Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids

L. A. Kunz-Schughart, R. C. Habbersett, and J. P. Freyer

Cellular and Molecular Biology and Cytometry Groups, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Two mitochondrion-specific fluorochromes, 10-N-nonyl acridine orange (NAO) and rhodamine 123 (Rh123), were used to determine the mechanism responsible for alterations in energy metabolism of transformed rat embryo fibroblast cells isolated from different locations within multicellular spheroids. Accumulation of Rh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thus represents mitochondrial distribution only. A reproducible selective dissociation procedure was used to isolate cells from different locations within the spheroids. After isolation, cells were simultaneously stained with one mitochondrial stain and the DNA dye Hoechst 33342, and several parameters, including cell volume, were monitored via multilaser-multiparameter flow cytometry. Our data clearly show a decrease in the uptake of Rh123 in cells from the periphery to the inner regions of the tumor spheroids, reflecting a persistent alteration in mitochondrial function. However, NAO staining experiments showed no reduction in the total mitochondrial mass per unit cell volume. Because cells were exposed to stain under uniform conditions after isolation from the spheroid, these data indicate that downregulation of mitochondrial function is associated with cell quiescence rather than a transient effect of reduced nutrient availability. This result, which is in accordance with data from two other cell lines (EMT6 and 9L), might reflect a general phenomenon in multicellular spheroids, supporting the hypothesis that quiescent cells in the innermost viable spheroid layer stably reduce their mitochondrial function, presumably to compensate for lower nutrient supply and/or decreased energy demand.

rat embryo fibroblasts; mitochondria; cell cycle; flow cytometry

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SINCE THEIR INTRODUCTION in 1971 by Sutherland and co-workers (37), multicellular tumor spheroids have gained experimental importance as an in vitro model of tumors (6, 28, 36). Particular interest has been placed on the role of the metabolic three-dimensional microenvironment, e.g., oxygen, glucose, and lactate distribution, in the development of cell quiescence and necrotic cell death (1-3, 13, 14, 27, 39). Many cultured cell lines undergo changes in cell volume and metabolism with increasing periods of growth in two- and three-dimensional culture. Quiescent cells in confluent monolayers or within the innermost region of the viable cell rim in large multicellular spheroids are often smaller than their counterparts in the exponential growth phase or in the superficial spheroid layer (e.g., Refs. 7, 12, 13, 18, 21, 26). For both growth conditions, a decline in cell size as a function of time in culture is paralleled by significant loss in proliferative activity. No systematic correlation between proliferative status or viability of cells within three-dimensional cultures and the distribution of microenvironmental factors has been recorded. For oncogene-transfected, tumorigenic rat embryo fibroblast spheroids, it was shown that oxygen deficiency coincided with the development of either centrally located necrosis or proliferation arrest (23). Reductions in cellular oxygen consumption while entering a quiescent state were demonstrated in monolayer culture for several cell lines, including Rat1-T1 and MR1 cells (3, 18, 21, 24, 38), yet these changes corresponded with a decrease in cell volume due to cell cycle arrest and were not comparable to those metabolic alterations recorded in spheroid cultures. Although restricted cellular energy metabolism due to nutrient deprivation does not seem to be the primary mechanism behind cellular quiescence in multicellular tumor spheroids (10), entry into a quiescent state in spheroids correlates with a variety of large metabolic modifications (3, 11, 13, 23, 38, 39) that would affect microenvironmental conditions within the spheroid.

Knowledge of the distribution of oxygen in spheroids is of particular relevance for tumor biology, therapy, and gene expression, and the oxygen gradient in spheroids is determined largely by the cellular consumption rate (QO2). The mean QO2 of cells in spheroids decreased with increasing spheroid size, whether QO2 was estimated from radial PO2 gradients in intact spheroids determined with recessed-type oxygen-sensitive microelectrodes (29) or was directly measured in intact spheroids (12, 13). In addition to changes in mean consumption rates, the oxygen gradient within spheroids can be drastically affected by alterations in QO2 across the spheroid rim. Estimations from measured oxygen gradients indicated a uniformly distributed respiratory activity within spheroids (e.g., Refs. 2, 14, 27, 30), whereas selective dissociation of the same spheroid types followed by in situ oxygen consumption measurement using Clarke-type electrodes showed a reduction in oxygen uptake per cell as a function of depth within the spheroid (7). If QO2 is reduced in inner spheroid regions, this must be due to either a loss of mitochondria or a downregulation of their respiratory function. Previous morphometric studies by Bredel-Geissler and co-workers (3) showed a relatively constant number of mitochondria across the viable cell rim of spheroids, without taking into account their functional status.

The objective of the present study was to determine both mitochondrial mass and activity of differently transformed rat fibroblasts in spheroid culture as a function of the location within the viable cell rim. Over the past decade, the cationic fluorescent dye rhodamine 123 (Rh123), which binds specifically to mitochondria of living cells (17), not only has been intensively studied as an effector of cell growth, ultrastructure, or related cell characteristics (e.g., Refs. 5, 15) but also has been used to investigate mitochondria during cell differentiation, antitumor drug treatment, and senescence (e.g., Refs. 31, 32, 34) and as a marker for multidrug-resistant cell populations (19). Because Rh123 uptake is due to the negative membrane potential of these cell organelles (16), quantitative measurements of the dye uptake into cells reflect both the number of mitochondria present and changes in mitochondrial activity. In contrast, 10-N-nonyl acridine orange (NAO) is spontaneously incorporated by mitochondria with high specificity and accumulated in the inner mitochondrial membrane, binding to cardiolipin independently of transmembrane potential (for review, see Refs. 25, 33). As a result, uptake of this fluorochrome can be used to identify changes in mitochondrial mass only. We also measured the volume and DNA content distributions of these cells, allowing correction of mitochondrial staining for changes in cell volume and correlation of these data with the proliferative status of the cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell lines and monolayer culturing. The two highly tumorigenic rat fibroblast cell clones Rat1-T1 and MR1 used in this study were derived from either spontaneously immortalized or myc-transfected Fisher 344 rat embryo fibroblasts by transfection with the point-mutated T24Ha-ras oncogene (24). Both cell clones are well characterized in monolayer and spheroid culture and are known to present the fully transformed phenotype within a two-step in vitro carcinogenesis model of rat embryo fibroblasts. Monolayer cultures were routinely maintained and subcultured for up to 20 passages (~120 cumulative population doublings) in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Gaithersburg, MD) containing 4.5 g/l D-glucose (Sigma, St. Louis, MO), 5% (vol/vol) fetal calf serum (Sigma), 100 IU/ml penicillin, and 100 µg/ml streptomycin (GIBCO).

Spheroid culturing and growth. Rat1-T1 and MR1 spheroids were cultured essentially as described in detail earlier (23, 28, 36). Spheroid aggregation and growth were initiated in 0.5% (wt/vol) agar-coated culture dishes (1-2 × 105 cells/100-mm dish). After a 4-day initiation interval, spheroids were transferred into 1-liter spinner flasks with continual stirring. Supplemented DMEM (see above paragraph) was used for spheroid culturing and was replaced every 24 h in the spinner flasks, and the number of spheroids was reduced each day to keep the total cell concentration relatively constant, ensuring a consistent environment throughout growth. Spheroid volume growth was routinely monitored as previously described (23). For individual spheroids, minor and major diameters were measured with a calibrated image-processing apparatus consisting of a microscope connected to a solid state camera with a monitor and a Macintosh-based computer system for image analysis using the National Institutes of Health program Image.

Selective dissociation of spheroids. For selective dissociation of cells from different locations within Rat1-T1 and MR1 spheroids, an automated procedure using a specially designed dissociation chamber connected to a perfusion system was applied (9). Dissociation conditions were 37°C, 100 revolutions/min (rpm) on the gyratory shaker, 0.125% trypsin, 0.5 mM EDTA, and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution (in Puck's saline) for MR1 spheroids and 37°C, 140 rpm on the gyratory shaker, a solution of 0.25% trypsin, 0.5 mM EDTA, 0.1% (wt/vol) collagenase/dispase (Sigma), and 25 mM HEPES (in Puck's saline) for Rat1-T1 aggregates (for more detail, see Ref. 22). Due to slightly different maximum average sizes reached by the spheroid populations investigated, two different dissociation protocols had to be used for each spheroid type to optimally and linearly dissociate the cultures. Two separate sets of spheroids were dissociated following each protocol. For each experiment, 20-50 spheroids with a mean diameter between 1,200 and 1,450 µm were hand selected. Cell suspensions were collected into cold supplemented medium and kept on ice after dissociation.

Staining of DNA and mitochondria. Viable cells dissociated from spheroids were simultaneously stained with one of the mitochondrial fluorescent dyes NAO and Rh123 and the DNA stain Hoechst 33342 for flow cytometric analysis. The staining procedure was as follows: 5 × 105 unfixed cells were centrifuged, resuspended in 1 ml medium supplemented with 25 mM HEPES and containing 5 µg/ml Hoechst 33342 and 5 µM NAO or 10 µg/ml Rh123. After an incubation interval of 10 min at 37°C, cells were spun down, washed with ice-cold medium, and postincubated with 5 µg/ml Hoechst 33342 in DMEM (10 min at 37°C). Immediately after staining, cells were held on ice until flow analysis to minimize stain efflux. To eliminate the time in dissociation enzyme cocktail as a factor influencing mitochondrial staining intensity, we incubated exponential phase and confluent Rat1-T1 and MR1 monolayer cells with the corresponding dissociation "cocktails" for 10-70 min at 37°C. For both cell lines, there was no significant alteration in cell viability, plating efficiency, cell volume, or staining intensity with either NAO or Rh123 at any of these time points. Given that the actual spheroid dissociations resulted in exposure to dissociation cocktails for no more than 30-60 min, this staining procedure reflects the intrinsic mitochondrial uptake parameters for the cells at the time they were isolated from the spheroid.

Flow cytometric measurement and analysis. Measurements were carried out with a multilaser-multiparameter flow cytometer with an integrated electronic orifice for direct cell volume measurement (35). Excitation wavelengths were 488 and 333-336 nm. Cell volume, forward light scatter, DNA distribution (via Hoechst 33342 dye), mitochondrial fluorescence (via mitochondrial dyes NAO or Rh123), and the ratio of mitochondrial fluorescence to cell volume were electronically detected as independent parameters and were analyzed via a Sun workstation, using the IDLYK program developed by the National Flow Cytometry Resource. Cell cycle distributions were calculated from the DNA histograms, using the MultiCycle AV program (Phoenix Flow Systems, San Diego, CA). A background correction was used to subtract debris from the DNA histograms before deconvolution. A population of 2.5 × 104 cells was examined for each sample. Remeasurement of the first sample after completion of each experimental series showed that there was no significant change in any parameter during the holding period on ice.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Three-dimensional growth and histological structure of the tumorigenic fibroblasts Rat1-T1 and MR1 were characterized in detail in previous studies (20, 23). Both cell lines show rapid spheroid growth while exhibiting an extremely different spheroid structure. For large spheroid sizes (>1,200 µm), central necrosis with dense pyknotic nuclei can be identified within both spheroid types, with a viable cell rim measuring some 210 µm in Rat1-T1 and 304 µm in MR1 aggregates (for schematic illustration, see Fig. 1A).


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Fig. 1.   Schematic illustration of morphological (A, top) and metabolic (A, bottom) characteristics and representative location of cell fractions (B; 1-6) dissociated from Rat1-T1 (left) and MR1 spheroids (right) with a diameter of ~1,300 µm. Shaded area, extent of central necrosis (N); C, spheroid center; VR, viable cell rim.

An automated dissociation procedure was used to selectively isolate cells from different regions within the viable cell rim of these spheroids (Fig. 1B). Two experimental designs each were performed, resulting in six or eight cell aliquots for Rat1-T1 and five or six samples for MR1 spheroids. Figure 2, A and B, documents the proportion of cells remaining in the spheroids as a function of the exposure time in the dissociation chamber. The average spheroid diameters, in two experiments, were 1,266 and 1,403 µm for Rat1-T1 and 1,352 and 1,415 µm for MR1 cultures, with standard deviations of <10%. A linear correlation (r > 0.995) between the percentage of cells remaining and the time in trypsin was observed for both spheroid types and in each experiment. From these data, the location of cells within the viable cell rim of Rat1-T1 and MR1 spheroids was calculated with the assumption of a homogeneous distribution of cells per spheroid volume (Fig. 2, C and D). For these estimations, the mean thicknesses of viable cell rim of 210 and 304 µm for Rat1-T1 and MR1 spheroids, respectively, were taken into account (23).


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Fig. 2.   Automated selective dissociation of a population of 20-30 Rat1-T1 (A and C) and MR1 (B and D) spheroids. Fraction of total spheroid cells remaining (A and B) and corresponding locations within viable cell rim of spheroids (C and D) are shown as a function of time of exposure to dissociation cocktail. Symbols (dotted lines and open symbols vs. solid lines and solid symbols) represent means from 2 separate experiments. bullet , Rat1-T1 spheroids with a diameter of 1,266 ± 80 µm; open circle , Rat1-T1 spheroids with a diameter of 1,403 ± 89 µm; black-triangle, MR1 spheroids with a diameter of 1,352 ± 90 µm; triangle , MR1 spheroids with a diameter of 1,415 ± 104 µm. Lines show linear (A and B; r > 0.995) or second-order polynomial (C and D; r > 0.998) fits to data points.

Figure 3 illustrates flow cytometric histograms of the following parameters: cell volume, forward light scatter, Hoechst 33342 fluorescence, mitochondrial fluorescence (NAO, left; Rh123, right), and the ratio of mitochondrial fluorescence to cell volume for cells in the outermost and innermost cell fractions. It was shown earlier that cell volume decreases from outer to inner spheroid layers for most cell lines, including Rat1-T1 cells, whereas it is relatively constant within MR1 spheroids (22). Because these changes in cell size will influence the fluorescence intensities of viable cells, we have focused on the ratios of NAO to cell volume and Rh123 to cell volume. Cell volume-corrected NAO fluorescence was slightly higher in cells isolated from the inner region of the viable cell rim (Fig. 3, bottom right), whereas Rh123 intensity per unit cell volume was reduced as a function of depth within Rat1-T1 spheroids (Fig. 3, bottom left). These results were confirmed by several independent experiments and were also true for MR1 aggregates, as documented in Fig. 4, which shows mean volume-corrected NAO or Rh123 fluorescence intensities relative to the corresponding cells in exponential monolayer culture. NAO fluorescence per unit cell volume was significantly higher in confluent compared with exponential monolayer cells, whereas Rh123 was reduced in plateau phase Rat1-T1 and MR1 cells. In general, mitochondrial mass per unit cell volume slightly increased as a function of depth within spheroids, whereas there was a systematic decline in volume-corrected mitochondrial activity by 30-40% for both spheroid types (r > 0.885; P < 0.05).


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Fig. 3.   Flow cytometric distributions of cell volume, forward light scatter, DNA fluorescence, mitochondrial fluorescence, and ratio of mitochondrial fluorescence to cell volume for cells isolated from superficial (solid lines) and innermost viable region (dots) within MR1 spheroids. Left: data from cell population tested for rhodamine 123 (Rh123) fluorescence. Right: data from cell population tested for 10-N-nonyl acridine orange (NAO) fluorescence. Both NAO and Rh123 staining are shown for clarity, although these were actually determined on separate cell samples. rel, Relative.


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Fig. 4.   Cell volume-related NAO (A and B) and Rh123 (C and D) fluorescence intensity of selectively isolated spheroid cells relative to exponentially growing monolayer cells as a function of location within viable cell rim. Symbols (dotted lines and open symbols vs. solid lines and solid symbols) represent means from 2 separate experiments. bullet , Rat1-T1 spheroids with a diameter of 1,266 ± 80 µm; open circle , Rat1-T1 spheroids with a diameter of 1,403 ± 89 µm; black-triangle, MR1 spheroids with a diameter of 1,352 ± 90 µm; triangle , MR1 spheroids with a diameter of 1,415 ± 104 µm. Lines show linear least-squares best fits to data (r > 0.870 and P < 0.05, except for NAO triangle ). Data is also shown for exponentially growing monolayer cells (E) and confluent monolayer cells (P).

Cell cycle distributions were calculated from the Hoechst 33342 DNA histograms, excluding acellular debris that was increasingly accumulated in the inner spheroid regions (e.g., Fig. 3). The number of S, G1/0, and G2/M phase cells are presented in Fig. 5 as a function of depth within Rat1-T1 and MR1 spheroids. A negative linear correlation (r = 0.947, P < 0.05) was observed for S phase cells, with values in the ranges of 11-22% in Rat1-T1 and 17-31% in MR1 spheroids. Compared with exponentially growing monolayer cells, the growth fraction was already reduced in the outermost spheroid layer by some 25% (MR1) and 50% (Rat1-T1), whereas the innermost spheroid cells had growth fractions similar to those in the corresponding unfed confluent monolayers. The number of cells in the G1/0 phase significantly increased from outer to inner viable cell rim (r = 0.953), reaching 85 and 75% in Rat1-T1 and MR1 spheroids, respectively. Changes in the number of cells in the G2/M phase were not consistent in the present study: although G2/M slightly decreased from the superficial toward the central spheroid area in one set of experiments (P < 0.05), it was stable in the other one for both spheroid types (P > 0.05). Additional dissociation experiments and flow cytometric DNA analyses on fixed cells, using mithramycin as DNA dye (data not shown), consistently found that the proportion of cells in G2/M phase is independent of the location within the spheroid. The slight but significant decline shown in some of the data sets presented here actually represent G2/M phase fractions decreasing from 8 to 4%, which is probably not biologically relevant. In general, the proportion of cells in the G2/M phase ranged between 4 and 10% in all spheroid regions.


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Fig. 5.   Proportion of cells in S (A and B), G1/0 (C and D), and G2/M phases (E and F) as a function of location within viable cell rim of Rat1-T1 (A, C, and E) and MR1 (B, D, and F) spheroids with a diameter of 1,250-1,450 µm. Symbols (dotted lines and open symbols vs. solid lines and solid symbols) represent means from 2 separate experiments. bullet , Rat1-T1 spheroids with a diameter of 1,266 ± 80 µm; open circle , Rat1-T1 spheroids with a diameter of 1,403 ± 89 µm; black-triangle, MR1 spheroids with a diameter of 1,352 ± 90 µm; triangle , MR1 spheroids with a diameter of 1,415 ± 104 µm. Lines show linear least-squares best fits to data (r > 0.947 and P < 0.05, except for G2/M phase data).

To analyze modifications in mitochondrial function associated with changes in the proliferative status of the cells, Fig. 6 shows the ratio of the volume-corrected mitochondrial fluorescence values (Rh123/NAO) as a function of the number of cells in S phase. This ratio represents mitochondrial activity per unit cell volume divided by mitochondrial mass per unit cell volume, giving an estimate of volume-corrected mitochondrial function. As indicated by linear regression analysis (r = 0.876), mitochondrial function systematically decreased with reduced number of S phase cells (P < 0.05) for both tumorigenic fibroblast spheroid types investigated. Finally, Fig. 7 shows that the NAO fluorescence per cell was relatively constant across the spheroid rim, even though the cell volume was decreasing.


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Fig. 6.   Mitochondrial activity (ratio of Rh123 to NAO volume-corrected fluorescence) of selectively isolated spheroid cells as a function of number of cells in S phase in Rat1-T1 and MR1 spheroids. Symbols represent means from 2 separate experiments. bullet , Rat1-T1 spheroids with a diameter of 1,266 ± 80 µm; open circle , Rat1-T1 spheroids with a diameter of 1,403 ± 89 µm; black-triangle, MR1 spheroids with a diameter of 1,352 ± 90 µm; triangle , MR1 spheroids with a diameter of 1,415 ± 104 µm. Line shows linear least-squares best fit to data (r = 0.876).


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Fig. 7.   Cellular NAO fluorescence intensity of selectively isolated spheroid cells relative to exponentially growing monolayer cells as a function of location within viable cell rim of Rat1-T1 (A) and MR1 (B) spheroids. Symbols (dotted lines and open symbols vs. solid lines and solid symbols) represent means from 2 separate experiments. bullet , Rat1-T1 spheroids with a diameter of 1,266 ± 80 µm; open circle , Rat1-T1 spheroids with a diameter of 1,403 ± 89 µm; black-triangle, MR1 spheroids with a diameter of 1,352 ± 90 µm; triangle , MR1 spheroids with a diameter of 1,415 ± 104 µm. Lines show linear least-squares best fits to data (0.714 < r < 0.780).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our data clearly show that incorporation of the mitochondrion-specific dye NAO per unit cell volume, which is independent of mitochondrial activity, increased as a function of depth within the spheroid. This was paralleled by enhanced cell quiescence and reduced number of cells in S phase. As documented in Fig. 7, NAO intensity per cell is rather uniform within the viable cell rim of these spheroids, with a slight but nonsignificant increase observed for MR1 cultures. These data confirm that changes in NAO per unit cell volume (Fig. 4) are primarily due to reductions in cell volume from the spheroid periphery to the inner edge of the viable rim. These data are consistent with the idea that, as the cells enter quiescence in spheroids, the cell volume shrinks but the mitochondria are retained. This result is in accordance with a previous report describing morphometric measurements performed on electron-microscopic photographs from median spheroid thin sections. On the basis of stereological considerations, Bredel-Geissler et al. (3) have estimated volume fractions of structures of interest with regard to viable spheroid "tissue." Although the spheroids investigated had a diameter of only 800 µm, they showed that the total cell volume fraction and the intracellular space decrease, whereas the mitochondrial fraction was constant from the surface toward the center of these EMT6 spheroids. From the fact that mitochondria are to be found in the intracellular area only, we conclude that mitochondrial density per cell stays relatively constant considering a homogeneous distribution of cells within the viable cell rim. As a result, the proportion of mitochondria per unit cell volume clearly increased with depth in the spheroid sections. Our results confirm that the volume density of mitochondria was homogeneously distributed within the viable rim of the spheroids investigated.

In contrast to the NAO distribution, staining of the cells with the fluorescent dye Rh123 demonstrated that mitochondrial activity per unit cell volume was much lower in those cells isolated from the inner viable cell rim than in cells isolated from the spheroid periphery. Because this decline in Rh123 accumulation was still evident after correcting for differences in cell volume and because the mitochondrial mass per unit volume did not change, these data together demonstrate that the respiratory activity of the mitochondria was downregulated. As a result, mitochondrial function described by the ratio of Rh123 to NAO fluorescence intensity is lowered from outer to inner viable spheroid layers. Comparison of this measure of mitochondrial activity with the cell cycle distribution data showed that quiescent cells in spheroids had a significantly lowered mitochondrial function, whereas quiescent cells in monolayer culture did not. The fact that changes in mitochondrial function and activity are retained even after spheroid dissociation strongly suggests a fundamental alteration in the mitochondria in quiescent cells in three-dimensional culture, rather than a transient response to altered nutrient supply. We assume that we have measured an upper estimate of the in situ mitochondrial function, since nutrient levels, pH, and/or some other microenvironmental factors may also reduce mitochondrial activity in situ but were eliminated due to dissociation before mitochondrial staining. One interpretation of these data is that the energy requirement of quiescent cells in spheroid culture is low and energy production might be downregulated to compensate both for decreased demand for ATP and for a lower nutrient supply in the inner spheroid region. The oxygen distribution is markedly different within Rat1-T1 and MR1 spheroids, yet similar alterations in mitochondrial function were observed, implying that induction of quiescence is more important to inherent mitochondrial function than is the oxygen supply. These results are in accordance with analogous investigations using two distinct spheroid types, EMT6 mouse mammary carcinoma and 9L rat gliosarcoma spheroids (Ref. 7 and unpublished observations), which are characterized by decreasing cellular oxygen uptake rates and reduced mitochondrial activities as a function of location within spheroids.

In contrast to the results of Bredel-Geissler et al. (3), our results confirm that respiratory activity decreases from outer to inner spheroid layers as shown by Freyer in 1994 (7). Also, a homogeneous spheroid-volume related or cellular mitochondrial distribution together with reduced mitochondrial function in the central viable cell layer does not explain uniform oxygen uptake per spheroid volume, in particular since extracellular space drastically increases. It is notable that reports of a constant respiratory activity per spheroid volume are only mathematical estimations based on oxygen tension gradients recorded stepwise on radial tracks toward the spheroid center (2, 14, 27, 30) and are not direct measurements of QO2. To obtain spheroid volume-related oxygen consumption rates within the viable cell rim of the spheroids, PO2 profiles were evaluated by nonlinear regression analysis and volume-related consumption rates as well as oxygen diffusion constants (Krogh's) were calculated. We agree that relatively exact mean values for these two parameters can be estimated by this procedure. However, many of the assumptions necessary for these calculations (e.g., a constant oxygen diffusion within the viable cell rim) are not confirmed, thus making it difficult to identify changes in respiratory activity along the oxygen gradient.

It has to be emphasized at this point that previous studies have shown a correlation between oxygen distribution and proliferative activity within the two different tumorigenic rat fibroblast spheroid types investigated. However, although the drop in PO2 to 0 mmHg was accompanied by centrally located necrotic cell death in Rat1-T1 spheroids, the emergence of necrosis occurs much later in three-dimensional MR1 cultures. With the latter, low PO2 (<1 mmHg) is paralleled by cell quiescence (24). The proliferative status within these Rat1-T1 and MR1 spheroids was determined via autoradiographic DNA labeling ([3H]thymidine; see Refs. 20 and 23) and using diverse DNA-staining fluorochromes combined with flow cytometric detection. A clear proliferation gradient was described for both spheroid types. However, the thymidine labeling index after pulse labeling was ~1% in the innermost viable region of Rat1-T1 and MR1 spheroids, whereas estimation from the DNA histograms showed 10-20% of the cells in S phase. We assume that either these cells were arrested in S phase or there was a drastic increase in the cell cycle time. To investigate this phenomenon in detail, studies need to be carried out using a bromodeoxyuridine-Hoechst 33342 fluorescence interaction technique (4).

For the two spheroid types investigated in this study, mean cellular oxygen consumption rates within spheroids were estimated from oxygen tension distributions indicating that respiration is two to three times higher in MR1 than in Rat1-T1 cells (20). Because mitochondrial fluorescence was monitored on an optimum scale for each cell line with respect to exponentially growing monolayer cells, our data do not allow for quantitative comparison of different cell lines. Therefore, correlation of cell line-specific oxygen consumption rates with mitochondrial function are not possible. Freyer (Ref. 7 and unpublished data) has reported that mitochondrial activity measured by a similar flow cytometric technique is correlated with independent measurement of QO2 for cells from spheroids and monolayers. Corresponding studies are needed to answer the question of whether mitochondrial function mirrors oxygen consumption within different cell lines.

For quiescent cells isolated from the innermost regions of spheroids of several cell types, a lag period of >20 h was described (8) for cells regrown in monolayer culture after separation. This delay in cell division, which was not recorded with cells originating from the superficial spheroid layer, is two to three times as long as the time required for one normal cell cycle transit. Our data suggest that reduced mitochondrial function and oxygen consumption, respectively, might be associated with limited cellular ATP production. As a consequence, entry into the cell cycle with the concomitant increase in energy production would require either recovery of function in existing mitochondria or mitochondrial replication. The fact that cellular mitochondrial distribution is relatively constant for cells from spheroids and for exponentially growing or confluent monolayer cells supports the first concept. However, further investigation is necessary to clarify this substantial pathophysiological aspect influencing tumor cell recovery from quiescence.

    ACKNOWLEDGEMENTS

We wish to acknowledge the technical assistance of Nicole Ballew.

    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft Grants Ku 917/1-1 and Ku 917/1-2 and National Cancer Institute Grant CA-51150.

Address for reprint requests: L. Kunz-Schughart, Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany.

Received 2 October 1996; accepted in final form 7 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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10.   Freyer, J. P., P. L. Schor, K. A. Jarrett, M. Neeman, and L. O. Sillerud. Cellular energetics measured by phosphorous NMR spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids. Cancer Res. 51: 3831-3837, 1991[Abstract].

11.   Freyer, J. P., and R. M. Sutherland. Selective dissociation and characterization of cells from different regions of multicell tumor spheroids. Cancer Res. 40: 3956-3965, 1980[Abstract].

12.   Freyer, J. P., and R. M. Sutherland. A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth. J. Cell. Physiol. 124: 516-524, 1985[Medline].

13.   Freyer, J. P., and R. M. Sutherland. Regulation of growth saturation and development of necrosis in EMT6/Ro multicell spheroids by the glucose and oxygen supply. Cancer Res. 46: 3504-3512, 1986[Abstract].

14.   Groebe, K., and W. Mueller-Klieser. Distributions of oxygen, nutrient, and metabolic waste concentrations in multicellular spheroids and their dependence on spheroid parameters. Eur. Biophys. J. 19: 169-181, 1991[Medline].

15.   Jaroszewski, J. W., O. Kaplan, and J. S. Cohen. Action of gossypol and rhodamine 123 on wild type and multidrug-resistant MCF-7 human breast cancer cells: 31P nuclear magnetic resonance and toxicity studies. Cancer Res. 50: 6936-6943, 1990[Abstract].

16.   Johnson, L. V., M. L. Walsh, B. J. Bokus, and L. B. Chen. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J. Cell Biol. 83: 526-535, 1981.

17.   Johnson, L. V., M. L. Walsh, and L. B. Chen. Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. USA 77: 990-994, 1980[Abstract].

18.   Kallinowski, F., G. Tyler, W. Mueller-Klieser, and P. Vaupel. Growth-related changes of oxygen consumption rates of tumor cells grown in vitro and in vivo. J. Cell. Physiol. 138: 183-191, 1989[Medline].

19.   Kessel, D., W. T. Beck, D. Kukuruga, and V. Schulz. Characterization of multidrug resistance by fluorescent dyes. Cancer Res. 51: 4665-4670, 1991[Abstract].

20.   Kunz, L. A., K. Groebe, and W. Mueller-Klieser. Oncogene-associated growth behavior and oxygenation of multicellular spheroids from rat embryo fibroblasts. Adv. Exp. Med. Biol. 345: 359-366, 1994[Medline].

21.   Kunz, L. A., and W. Mueller-Klieser. Influences of recombinant human tumor necrosis factor-alpha on growth and oxygen consumption of MCF-7 and HT-29 cells. Cell. Physiol. Biochem. 1: 214-225, 1991.

22.   Kunz-Schughart, L. A., and J. P. Freyer. Adaptation of an automated selective dissociation procedure to multicellular spheroids of oncogene-transformed fibroblasts. In Vitro Cell. Dev. Biol. Anim. 33: 73-76, 1997[Medline].

23.   Kunz-Schughart, L. A., K. Groebe, and W. Mueller-Klieser. Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation. Int. J. Cancer 66: 578-586, 1996[Medline].

24.   Kunz-Schughart, L. A., A. Simm, and W. Mueller-Klieser. Oncogene-associated transformation of early passage rodent fibroblasts is accompanied by large morphologic and metabolic alterations. Oncology Rep. 2: 651-661, 1995.

25.   Maftah, A., J. M. Petit, and R. Julien. Specific interactions of the new fluorescent dye 10-N-nonyl acridine orange with inner mitochondrial membrane. FEBS Lett. 260: 236-240, 1990[Medline].

26.   Marx, E., W. Mueller-Klieser, and P. Vaupel. Lactate-induced inhibition of tumor cell proliferation. Int. J. Radiat. Oncol. Biol. Phys. 14: 947-955, 1988[Medline].

27.   Mueller-Klieser, W. Microelectrode measurements of oxygen tension distributions in multicellular spheroids cultured in spinner flasks. In: Spheroids in Cancer Research: Methods and Perspectives, edited by H. Acker, J. Carlsson, R. Durand, and R. M. Sutherland. New York: Springer-Verlag, 1984, p. 24-49.

28.   Mueller-Klieser, W. Multicellular spheroids. A review on cellular aggregates in cancer research. J. Cancer Res. Clin. Oncol. 113: 101-122, 1987[Medline].

29.   Mueller-Klieser, W., B. Bourrat, H. Gabbert, and R. M. Sutherland. Changes in O2 consumption of multicellular spheroids during development of necrosis. Adv. Exp. Med. Biol. 191: 775-784, 1985[Medline].

30.   Mueller-Klieser, W., J. P. Freyer, and R. M. Sutherland. Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids. Br. J. Cancer 53: 345-353, 1986[Medline].

31.   Myc, A., P. DeAngelis, M. Kimmel, M. R. Melamed, and Z. Darzynkiewicz. Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle. Exp. Cell Res. 192: 198-202, 1991[Medline].

32.   Pagliacci, M. C., F. Spinozzi, G. Migliorati, G. Fumi, M. Smacchia, F. Grignani, C. Riccardi, and I. Nicoletti. Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival. Eur. J. Cancer 29: 1573-1577, 1993.

33.   Ratineau, M. H., P. Leprat, and R. Julien. In situ flow cytometric analysis of nonyl acridine orange-stained mitochondria from splenocytes. Cytometry 9: 206-212, 1988[Medline].

34.   Shinomiya, N., S. Tsuru, Y. Katsura, I. Sekiguchi, M. Suzuki, and K. Nomoto. Increased mitochondrial uptake of rhodamine 123 by CDDP treatment. Exp. Cell Res. 198: 159-163, 1992[Medline].

35.   Steinkamp, J. A., R. C. Habbersett, and R. D. Hiebert. Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles. Rev. Sci. Instrum. 62: 2751-2764, 1991.

36.   Sutherland, R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240: 177-184, 1988[Medline].

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38.   Walenta, S., A. Bredel, U. Karbach, L. Kunz, L. Vollrath, and W. Mueller-Klieser. Interrelationship among morphology, metabolism, and proliferation of tumor cells in monolayer and spheroid culture. Adv. Exp. Med. Biol. 248: 847-853, 1989[Medline].

39.   Walenta, S., J. Doetsch, B. Bourrat-Floeck, and W. Mueller-Klieser. Size-dependent oxygenation and energy status in multicellular tumor spheroids. Adv. Exp. Med. Biol. 277: 889-893, 1990[Medline].


AJP Cell Physiol 273(5):C1487-C1495
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