ARTICLE |
Correspondence to: Leoni A. KunzSchughart, Institute of Pathology, University of Regensburg, 93042 Regensburg, Germany. E-mail: leoni.kunz-schughart@klinik.uni-regensburg.de
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Summary |
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Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 µm. In contrast, the histomorphological emergence of cell death at a diameter of around 500 µm in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture. (J Histochem Cytochem 48:509522, 2000)
Key Words: imaging bioluminescence, multicellular tumor spheroids, rat embryo fibroblasts, myc/ras co-transfection, metabolism, ATP, glucose, lactate
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Introduction |
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In vitro cultivation of tumor cells as multicellular tumor spheroids (MCTS) has greatly contributed to the understanding of the role of the cellular microenvironment in tumor biology (for review see
Despite the importance of understanding metabolic alterations associated with tumorigenic conversion for the development of novel therapeutic strategies, only a few systematic studies have been undertaken using differently transformed cell lines in 3D culture. In 1996, the classical two-step carcinogenesis model of myc/ras-co-transfected rat embryo fibroblasts (REFs) proposed by
The model system used here consists of four different cell lines derived from Fisher 344 REFs with two immortalized cell clones (Rat1 and M1) representing early stages of transformation and two ras-transfected cell clones (Rat1-T1 and MR1) that are highly tumorigenic and aggressive in vivo. The immortalized cell types have an extended lifespan but demonstrate normal contact inhibition of proliferation and anchorage dependence in vitro, and they are poorly (Rat1) or nontumorigenic (M1) when injected into nude mice. T24Ha-ras transfection of Rat1 cells and c-myc1/T24Ha-ras co-transfection of REFs resulted in the two cell clones Rat1-T1 and MR1, respectively, which represent advanced stages of transformation. All four cell lines form multicellular aggregates, when attachment to an artificial substratum is prevented, but they exhibit large differences in proliferative behavior. Rat1 and M1 aggregates cease to grow in spheroid culture, whereas Rat1-T1 and MR1 spheroids show high proliferative activity, reaching maximal sizes of >1200 µm (
Although genetically closely related, the two tu-morigenic cell types dramatically differ in their sensitivity to oxygen deficiency in 3D culture. Our present investigations were inspired by the observation that oxygen deficit coincided with central cell death in Rat1-T1 spheroids, whereas MR1 cells appeared to be highly resistant to hypoxia. Minimum pO2 values accompanied cell quiescence, which raises the question of whether oxygen deficiency may lead to a reduction in ATP content. The aim of our study was to systematically investigate the relationship between parameters reflecting metabolism, such as ATP content, and oxygen availability, proliferative activity, and cell death in the 3D culture system described. In addition, the impact of ras-associated tumorigenic conversion on metabolic behavior was studied by involving the nontumorigenic ancestors Rat1 and M1 that exhibit cell- cycle arrest in aggregate culture despite an optimal oxygen supply.
We analyzed ATP, glucose, and lactate distributions in the four different aggregate types by imaging bioluminescence. This technique has been progressively applied in the past few years to quantitatively localize metabolites in cryosections of various biological tissues (
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Materials and Methods |
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Spheroid Types and Culturing
Studies were carried out on aggregates of four differently transformed Fisher 344 REF cell lines. Rat1 and M1 cells were immortalized either spontaneously or by myc transfection as described earlier (
Spheroid Volume, Growth, and Histology
Spheroid growth kinetics were routinely monitored by measuring two orthogonal diameters of 50 individual spheroids every 48 hr using a calibrated eyepiece reticule of an inverted microscope, and were analyzed by fitting the Gompertz equation to the experimental data as detailed earlier (
Immunohistochemical Determination of Apoptotic Cells
Apoptotic cells were observed in 5-µm median sections of paraffin-embedded MCTS of different sizes (with and without necrosis) by detecting single- and double-stranded DNA breaks using the TUNEL assay (BoehringerMannheim; Mannheim, Germany). All sections were double stained using DAPI as a marker for nuclei and fluorescein-dUTP for DNA strand breaks (BoehringerMannheim). A standard staining protocol was used. Stained sections were analyzed in a fluorescence microscope equipped with the appropriate filter sets at a magnification of x100250 (Zeiss; Oberkochen, Germany). This test allows the detection of apoptotic cells in the viable cell rim only, because necrotic cell destruction in the spheroid center is finally accompanied by the occurrence of DNA strand breaks resulting in fluorescein-dUTP positivity.
Spheroid Conservation and Cryosectioning
To investigate ATP, glucose, and lactate concentrations using the bioluminescence technique, spheroids were shock-frozen in liquid nitrogen and were stored at -80C to avoid or minimize diffusion processes influencing local substrate concentrations. For measurements, serial cryostat sections with a thickness of 5 µm through the center region of aggregates with defined sizes were made. One section was used for histological processing and observation, and three adjacent sections allowed immediate assessment of the substrate concentrations.
Imaging Bioluminescence
Regional distributions of ATP, glucose, and lactate in cryosections of spheroids were registered via imaging bioluminescence (for review see
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For each measurement, a central cryosection of a spheroid was adhered to a coverglass and kept frozen until use. The coverglass was placed upside down on a casting mold filled with a frozen solution of enzymes and co-enzymes (see below) to link the metabolite of interest to the luciferase light reaction. The mold carrying the slide was placed in a temperature-controlled chamber fixed on a microscope stage; the temperature was adjusted to 10 ± 1C for reproducible kinetics of the enzyme reaction. Subsequently, the enzyme solution and the tissue section melted and the enzyme reaction started. Light emission was registered by the video system over a period of 60 sec. Within this time interval, light intensities were digitized and accumulated in a frame memory of the computer. The intensity values of the resulting images were converted into concentration values (µmol/g tissue fresh weight) using appropriate tissue standards. These frozen standards were prepared from homogenized, heat-inactivated liver tissue containing defined metabolite concentrations and were processed similarly to the spheroid samples.
Three different enzyme solutions were applied to image ATP, glucose, and lactate levels. Each solution contained 60 g/liter gelatin, 0.3 M glycerol, and 30 g/liter polyvinylpyrrolidone (PVP) in a buffer solution. For ATP imaging, substances were dissolved in a 0.2 M HEPES buffer (pH 7.6) supplemented with 0.1 M sodium hydrogen arsenate. Subsequently, 2 mM MgCl2 and 50 g/liter lyophilized and powdered firefly lanterns were added for enzymatic reaction. Additional ingredients of the enzyme solution for glucose measurements were 100 mM ATP, 75 mM NADP, 8 mM MgCl2, 0.5 mM 1,4-dithiothreitol (DTT), 0.4 mM FMN, 8 mM capric aldehyde dissolved in 0.3 M PBS (pH 7.0). Enzymes added to link glucose to the light reaction of luciferase were 44 U/ml hexokinase, 55 U/ml glucose-6-phosphate dehydrogenase, 13 mU/ml luciferase, and 8 U/ml NAD(P)H-FMN-oxidoreductase. Lactate was determined using a solution of 50 mM L-glutamic acid, 160 mM NAD, 0.5 mM DTT, 0.4 ml FMN, 8 mM capric aldehyde, 463 U/ml glutamate-pyruvate transaminase, 67 U/ml lactate dehydrogenase, 13 mU/ml luciferase, and 8 U/ml NAD(P)H-FMN-oxidoreductase in 0.1 M PBS (pH 7.0) plus supplements mentioned above. All chemicals were obtained from Merck (Darmstadt, Germany) or Sigma. Enzymes and coenzymes were from BoehringerMannheim.
Glucose Uptake and Lactate Release
Glucose and lactate concentrations were assessed using standard enzyme reagent kits (Glucotest, Monotest Lactate; BoehringerMannheim), both based on the production of NADPH+H+ that can be detected photometrically. Glucose uptake and lactate release rates were measured in rotated suspension cultures by incubating 102104 spheroids of defined average sizes in 50 ml of fresh DMEM. The number of spheroids to be incubated was estimated from the mean spheroid size and the average number of cells per spheroid (
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Results |
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ATP Distribution in Spheroids
Representative color-coded ATP distributions in central cryosections of ras-transformed tumorigenic Rat1-T1 and MR1 spheroids with a diameter of 920 and 930 µm, respectively, are shown in Fig 2A and Fig 2B. The borderlines between the nonviable core and the viable cell rim are indicated by dashed circles. Rat1-T1 spheroids showed a steep drop of ATP towards the center during growth. As shown in Fig 2A, ATP is homogeneously distributed in the viable cell layer in these spheroids, whereas at a certain size almost no ATP could be found in the nonviable core. MR1 spheroids of the same size showed a different behavior: ATP in the nonviable center was reduced to only around 30% of the value found in the viable cell layer (Fig 2B).
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To further analyze the relationship between ATP concentrations, cell quiescence, and viability in MCTS, ATP values from the center (innermost region of 50 x 50 µm of the aggregates) were determined as a function of spheroid size (Fig 3A). For Rat1-T1 spheroids, the ATP distribution reflected not only the thickness of the viable cell rim, which was some 200 µm in large spheroids, but also the development of central cell death. Here, ATP concentration drastically decreased from values of 1.52.0 µmol/g in the center of Rat1-T1 spheroids with a diameter of around 500 µm to almost zero in central regions of 800-µm large aggregates. These data imply the onset of ATP reduction with or shortly after the development of cell death, which has been documented at spheroid sizes of 500600 µm in this particular spheroid type. In contrast to Rat1-T1 spheroids, the decline in central ATP as a function of spheroid size was much more gradual in MR1 aggregates, reflecting a different relationship between cell viability and energy status in the two spheroid types. The ATP level was 1.52.0 µmol/g in MR1 spheroids with a diameter of 400600 µm, dropped to approximately 50% of the initial values in the center of spheroids with developing necroses at sizes of approximately 800 µm, and finally reached minimum ATP of 0 µmol/g in the center of 1200-µm large MR1 spheroids.
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The immortalized but non- or poorly tumorigenic ancestors M1 and Rat1 were both characterized by a relatively homogeneous ATP distribution in aggregate culture. Representative ATP images and H&E-stained histological sections are shown in Fig 4A and Fig 4B, showing that ATP concentration in these aggregate types with maximal mean diameters of approximately 200 µm measured around 0.6 µmol/g, which is considerably lower than the central ATP level in small Rat1-T1 and MR1 spheroids by a factor of 34, as detailed in Fig 3A. However, taking into account the lower overall cell density of M1 aggregates compared to the other spheroid types, the actual intracellular ATP concentration could be as high as in the ras-transfected tumorigenic counterparts (for details see Appendix). For both immortalized aggregate types, ATP concentrations did not change significantly as a function of location in the spheroids. A slight decrease from the periphery towards the center was seen in some but not all M1 aggregates. This was unexpected because a dramatic loss in cell density from outer to inner aggregate regions was shown for this aggregate type (Fig 4A).
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Glucose Distribution and Uptake in Spheroids
Fig 5A represents the color-coded glucose distribution in a Rat1-T1 spheroid with a diameter of 920 µm (parallel section to Fig 2A). Unlike the results of the ATP concentration measurements, glucose distributions in frozen central sections of Rat1-T1 and MR1 spheroids were quite similar. Whereas small Rat1-T1 and MR1 aggregates with a diameter of <600 µm and the periphery of large spheroids showed glucose concentrations close to the medium level between 20 and 25 µmol/g, glucose in the center of large 3D cultures was significantly reduced. This is summarized in Fig 3B, documenting central glucose concentrations as a function of the spheroid diameter. Minimal glucose level in central spheroid regions was 35 µmol/g and was detected in spheroids with sizes >9001000 µm. For the two tumorigenic spheroid types, no clear correlation between development of cell death and/or thickness of the viable cell rim and the glucose distribution could be determined. Despite an earlier emergence of central cell death in Rat1-T1 spheroids, central glucose concentrations were not significantly different in the two spheroid types at similar sizes. Although there was a substantial drop in central glucose concentration with increasing spheroid size for both cell types, values did not fall below a range of 36 µmol/g, i.e., a concentration range in which these cells grew well in monolayer culture.
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The small Rat and M1 aggregates had central glucose concentrations of 2025 µmol/g, similar to the ras-transfected descendants and almost as high as the glucose level in the culture medium (Fig 3B). Accordingly, there was hardly any glucose gradient within the aggregates.
In addition to similar glucose distribution patterns, determination of glucose consumption rates per viable spheroid volume indicated that Rat1-T1 and MR1 spheroids may differ in their glucose metabolism at sizes of <500 µm. As shown in Fig 6A, glucose uptake per viable volume was substantially higher in small MR1 spheroids with values of (1012) x 10-8 mol/(s cm3) as opposed to Rat1-T1 spheroids with a diameter between 200 and 400 µm taking up approximately 6 x 10-8 mol/(s cm3). Spheroid type-specific variations in cell number per spheroid, cell volume, and the ratio of intra- to extracellular space do not account for this metabolic difference as documented earlier (
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For the immortalized Rat1 and M1 aggregates glucose uptake per volume unit ranged around 4 x 10-8 mol/(s cm3), which was significantly lower (p< 0.001) than that of the corresponding ras-transfected counterparts at similar sizes. With M1 aggregates, this difference is partly due to the low cell density.
Lactate Distribution and Release in Spheroids
Central lactate concentrations in Rat1-T1 and MR1 spheroids measured as a function of the spheroid size were not significantly different (p>0.05). As shown in Fig 3C, lactate concentration in the center of these two spheroid types increased in a similar way with increasing spheroid diameter, reaching maximal values of 711 µmol/g in spheroids that were 700900 µm in diameter. Consequently, high lactate levels coincided with the emergence of cell death in MR1 spheroids, whereas the development of necrosis in Rat1-T1 spheroids preceded maximal lactate accumulation. A representative bioluminescence image of lactate in a Rat1-T1 spheroid with a diameter of 920 µm is shown in Fig 5B (parallel section to Fig 2A and Fig 5A).
Lactate concentration the small non-ras-transfected Rat1 and M1 aggregates was 56 µmol/g. This is slightly higher than the lactate level in the tumorigenic counterparts at the same spheroid size but is clearly lower than the value determined in the nonviable center of large Rat1-T1 and MR1 spheroids (Fig 3C).
Lactate release rates were in accordance with glucose consumption data showing that MR1 spheroids are characterized by a drastic decrease in lactate release per viable spheroid volume as a function of spheroid diameter, as detailed in Fig 6B. Lactate production was reduced from 16 x 10-8 mol/(s cm3) in MR1 aggregates with a diameter of 200 µm to (56) x 10-8 mol/(s cm3) in 1000-µm large spheroids. In contrast, lactate release was relatively constant during growth of Rat1-T1 spheroids, measuring some 8 x 10-8 mol/(s cm3). Lactate production of both Rat1 and M1 aggregates measured approximately (68) x 10-8 mol/(s cm3) and was not significantly different from that in the viable cell rim of large Rat1-T1 and MR1 spheroids (Fig 6B).
Apoptotic Cell Death in Spheroids
To determine if the phenomenon of programmed cell death accounts for differences in the metabolite concentrations, particularly in the ATP distribution, of Rat1-T1 and MR1 spheroids, central sections of these two spheroid types with various diameters were analyzed by TUNEL assay. Fig 7A shows a typical result for a Rat1-T1 spheroid (diameter 900 µm). The section was double stained for nuclei (Fig 7A, DAPI fluorescence) and for apoptotic cells (Fig 7B, TUNEL fluorescence). The following observations are to be emphasized. (a) No evidence could be found for enhanced apoptotic cell death in MR1 spheroids compared to Rat1-T1 aggregates. Single cell death via apoptosis occurred in the viable cell rim (arrows) of both spheroid types despite sufficient nutrient availability. We therefore conclude that modifications in the ATP distribution are not due to a different apoptotic behavior of the cells. (b) For both Rat1-T1 and MR1 spheroids, central staining accompanied central necrotic cell death. Here, the TUNEL assay did not allow discrimination of apoptotic and necrotic cells in the center.
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Discussion |
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It has been controversially discussed in the literature that changes in tumor energy metabolism, as indicated by the cellular ATP level, may be correlated with oxygen and glucose supply and/or with cell cycle arrest and emergence of cell death in vivo and in vitro (
The tissue oxygen partial pressure (pO2) is an important parameter affecting and reflecting tumor metabolism. In a previous study, oxygen-sensitive microelectrodes were used to register pO2 gradients in the four spheroid types investigated in the present study (
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The transition of viable cells to a nonviable state, indicating the onset of cell death, emerges in the center of Rat1-T1 spheroids in the presence of ATP concentrations that can be measured in viable tumor and normal tissues (
These data are in accordance with findings obtained in EMT6/Ro spheroids, a cell line originating from a mouse mammary sarcoma, showing rather high ATP concentrations at the emergence of central cell death (
In contrast to Rat1-T1 spheroids, myc/ras-co-transfected MR1 spheroids show a pronounced reduction in the ATP concentration. This decrease occurs not only in the viable regions close to the nonviable core but also in the spheroid center as a function of the spheroid size, and it precedes the emergence of a histomorphological nonviable region (Fig 8B). The decrease in ATP is paralleled by enhanced cell quiescence (see above), indicating that a 50% reduction in central ATP level in MR1 spheroids accompanies cell-cycle arrest, mainly in the G1/0-phase (
In summary, spheroids of the two tumorigenic cell lines showed a different metabolic milieu at the onset of central cell death, which is true despite identical growth conditions, i.e., identical nutritional supply throughout all experiments. Obviously, differences in the morphological structure, the proliferation and growth behavior, and the metabolism of the spheroids investigated are the result of relatively small differences in the transformation process, i.e., two different modes of immortalizing the precursor cell. The present results, together with previous data, suggest that myc-transfection compared to spontaneous immortalization in fully transformed cell lines (MR1 vs Rat1-T1) confers a higher proliferation rate and a substantial resistance to hypoxia by the tumor cells (
Giaccia and associates showed that the myc oncogene is involved in the balance between cell proliferation and cell death by apoptosis (
In Rat1 and M1 aggregates, relatively low ATP concentrations of around 0.6 µmol/g were registered, although central cell destruction has been detected solely in M1 spheroids. Because of the lower cell density in M1 spheroids, it is likely that the intracellular ATP concentration could be as high as in the ras-transfected counterparts. Because ATP concentration did not change as a function of location in M1 aggregates despite a mean viable cell rim of only 30 µm, we conclude that cell destruction is not complete with regard to function, i.e., that there are still some metabolic activities in the center of these 3D cultures. This hypothesis is supported by pO2 measurements showing that oxygen profiles decrease towards the center of M1 aggregates, which can only result from oxygen-consuming processes. However, central pO2 in M1 aggregates is relatively high, with values of 80100 mmHg. There are two potential explanations for this behavior. (a) Some cells in the histologically destroyed area survive, accounting for a basic metabolic rate, or (b) cell destruction in M1 aggregates results from programmed cell death, which has been shown to be induced in fibroblasts by myc transfection (e.g.
Aggregates of both non-ras-transfected cell lines showed sufficient glucose and oxygen supply. However, the metabolic rates, as indicated by the glucose consumption, lactate production, and the proliferative activity (TLI <10%;
Because knowledge about the correlation between oncogene expression and metabolism is still sparse (
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft (grants Mu 576/2-4, Mu 576/4-1, Ku 917/1-1, and Ku 917/2-2).
We thank Dr A. Simm (Institute of Physiological Chemistry II, University of Wuerzburg, Germany) for providing oncogene-transfected rat embryo fibroblasts.
Received for publication October 16, 1999; accepted November 4, 1999.
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Appendix |
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Methodological Aspects
The reliability of quantitative enzyme histochemical assays, such as imaging bioluminescence, is influenced by several factors, including section thickness, cell density, availability of the metabolite in the tissue section for the exogenous enzymes in the bioluminescence solution, and endogenous enzyme activities in the tissue interfering with the detection step (
The accurate calibration of the light intensities is based on a more or less constant cell density of the tissues. The cell densities of the spheroids were calculated using the viable volume and the corresponding cell number of the cell aggregates. The cell densities in Rat1, Rat1-T1, and MR1 spheroids are largely the same (5.2, 4.8, and 4.1 x 108 cells/ml), whereas in M1 spheroids the cell density in the viable cell layer is approximately two to three times lower compared to the other types. As a consequence, ATP values will be underestimated in M1 spheroids, because ATP is located only intracellularly.
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