sections is sufficiently maintained and major cutting artifacts can usually be avoided. which we considered to be disadvantageous because incubation intervals that are too short may introduce a source of variation from timing error.e. the “nothing dehydrogenase” reaction (Van Noorden et al. Control reactions in the absence of lactate. De Witt Hamer. 2012 . Although the contrast may have increased further beyond 30 min. Some nonspecific formazan formation was observed in media containing lactate and NAD. which unambiguously ascer- Downloaded from jhc. Viable and nonviable tissue areas in HE-stained OMS sec- Figure 4 Time-dependent increase in absorbance values of formazan produced by LDH activity at RT in sections of human glioma spheroids. (B) Kinetic curves for ( ) LDHactive tissue minus control reaction. Ruijter. ( ) no tissue.com by guest on May 4. 1985. we concluded that the maximal contrast between LDH-active and LDH-inactive tissue was obtained after 30 min of incubation at RT. Leenstra. Van Noorden Results Kinetic Measurements of LDH Activity The Journal of Histochemistry & Cytochemistry Reaction curves describing formazan precipitation due to LDH activity in LDH-active tissue increase nonlinearly in time (Figure 4A) as described previously (Van Noorden and Vogels 1989a). a Kruskal-Wallis test was performed to test differences of the three VI group means. (A) Reaction curves for circular regions of interest (size 200 m2) containing ( ) LDH-active tissue. and ( ) LDH-inactive tissue minus control reaction. i. OMSs were incubated in the presence of 10 mM sodium azide in culture medium for 1 week before snap-freezing and analysis. indicating that endogenous substrate levels were very low. When the HE-stained sections are compared with the LDH activity-stained sections.sagepub.

This outlier contributes considerably to the average VI of 0. a single outlier with a VI of 0. and p 0. 5F.001. The ideal tumor model for this purpose would be a system that is rapid in providing results in the short term. efficient. and 5L reflect the decreasing extent of LDH-active tissue areas in the successive OMS sections. we conclude that. These specific OMSs harbor reduced levels of LDH activity. First.01). Based on these observations. 5I. LDH activity levels and morphological aspects after HE staining were in agreement with LDH-inactive tissue with low viability. the viability can be adequately quantified using the VI. Even complex contours of the LDH-inactive tissue areas in Figures 5E and 5H are detected correctly. On the one hand. Detection of Viability Reduction by the VI The frequency plot of untreated and sodium azidetreated samples is shown in Figure 7. However. The large LDH-inactive tissue area is included in the OMS section enclosed by the red line. The Kruskal-Wallis test shows a -square of 22.8. Again. However. Second. Furthermore. The value of the rim exclusion algorithm can be appreciated from the way the LDHinactive tissue area bordering the artifactual rim is handled in Figure 5K. and allows analysis of multiple aspects of tumor biology. Kunz-Schughart et al. 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. presumably due to spontaneous necrosis. as a consequence.02. Closer inspection indicated that the sections involved were correctly stained and processed to determine VIs. This effect is attributed to the significantly reduced VI in the sodium azide group. Second. 2000.48 was found in the sodium azide- Discussion This report describes the quantification of viability in cryostat sections of organotypic multicellular spheroids (OMSs) from human malignant glioma on the basis of LDH activity using image cytometry. the entire procedure of histochemical localization of LDH activity. Bates et al. 2000). vascular elements. On the other hand. 1998. 1999). the two untreated OMS groups have closely similar average VIs (0.79) and distributions. localization of LDH activity reliably separates LDH-active and LDH-inactive tissue areas. Third. is slow. and fourth. Hamilton 1998. Comparison of the LDH activity-stained sections with the digital images indicated that the reliability of the contour finding cytometry is manifest.f. On the one hand. The red line in Figure 5L outlines the OMS section contour that was used for the VI calculation after application of the rim exclusion algorithm. Perhaps this OMS was less dependent on oxidative phosphorylation for its metabolic needs by having switched to anaerobic glycolysis and. 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. 2. The Journal of Histochemistry & Cytochemistry tains viability of this tissue area (Figure 5B). closer inspection of the sections indicated that staining and processing to determine the VI appeared to be correct. some overlap is observed in VI extremes for treated and untreated OMSs. valid with respect to the original responsiveness of tumors. first. 1995. and ethically unacceptable. and cell–cell interactions (Sutherland 1988. decreasing VIs of tissue sections in Figures 5C. 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. inefficient. p 0. Kaaijk et al. it is obvious that assessment of the anticancer potential of an unselected panel of promising novel agents in malignant glioma patients. four of eight treated OMSs express a VI less than 0.50 in the two untreated samples.72 vs 0. although highly biologically valid. These data highlight the detection of the cytotoxic effects of sodium azide by the VI. 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. A number of interesting details arise from this frequency plot. a test to evaluate drug responsiveness is required for the OMS tumor model that under the best circumstances is rapid. the impact of the rim exclusion algorithm is nontrivial. Santini et al.sagepub. 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. there are respectively two and three OMSs with a VI less than 0. 2012 .com by guest on May 4.09 for sodium azidetreated OMSs. these areas can be automatically segmented by image cytometry independently of the complexity of the tissue areas. Mueller-Klieser 2000. the mean AI (SD) for the analyzed sections all together was 23% (17%). became less susceptible to the toxic effects of sodium azide. digital image acquisition and cytometric analysis to determine the VI are reproducible (Pearson’s correlation coefficient 0. accurate in terms of quan- Downloaded from jhc. In fact. This high significance is interpreted as strong statistical evidence for unequality of the three means. d. Reproducibility of the VI According to the scatter diagram (Figure 6).935.Quantification of Viability in Glioma Spheroids 29 treated OMS group. In addition to this. On the other hand.

30 De Witt Hamer.sagepub.com by guest on May 4. Jonker. Van Noorden The Journal of Histochemistry & Cytochemistry Downloaded from jhc. Leenstra. 2012 . Ruijter.

Figure 7 Frequency plot of viability indices for untreated spheroids from two tumors (n 32 for each) and sodium azide-treated spheroids (10 mM in culture medium for 1 week) from one tumor (n 8). Narla et al. 2000. there are OMSs with an architecture native to the surgical specimens from which they are grown. the biological behavior of these aggregated spheroids is. and cytometric analysis in separate sessions. Kaaijk et al. 0. there are multicellular aggregated spheroids that evolved from monolayer cell cultures by aggregation and.J). 1996. 1999.E.G. image acquisition. In the first place. 2000). Other approaches to quantify responsiveness in OMS models are in use. 1998. in the second place. tifiability. 1990. 2000. 1990.01. d. de Ridder et al.001. chemical dissociation to provide cell suspensions that are amenable to conventional monoclonal cell culture cytotoxicity assays (Freyer and Sutherland 1980. Downloaded from jhc. Marker (*) locates tissue area that was difficult to interpret as either viable or nonviable in HE-stained section on the basis of morphological aspects. 1997. 2000). and after digital image acquisition and cytometric processing with outlines that color the entire spheroid contour (yellow). Before weighing the merits of these approaches. Kruskal-Wallis -square of 22. Weber et al. Wharton et al. Bars 250 m. Although these dissociative characteristics provide an opportunity for quantification of drug responsiveness in aggregated spheroids. the adjusted contour resulting from the rim exclusion algorithm (red) and LDH-active tissue contours (green). and p 0. p 0. histochemical localization of LDH activity (B. 1998. 1994). Each dot depicts values obtained from an average of 5.com by guest on May 4.Quantification of Viability in Glioma Spheroids 31 The Journal of Histochemistry & Cytochemistry Figure 6 Scatter diagram of viability indices in two serial sets of the same 32 spheroids with histochemical detection of LDH activity. Kolchinsky and Roninson 1997.D.K). Walenta et al. and allows multiple comparative analysis of different aspects of tumor responses in the same tissue material. Bell et al. including growth in diameter (Bjerkvig et al. it is of importance to discern two categories of tumor spheroids. Santini et al. 1994.1995.935. and immunohistochemical detection of proliferation and apoptosis markers (Kaaijk et al.29 in L. Pearson’s correlation coefficient 0. Wartenberg et al. confocal laser scanning microscopy after fluorescence probing (Wartenberg and Acker 1995. The horizontal lines determine the average VI per spheroid group.sagepub. 2001). takes advantage of the spatial information available from the three-dimensional structure. Chignola et al. cell adhesion and migration assays (Bjerkvig et al.78 in both F and I and 0. 2. reproducible. Jung et al. Each dot represents one spheroid.98 in C.H.f. 1995) and are not susceptible to chemical dissociation (unpublished data from our laboratory) in contrast to the aggregated spheroids. Mahesparan et al.5 (SD 1. Ohnishi et al. again. based on transparent principles. 1998. The latter OMSs stabilize in volume over time (Bjerkvig et al. Freyer and Schor 1989). 2012 . 1991. probably affected Figure 5 Sections of human glioma spheroids with various appearances of LDH-active and LDH-inactive tissue after HE staining (A.5) sections of a spheroid.1997. The VI is 0.8.

Legrand et al. an example of sodium azide responsiveness has provided proof of principle for the validity of the VI to detect a cytotoxic response in OMSs. TOX71KT. Acknowledgments Our gratitude is expressed to Professor Dr D. Poor penetration of fluorescence probes in OMSs [up to 60 m from the margin (unpublished data)] impedes confocal laser scanning microscopy. 1992. Allen et al. 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.32 by clonal selection and by deficiency of extracellular matrix and vascular elements. Frederiks. 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. Apparently. 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. In the first place. Hence. ATCC (Rockville. according to preliminary analysis (unpublished data).e. This test provides an assay that is rapid. Van Noorden model and the LDH activity assay.e. Chemicon (Temecula. head of the Department of Neuropathology. Leenstra. MTT Cell Proliferation Assay. In general.O. and significance level). However. for his contribution to interpretation of the HE-stained sections of OMSs.com by guest on May 4. accurate in quantification. Sigma Aldrich] or directly by demonstration of dehydrogenase activity in viable cells [i. Cell Proliferation Assay Kit. the consequence of heterogeneity of tumor tissue needs to be addressed for a tumor model to be efficient. which is an early event in cell death (Frederiks et al. part of the LDH-inactive tissue stains positive for picrosirius red. Nevertheless. reproducible. Vischer. before relevant results can be provided with this OMS tumor De Witt Hamer. II (XTT). IHC markers are characterized by the fact that they provide information on one specific protein. from the Department of Cell Biology and Histology. enables the use of spatial information. for facilitation of the culture incubators and reagents. We also wish to thank K. Troost. Dawes E. head of the research laboratory of the Department of Pathology. Other parts stain positive for vimentin IX.J. however. This is an important advance towards relevant drug screening in this human malignant glioma tumor model. In the present study. Brandes AA (2002) Non-cytotoxic therapies for malignant gliomas. transparent by being founded on generally established concepts of tissue viability. also referred to as viability. Clin Mater 16:189–194 Basso U. J Neurooncol 58:57–69 Downloaded from jhc. Decker and Lohmann-Matthes 1988. 1996). Sepp et al. In the third place.. We conclude that the viability of OMSs can be quantified in a rapid. and in essence provides an in situ application of the MTT assay. 1994. which is of primary concern in the malignant glioma patient. software engineer at the Faculty of Biology. Therefore. 2012 . Korzeniewski and Callewaert 1983. Vastola F. PhD. Furthermore. and is available for further analysis of other aspects of tumor responsiveness in serial sections from the same tissue material. Bosch and W. 1995. MoBiTec. a more general end point was pursued to determine the balance between cell proliferation and (programmed) cell death. Rushton N (1994) Lactate dehydrogenase activity as a rapid and sensitive test for the quantification of cell numbers in vitro. reliable. III (WST1). van Amstel. These issues need to be addressed for this tumor model in succeeding studies. Jonker. Cell Proliferation Kit I (MTT). eight OMSs sufficed for statistical analysis in the sodium azide experiment. The superior validity of the OMS model compared with the monolayer cell culture has only been theoretically deduced. CA)]. The Journal of Histochemistry & Cytochemistry Literature Cited Allen M. for their contribution to the enzyme histochemical laboratory protocols and facilities. 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. the LDH-inactive tissue areas do not entirely consist of necrosis. LDH Cytotoxicity Detection Kit. 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. and to Ing P. Some issues remain to be resolved. power. 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. Ruijter. 1983. Millett P. localization of LDH activity in cryostat sections of OMSs is described as a tumorresponsiveness test.S..sagepub. is kindly appreciated. MD). another important issue is whether the OMS model proves to be sufficiently biologically valid. One end point meeting this requirement is metabolic activity of cells. Subsequent quantification can be accomplished by colorimetry of the supernatant in an ELISA plate reader for cell culture suspensions. indicating collageneous elements of the extracellular matrix. Here we show that tissue viability can be assessed in OMSs in analogy to various tetrazolium salt reduction assays available for cell cultures. Apparently. Support with the software development provided by N. Cytotoxicity Detection Kit (LDH). In the second place. Roche Applied Science. indicating vascular elements. Ermani M.E. and reproducible way using localization of LDH activity in cryostat sections with automated image cytometric analysis. Roche Applied Science.M. PhD. which may be expressed only transiently.

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