1 Institute of Pathology, University of Regensburg, 93042 Regensburg, and 2 Institute of Physiology and Pathophysiology, University of Mainz, 55128 Mainz, Germany
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ABSTRACT |
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Oxygen consumption, glucose, lactate, and
ATP concentrations, as well as glucose and lactate turnover rates, have
been studied in a three-dimensional carcinogenesis model of differently
transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and
myc-transfected M1, and the ras-transfected,
tumorigenic descendants Rat1-T1 and MR1) to determine metabolic
alterations that accompany tumorigenic conversion. Various
bioluminescence techniques, thymidine labeling, measurement of
PO2 distributions with
microelectrodes, and determination of cellular oxygen uptake rates
(cO2)
have been applied. In the ras-transfected, tumorigenic spheroid
types, the size dependencies of some of the measured parameters
exhibited sharp breaks at diameters of ~830 µm for Rat1-T1 and
~970 µm for MR1 spheroids, respectively, suggesting that some
fundamental change in cell metabolism occurred at these characteristic
diameters (denoted as "metabolic switch").
cO2
decreased and lactate concentration increased as functions of size
below the characteristic diameters. Concomitantly, glucose and lactate
turnover rates decreased in MR1 spheroids and increased in
Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting
cell quiescence and lactate accumulation) showed an enhancement of
cO2
with size. Systematic variations in the ATP and glucose levels in the
viable cell rim were observed for Rat1-T1 spheroids only. Proliferative
activity,
cO2,
and ATP levels in small, nontumorigenic Rat1 and M1
aggregates did not differ systematically from those recorded in the
largest spheroids of the corresponding ras transfectants.
Unexpectedly, respiratory activity was present not only in viable but
also in the morphologically disintegrated core regions of M1
aggregates. Our data suggest that myc but not ras
transfection exerts major impacts on cell metabolism. Moreover, some
kind of switch has been detected that triggers profound readjustment of
tumor cell metabolism when proliferative activity begins to
stagnate, and that is likely to initiate some other, yet
unidentified energy-consuming process.
tumor cell metabolism; metabolic readjustment; carcinogenesis model; ras/myc transfection; oxygen-sensitive microelectrodes; bioluminescence; glucose; lactate; adenosine 5'-triphosphate; spheroid culture; three-dimensional culture
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INTRODUCTION |
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WITHIN THE PAST FIFTEEN YEARS, research on oncogenesis has become one of the most active areas of biological investigation. To date, the majority of this work has been molecular in focus, and little has been done to discover and understand pathophysiological variations of tumor vs. normal cell phenotypes that are relevant for the design of nonsurgical therapeutic treatments such as radio- and chemotherapy. Studies on tumor cell energetics, for example, have been a field of controversial discussion since Warburg et al. (45) hypothesized respiratory deficiency and elevated nonaerobic glycolysis to be involved in carcinogenesis. Today, it is generally accepted that many but not all cancers are characterized by a highly deviated metabolism, and some modifications have been considered as diagnostic factors. However, it remains unclear whether these energetic alterations are primarily and directly due to the extent of transformation or rather accompany enhanced proliferative activity of these cells.
The biological significance and clinical relevance of tumor spheroids have been well documented in the literature and have been summarized in several comprehensive reviews (e.g., Refs. 23, 30, 31, 40). It is well accepted that spheroids mimic the specific environment of malignant cells in solid tumors more closely than conventional monolayers. This includes cell-cell and cell-matrix interactions, metabolic gradients, cellular viability, and differentiation. Mechanisms involved in gene regulation, such as cell cycle control or DNA repair, have been demonstrated to be very similar to those found in vivo and often different from those observed in adherent monolayers. From these findings it can be concluded that spheroids represent an appropriate model system for the registration of metabolic effects that are associated with malignant transformation. Multicellular spheroids have therefore been widely applied as an in vitro system to investigate the association of specific microenvironmental factors with success or failure of tumor therapy. Previous studies using a variety of tumor spheroids have shown that the oxygen uptake of tumor cells at advanced growth stages is considerably lower than that of rapidly dividing cells despite optimal oxygen availability (e.g., Refs. 8, 9, 15, 32). This indicates that oxygen consumption in three-dimensional (3-D) culture correlates with the proliferative activity supporting the hypothesis that oxygen metabolism rather reflects tumor cell growth characteristics than transformation state. However, tumorigenic spheroid types have never been directly compared with their nontumorigenic ancestors, and conclusions on metabolic changes associated with either aggressive tumor growth and/or tumorigenic conversion of spheroid cells are rather ambiguous.
In an attempt to examine cellular metabolism of cells transformed to
different extents in 3-D culture, we have previously established and
characterized a myc/ras-dependent two-step carcinogenesis spheroid system (21). This system is based on Weinberg's generalized model for tumorigenic conversion of rodent cells, with the first step
being immortalization, conferred by the activity of a nuclear oncoprotein, and with the second step being transformation, conferred by the activation of a cytoplasmic oncoprotein (25, 47). Cell types
involved in this study were spontaneously immortalized Rat1 and
myc-transfected M1 cells that are non- or poorly tumorigenic in
vivo and highly aggressive ras-transformed Rat1-T1 and MR1 cells. The nontumorigenic cell types show poor 3-D aggregation and
proliferation, whereas ras transfectants are characterized by
anchorage independence, growth in semisolid media, spheroid formation
in vitro, and rapid spheroid growth. In addition, spheroid types
extremely differ in cell morphology, viability, and proliferative activity (21). For example, Rat1 cultures consist of viable cells only,
whereas M1 aggregates are typically characterized by centrally located
cell debris (Fig. 1). Also,
the viable cell rim is ~100 µm thicker in MR1 spheroids than in
Rat1-T1 spheroids (304 ± 24 vs. 204 ± 28 µm), and necrosis
develops at a larger size (800-900 µm in MR1 spheroids vs.
500-600 µm in Rat1-T1 spheroids) (21).
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The objective of the present study was to clarify whether ras transfection and tumorigenic conversion of fibroblasts directly leads to systematic changes in metabolic milieu and cell metabolism, as indicated in monolayer culture and tumor xenografts (13, 14, 20, 24). One particular aim was to analyze average cellular oxygen consumption rates as well as glucose, lactate, and ATP levels within the viable region of individual spheroids of the immortalized fibroblast cell lines Rat1 and M1 and of the highly tumorigenic ras-transformed fibroblast clones Rat1-T1 and MR1 relative to their growth/proliferation pattern and to their tumorigenic state.
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MATERIALS AND METHODS |
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Cell Lines
Four differently transformed, diploid fibroblast cell lines derived from primary/secondary Fisher 344 rat embryo fibroblasts (REF) were used for the present investigation (24). Spontaneously immortalized (Rat1) and c-myc-transfected (M1) REF represent early, nontumorigenic stages in the transformation process and are characterized by poor cellular aggregation and aggregate growth in vitro (maximum diameters 150-300 µm) (21). Transfection of Rat1 cells with a point-mutated Ha-ras oncogene and c-myc/T24Ha-ras cotransfection of REF resulted in the development of the two highly tumorigenic and aggressively growing cell clones Rat1-T1 and MR1, respectively. Both ras transfectants show rapid spheroid formation with high proliferation rates (21) and maximum spheroid diameters of >1,200 µm (Fig. 1). Transfections were carried out via calcium phosphate precipitation with plasmids pHO6T1 containing an Ha-ras oncogene isolated from a human bladder carcinoma and pSVc-myc1 detected first in a mouse plasmacytoma. Routine methods for cell preparation, oncogene transfection, and oncogene protein expression have been detailed previously (24).Spheroid Culturing
Aggregates were cultured under identical physiological conditions using a spinner flask technique. DMEM (Sigma Chemical) supplemented with 25 mM glucose, 5% (vol/vol) FCS, 10,000 IU/l penicillin, and 10 mg/l streptomycin (Flow Laboratory/GIBCO) was used as culture medium. Cells were maintained in a humidified incubator equilibrated with 5% (vol/vol) CO2 in air at 37°C. Spheroids were initiated from trypsinized, exponentially growing monolayer cultures by inoculating (1-2) × 105 T24Ha-ras-transfected Rat1-T1 or MR1 cells and (1-2) × 106 Rat1 or M1 cells in 15 ml medium/100 mm nonadherent petri dish. After an initiation phase of 4 days, aggregates were transferred into a 1-liter-spinner flask containing 300 ml medium according to a procedure detailed earlier (21). All spinner cultures were grown at 75-100 rpm thereafter, and medium was replaced daily. The number of spheroids per flask was reduced gradually with increasing spheroid size to maintain a relatively constant cell count ofFor each spheroid investigated, the total spheroid volume was calculated from two orthogonal diameters that were quantified using an inverted microscope equipped with a calibrated reticule. Spheroid volume growth was routinely recorded and analyzed for a representative spheroid population.
Thymidine-Labeling Index and Cell Count
Thymidine labeling indexes (TLI) in spheroids were determined using conventional autoradiography (for details, see Refs. 17 and 21). Aggregates were pulse labeled at 37°C for 15 min in complete medium containing 4 µCi/ml [3H]thymidine (sp act 20 Ci/ml; New England Nuclear, Dreieich, Germany), fixed, histologically processed, and prepared for autoradiography using a standardized dipping technique and processing of the film (film emulsion K2; Ilford, Mobberley, UK). The TLI was determined on hematoxylin and eosin-stained central sections of the spheroids by microscopic observation as the proportion (%) of labeled nuclei to total nuclei count. Cells were considered labeled if the grain count per nucleus exceeded 10, with an average background ofIn addition to automated cell counting following spheroid dissociation, cell volumes of cells isolated from aliquots of spheroids with defined sizes have been analyzed using the Schaerfe System Casy-1 (Schaerfe System, Reutlingen, Germany) to allow for the calculation of cell volume-related oxygen consumption rates (21). Cell counts per spheroid were also calculated in histological sections from the individual values of the viable spheroid volume and the cell number per section surface according to Weibel and Gomez (46).
ATP, Glucose, and Lactate Concentration and Glucose and Lactate Turnover
Local metabolite concentrations within spheroids were assessed via metabolic imaging based on quantitative bioluminescence combined with single-photon imaging as described earlier (34, 35). ATP and glucose concentrations were determined in 5-µm thick cryostat sections through the center of rapidly frozen spheroids. Each frozen cryostat section was covered with a specific enzyme cocktail. Enzyme reactions linked the substrate of interest to the luminescence of different luciferases. For ATP detection, luciferase from firefly lantern was used, whereas glucose and lactate were identified via luciferase from marine bacteria (for details see Ref. 34). Bioluminescence intensities were registered using an appropriate microscope (Axiophot; Zeiss, Oberkochen, Germany) and an automated imaging photon counting system (ARGUS 100; Hamamatsu, Herrsching, Germany). Calibration was carried out as detailed previously by assigning photon counts to luminescence of defined tissue homogenate standards. Metabolite concentrations of these standards were quantified either by HPLC or by standard enzymatic assays (32). Substrate concentrations were obtained in micromoles per gram with respect to tissue net weight.For the determination of glucose and lactate turnover rates, 102 to 104 spheroids with a defined size were transferred into 100-ml spinner flasks and were incubated in 50 ml of supplemented medium under culture conditions. After 10 min and every 60 min thereafter over a period of 6 h, two independent samples of 100 µl of medium were taken from the spinner flasks containing Rat1-T1 or MR1 cultures. For Rat1 and M1 aggregates, 100-µl medium aliquots were collected every 12 h over a period of 3 days due to poor glucose consumption and lactate release. Aliquots were immediately mixed with 1 ml and 0.5 ml ice-cold 0.6 M perchloric acid for glucose and lactate measurements, respectively. Samples were pelleted after 15-20 min, and metabolite concentrations were analyzed using routine test kits (test combination Glucoquant and Monotest lactate; Boehringer Mannheim), utilizing the metabolites for the production of NADH and NADPH to be measured photometrically at 365 nm. Mean turnover rates were calculated from the increase and/or decrease in metabolite concentration per time interval.
Oxygen Consumption
Oxygen consumption rates (Statistical Analysis
Group differences were evaluated using a two-tailed t-test for unpaired observations. Correlations between two parameters were obtained via linear regression analysis. The dependence of some of the assessed parameters (i.e., thymidine labeling index, O2 consumption rate, and lactate concentration; see Joint Biphasic Diameter Dependence of TLI, ![]() |
RESULTS |
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Spheroid and Cell Characteristics
In addition to the characteristic differences between the four cell lines mentioned in the Introduction, cell count in small M1 aggregates is four to six times less than in Rat1, Rat1-T1, and MR1 spheroids of the same size (Fig. 2A), which is partly due to centrally located cell destruction. Within Rat1-T1 and MR1 cultures, cell count per spheroid increased with spheroid size being significantly higher in Rat1-T1 vs. MR1 spheroids of equal diameter (21). Concomitantly, the cell volume measured 734 ± 109 µm3 in Rat1-T1 spheroids and was consistently less (P < 0.001) than in MR1 spheroids (931 ± 113 µm3), as documented in Fig. 2B. In neither case, cell volume changed significantly (P > 0.05) as a function of spheroid diameter. Cell volume of Rat1 cells isolated from aggregates was comparable with that of Rat1-T1 cells, and M1 cells were similar to MR1 spheroid cells; hence cell volume in 3-D cultures was not altered by ras transfection.
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Figure 2C presents the thicknesses of the viable cell rim in Rat1-T1 and MR1 spheroids as a function of spheroid size, showing a slight decrease within Rat1-T1 spheroids (R = 0.413; P < 0.01) but no change with diameter in MR1 3-D cultures. M1 aggregates with central cell death were characterized by a thin, no more than 29 ± 7 µm thick outer layer of morphologically intact cells.
From cell counts as well as cell and viable rim volumes, the ratio of intra- to extracellular space was estimated. Resulting average extracellular space was 68 ± 8% in the viable regions of MR1 spheroids (n = 24) and 50 ± 14% in Rat1-T1 spheroids (n = 23). Unfortunately, the scatter in these data was too large to allow for any predictions about systematic changes during spheroid growth. Interestingly, cell density within the viable rims of M1 spheroids turns out to be two to three times less than in Rat1, Rat1-T1, and MR1 spheroids of the same size.
[3H]thymidine labeling within the four aggregate types had shown earlier (21) that 1) the proliferative activity is much smaller in Rat1 and M1 aggregates compared with the highly tumorigenic Rat1-T1 and MR1 fibroblast spheroids at diameters of 150-300 µm, and 2) with growth, volume-averaged TLI in the tumorigenic spheroid types drops from 30-40% to values <20% (P < 0.05), with MR1 spheroids exhibiting higher TLIs (and faster spheroid volume growth) than Rat1-T1 spheroids. The diameter dependence of the individual volume-averaged TLI values (%) is further analyzed in Fig. 2D. It demonstrates that thymidine labeling of Rat1-T1 and MR1 tumor spheroids continuously decreases with increasing spheroid size up to a characteristic diameter of ~830 µm for Rat1-T1 and some 970 µm for MR1 spheroids. For larger aggregate sizes, TLI remains essentially constant.
ATP, Glucose, and Lactate Levels and Glucose and Lactate Turnover
ATP concentration within the viable region of 3-D MR1 cultures remained constant (P > 0.05) as a function of spheroid size, whereas a significant (P < 0.001) decline in the ATP level was observed for Rat1-T1 spheroids (Fig. 3A). In Rat1 and M1 aggregates, ATP concentrations were lower by a factor of 2-3 compared with the fully transformed counterparts (P < 0.0001), even though the proportion of intracellular space was two to three times lower solely in M1 aggregates. However, Rat1-T1 spheroids with diameters of 1,000-1,300 µm, which exhibited a larger fraction of quiescent cells, had ATP concentrations in the viable cell rim almost as low as determined for the small aggregates of the corresponding precursor Rat1.
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In parallel with the ATP levels, glucose concentration was significantly decreasing in Rat1-T1 (P < 0.01) but not in MR1 spheroids as a function of spheroid size (Fig. 3B; glucose concentration in the medium was 25 mM). Yet, no biologically relevant lack in glucose could be observed in any of the fibroblast spheroid types investigated. The average glucose level ranged from 18 to 20 µmol/g.
In contrast to the relatively moderate changes in ATP and glucose
levels, lactate accumulated in the viable cell rim of both Rat1-T1 and
MR1 cultures during spheroid growth (P < 0.01), increasing from 2-4 µmol/g in small nonnecrotic spheroids to 7-9
µmol/g in the viable cell rim of spheroids with a diameter of 1,000
µm (Fig. 3C). Within the size range of ~200-300 µm,
lactate concentrations in Rat1 and M1 aggregates were 6-7 µmol/g
and tended to be higher than in the ras transfectants.
Other than the metabolite concentrations (that at least exhibited
similar tendencies), glucose and lactate turnover rates showed very
striking differences between Rat1-T1 and MR1 spheroids (Fig.
4). Although glucose uptake and lactate
release per cell both moderately increased with diameter in Rat1-T1
spheroids (P < 0.025 and P < 0.0025, respectively), these turnover rates in MR1 spheroids were found to
decrease some 50% up to a diameter of ~1,000 µm (P < 0.005 and P < 0.025, respectively) and to remain essentially
constant in larger aggregates. Absolute turnover rates in MR1 compared
with Rat1-T1 cells were about two to three times larger in small
aggregates and very close to each other in large ones. Due to the poor
aggregation characteristics and the small average spheroid volume,
glucose uptake and lactate release in Rat1 and M1 aggregates were
extremely hard to measure and were found to be in the lower range of
their corresponding tumorigenic descendants.
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Oxygen Diffusion and Consumption
Krogh's diffusion constants within aggregates are documented in Fig. 5A as functions of spheroid size. There was no significant correlation between oxygen diffusion coefficients and spheroid size (for details see Ref. 20).
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Oxygen consumption rates per viable spheroid volume
(svO2)
slightly but insignificantly decreased as a function of spheroid size
in MR1 spheroids, whereas
svO2
significantly dropped by some 50% (P < 0.01) in Rat1-T1
spheroids over a diameter range of 200 to ~800-900 µm (Fig.
5B). Rat1 and Rat1-T1 aggregates of comparable size did not
significantly differ in their volume-related oxygen uptake rates.
However, if aggregates with similar proliferation rates (Rat1
aggregates vs. Rat1-T1 spheroids with a diameter of
1,000 µm) were
compared, a significant (P < 0.001) decrease in the oxygen
consumption rate per viable volume was observed after ras
transfection, which is not due to limited oxygen availability as shown
earlier (21).
svO2
calculated in M1 aggregates was significantly lower than in their
highly tumorigenic counterpart MR1 (P < 0.001). However, M1
aggregates are special, in that cell density in their viable rim is
exceptionally low.
As mentioned above, M1 spheroids consist of an extraordinarily thin
viable cell rim surrounding a central region that is characterized by
morphological disintegration in routine paraffin histology (21) but
that does not represent the classical type of necrosis in spheroids.
Most surprisingly, the radial oxygen partial pressure profiles in M1
aggregates continually dropped from the surface of the aggregates up to
their centers and appeared to be absolutely smooth across the boundary
between viable rim and region of cell debris (Fig.
6). Similarly, ATP gradients extended
beyond the viable cell rim as well (data not shown). Mathematical
analysis of PO2 profiles consistently
indicated that oxygen was consumed within the entire volume of M1
aggregates, even in the central region. We conclude that the latter is
characterized by some unexpected respiratory activity, the rate of
which was not distinguishable by our regression analysis from
svO2 in
the viable rim. Ongoing energy metabolism as well as the unusual
morphological appearance suggest that the material found in M1 spheroid
centers is different from necrotic cells indeed. Rather, cell
destruction in M1 aggregates may result from programmed cell death,
which has been shown to be induced by myc transfection in
epithelial (e.g., 38) as well as in fibroblast (e.g., Refs. 2, 12, 37) cells. The apoptotic process produces membrane-bound cellular fragments
with intact, functional mitochondria, which may be metabolically active
despite the fact that the cells are "dead" and which therefore may explain the presence of respiration and ATP in regions without intact cells.
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Interestingly, MR1 spheroids show a phenomenon that, in a way, is complementary to the one just described for M1 spheroids. Next to the central necrosis in large MR1 aggregates there is an ~75-µm-thick layer of viable cells that is completely depleted of oxygen and exhibits zero oxygen consumption (21).
To eliminate the effects of differences in cell density on
volume-related oxygen uptake, Fig. 5C presents cellular oxygen consumption rates
(cO2)
that are based on individual
O2 values per spheroid and the cell count per spheroid estimated from the
formerly described relation between spheroid size and cell count (21).
In M1 aggregates, oxygen consumption of the viable cell rim had to be
used instead of
O2
in the entire spheroid because oxygen consumption was found not only in
the viable rim but also in the structurally disintegrated center.
In Rat1-T1 and MR1 spheroids
cO2
decreased up to a diameter of ~830 and ~970 µm, respectively, by
35-45% (P < 0.001). Further spheroid growth (>830 and
970 µm, respectively) was associated with enhanced cellular oxygen
uptake (P < 0.001). Comparison of partly vs. fully
transformed cell lines showed that
cO2 was not significantly different if spheroids within the same size range
were taken into account. However, due to the reduction in
cO2 as
a function of spheroid diameter in Rat1-T1 and MR1 spheroids (which was
paralleled by a decline in the TLI), cellular respiration in Rat1 and
M1 aggregates was significantly higher (P < 0.001) than that of the corresponding descendants at growth stages at which
comparably low TLI values are reached (spheroid diameter ~830 µm
for Rat1-T1 and ~970 µm for MR1 spheroids). This difference in
cO2
between immortalized ancestors and their ras-transfected descendants vanished more and more with further growth of the transfectants (occurring at constant TLI; see Fig. 2D), so the very large aggregates statistically differed in neither TLI nor (in the
case of M1/MR1 only)
cO2
from their nontumorigenic ancestors.
Differences between the correlations of
svO2
and
cO2
with spheroid size are mainly due to variations in the cell count per
unit of viable spheroid volume during spheroid growth and are more
prominent in the large diameter range and in MR1 spheroids. Because
cell volume of Rat1-T1 and MR1 cells in spheroids is not correlated
with spheroid diameter (Fig. 2B), it must be the ratio of
extracellular to intracellular space that changes as a function of
spheroid size. Histological observations qualitatively confirm this result.
Joint Biphasic Diameter Dependence of TLI,
O2,
and Lactate Levels
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DISCUSSION |
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The present, well-established cell system allows for systematically
studying the impacts of spontaneous immortalization vs. myc
transfection as well as additional ras transfections on
cell metabolism. As a third independent variable, the effects of 3-D growth in tumor spheroids are assessed. To facilitate understanding of
the complex interrelations between the four cell lines studied, Fig.
7 summarizes the results (along with
corresponding monolayer data from Ref. 24) as percent changes in
TLI, cell volume-related oxygen uptake
(cvO2),
lactate release (
cvLac), and
concentrations of ATP ([ATP]), glucose
([Glc]), and lactate ([Lac]) as function of
days in monolayer culture or of spheroid diameter, respectively.
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Metabolic Effects of Spheroid Growth Stage and Proliferative Activity: Biphasic Dependence of Metabolism on Diameter
It was demonstrated with extremely high statistical significance (P < 10The mechanisms that have been proposed to bring about the first phase
of dropping TLI in ras-transformed fibroblasts are numerous and
not well understood (19, 23, 26, 41, 43). The accompanying decrease in
O2
as well as lactate accumulation (which is similar to observations from
former studies) may be accounted for by changing energy demand with
falling cellular proliferative activity (combined with altered supply
conditions) and are discussed below. For the second phase as well as
for the nature of the metabolic switch, more complex explanations need
to be developed thereafter.
ATP concentration and proliferative activity.
ATP concentration (and, in correspondence, volume-related
svO2;
Fig. 5B) decreased ~50% during spheroid growth
in Rat1-T1 cultures, whereas it remained constant in
MR1 spheroids (Fig. 7, l and p). Thus the course of
[ATP] follows one of two patters described earlier.
1) The ATP distribution is independent of tumor growth activity
(33) and hence of spheroid size and position within the viable rim.
This behavior of MR1 aggregates was also seen in EMT6 spheroids (Ref. 7
and Kunz-Schughart LA, Walenta S, and Mueller-Klieser W, unpublished
observations). 2) On the contrary, data on
Rat1-T1, WiDr (human colon carcinoma), and different human
rhabdomyosarcoma spheroids show a decrease of ATP concentration in the
viable cell rim with spheroid size (unpublished observations). This may
result from a factual dependence of intracellular [ATP] on
proliferative activity (36) or may be an artifact caused by falling
cell packing density during growth. Alternatively, a fall in
[ATP] may be indicative of beginning energy depletion enforcing adaptive metabolic actions for further growth (see
Metabolism in large spheroids and metabolic
trigger).
Oxygen consumption and proliferative activity.
In Rat1-T1 and MR1 spheroids smaller than
dL,
svO2
decreased as a function of size (Fig. 7, k and o). This
result corresponds with previous investigations, describing
correlations of cellular respiration and proliferative activity during
spheroid growth (1, 4, 8, 9, 20, 29, 44) as well as across viable rims
of spheroids at given growth stages (6). In aggregates larger than
dL, cellular respiration increases with spheroid
diameter,2 which came
unexpectedly and is at variance with earlier studies (1, 8, 9, 29, 44).
Although the fall in oxygen consumption for smaller spheroids is
attributable to the reduced proliferative activity (Figs. 2D
and 7, k and o), the same rationale would suggest
cO2 to
stagnate as TLI remains fairly constant at ~15% (Fig. 2D).
Yet, this is not the case. Moreover, a strict dependence of
O2
on TLI is not supported by a regression analysis of
cO2 vs.
TLI (taken from Fig. 5C and calculated from the regressions given in Table 1, respectively). While there is a weak (R = 0.65) but statistically significant correlation in Rat1-T1
spheroids,
cO2 and
TLI are entirely unrelated in MR1 aggregates (R =
0.007, P > 0.95).
Glucose/lactate metabolism and proliferative activity.
TLI and hence energy demand both become less during spheroid growth
(Fig. 7, k and o). Therefore, not only
O2
but also glucose uptake rate should fall as well. With lengthening diffusion distances, diffusion limitation for lactate may occur (this
is somewhat different for
glucose).3 As a consequence,
one should expect lactate to accumulate, which, in turn, may obstruct
glucose turnover and energy production, may lower ATP levels, and may
ultimately arrest proliferative activity (Ref. 3 and see
below) and suppress cell viability. In the present study,
lactate concentration ranged to 6-9 mM in the viable cell rim of
Rat1-T1 and MR1 spheroids and to ~6 mM in Rat1 and M1 aggregates
(Figs. 3C and 7, l and p). Lactate accumulation of comparable magnitude is known to impede cellular proliferation and
energy metabolism [i.e., glycolysis (3, 11)] and to affect viable and proliferative rim thicknesses (3, 27). Despite similar
lactate concentrations, only MR1 spheroids conform to the above pattern
of attenuated glucose and lactate turnover with growth (Fig.
7o). In contrast, Rat1-T1 turnover rates increase (Fig.
7k), which may be interpreted as an adaptive mechanism as discussed below.
Metabolism in large spheroids and metabolic trigger.
In large Rat1-T1 (>830 µm) and MR1 aggregates (>970
µm), oxygen consumption increases with spheroid size (Fig. 7, k
and o). This may be interpreted as a response to blockade
of glycolysis by increased spheroidal lactate concentration (Figs.
3C and 7, l and p) that forces energy
metabolism to favor oxidative pathways (in combination with rising
energy demand for maintaining cellular integrity in an increasingly
acidotic environment). Raising the proportion of aerobic vs. anaerobic
energy production should, vice versa, attenuate further elevation of
lactate concentration. This hypothesis is supported by Ref. 3, showing
an increase in
O2
and even a net uptake of lactate with rising external lactate
concentration. On the other hand, this reasoning is not completely
satisfactory, since, below the limiting diameter,
O2
and lactate concentration are indirectly related, and, simultaneously
with the rise in
O2,
lactate concentration begins to stagnate rather than continue to
increase, as postulated above (slope statistically indistinguishable
from zero; Fig. 7, k, l, o, and p).
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Metabolic Effects of Myc and Ras Transfections
Metabolism and tumorigenic conversion.
In monolayers during early exponential growth and
confluence,4 cellular oxygen
consumption rates
(cO2)
were lower in Rat1-T1 and MR1 cells than in the
non-ras-transformed precursors (P < 0.001; Ref. 24;
Table 2). The values of
cvO2
become equally smaller by ras transfection (Fig. 7, a
and e vs. i and m). Hence there must be some
extra energy sink in Rat1 and M1 cells that is unrelated to
proliferation, suggesting that ras transfection per se corrupts
differentiation and synthesis, which, in turn, may explain why the
metabolic readjustment is fully effective even in Rat1 and M1
monolayers and small spheroids.
Metabolism and myc transfection.
Although by the current data major differences between Rat1 and M1
metabolisms cannot be identified, there are pronounced changes with
myc transfection in the tumorigenic descendants. Most
obviously, the anaerobic pathways of energy metabolism are more
prominent in MR1 compared with Rat1-T1 spheroids, e.g., for diameters
500 µm, (volume-related)
svO2
and
cvO2 in
MR1 aggregates are significantly smaller than in Rat1-T1, and glucose
and lactate turnover rates are larger (P < 0.001; Fig. 7,
k and o). Moreover, MR1 cells are able to survive
without any oxygen (which is true in a layer of ~75 µm thickness
next to the central necrosis in large MR1 aggregates), whereas Rat1-T1
oxygenated and viable cell rims are of equal thickness (21). There is
also excessive glucose uptake and lactate release in MR1 monolayer
cultures (Fig. 7, m vs. i). Therefore, lactate
concentration in MR1 supernatant grows much higher than in Rat1-T1,
which may, in part, explain why
O2
increases in confluent MR1 but not Rat1-T1 monolayers (24).
Methodological Considerations
Errors in ATP, glucose, and lactate levels determined by bioluminescence. There is a large scatter in most of the bioluminescence data shown in Fig. 3, which calls for some comments. 1) Most of the scatter is due to a biological/histomorphological variability among spheroids of one type, which is confirmed by the data underlying Figs. 2, C and D, and 5 that have been measured with distinct techniques. Variations in the bioluminescence data do not only result from variable intra- or extracellular concentrations but may also be due to modifications in the ratio of intracellular to extracellular space (ECS) which may vary not only between but also within individual spheroids of a given cell line. The latter type of error is of particular importance for measurements of (intracellular) ATP in spheroids with a high and variable proportion of ECS such as MR1 (~70%) and may explain the large scatter in the ATP data from MR1 as opposed to Rat1-T1 spheroids. 2) Although measurements have been performed in the center of the viable cell rim where concentration gradients are generally small, some degree of diffusion of both glucose and lactate may occur. This may result in an underestimation of the glucose and lactate levels. 3) Random inaccuracies in sectioning spheroids may add some further scatter, since the emitted light intensity changes with section thickness. Similarly, small variations may arise from scattering of the emitted light. 4) Any conclusions derived from the above bioluminescence data are, of course, valid despite their large scatter, because variations were accounted for in the statistical analysis. It is but a matter of speculation if any more advanced inferences could have been drawn, had the scatter been smaller.
Radial gradients in
svO2
and present methodology.
In the present study, average respiration rates in the viable
portions of spheroids were computed, hence changes in
svO2 with radial position are not quantified. This restraint was
deliberately implemented in the analysis of experimental
PO2 profiles, because the method's
sensitivity for determining spatially resolved
svO2
distributions is not satisfactory, given the limited precision of
currently available input data. Nevertheless, substantial radial gradients in
svO2
can be detected because, if present, they ensue pronounced systematic
deviations of the best approximations from the measured
PO2 profiles. No such deviations were observed, hence
svO2
most likely was rather homogeneous, although there is
experimental evidence suggesting a drop in mitochondrial function and
possibly also
O2
from outer to inner regions in Rat1-T1 and MR1 spheroids (22).
Cell volume and cO2 in
spheroid vs. monolayer culture.
Spheroid cell volume has been found to be independent of time in
culture, proliferation rate, and malignant transformation (not,
however, of myc transfection). In monolayers of each cell line, cell volume is directly related to TLI and decreases with time in
culture (which may be caused by changing intensity of contact
inhibition with number of cells per dish). If cell volume-TLI relations
of different cell lines are compared, there is an indirect relation,
i.e., larger cells are associated with lower TLI and vice versa. These
observations suggest that, in early monolayers (with plenty of space
available), there is little control of cell volume by contact
inhibition but rather by the demands of proliferation, whereas, at
confluence, the available space seems to be a major determinant.
Metabolic readjustment in spheroid vs. monolayer culture.
As already mentioned, the fall and, later on, rise of
cO2 in
growing M1 and Rat1 monolayer cultures parallels the courses of
cO2 in
MR1 and Rat1-T1 spheroids, suggesting that in both cases
similar metabolic readjustment occurs. It seems that the more malignant
the cell line, the more intense cell-cell interactions and/or hostile
metabolic milieu is necessary to trigger the metabolic switch. Although
in M1 and Rat1 cells the 2-D contact inhibition in monolayers suffices
to induce complete readjustment, this occurs for the
ras-transformed descendants only in 3-D culture and under rather adverse environmental conditions. In consequence, some cell line
specific characteristics of malignant cells obviously become expressed
only under very particular conditions that are present, e.g., during
volume growth in large spheroids and probably also in vivo (3-D contact
inhibition and/or detrimental metabolic milieu), but that are never
attained in monolayers.
Relevance for tumors in vivo. In the preceding subsections on cell volume and metabolism in spheroids vs. monolayers, it was shown that, under 3-D growth conditions (present in vivo and in multicellular spheroids), a number of important tumor cell properties are different from those found in 2-D culture. More generally, it is well known that spheroids (other than monolayers) closely mimic tumors in vivo in many biological and clinical aspects. Yet, it cannot necessarily be expected that findings from the present in vitro model are directly applicable to the metabolism of ras-transfected human tumors because, among other reasons, the latter are generally of epithelial origin, whereas the former are derived from fibroblast cells.
In conclusion, our investigations have shown that myc but not ras transfection per se seems to have a major impact on fibroblast metabolism. In the late growth stages, some striking metabolic readjustment was identified that comprised reversal of the observed fall in ![]() |
APPENDIX |
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Oxygen Diffusion and Consumption
PO2 distributions in and around multicellular tumor spheroids suspended in stirred media may be recorded by using oxygen-sensitive microelectrodes and are characterized by constant PO2 in the stirred medium and by a continuous PO2 decline in the unstirred layer surrounding the spheroid (oxygen-depleted zone) as well as in the spheroid itself. This type of PO2 distribution may be described mathematically by diffusion theory. PO2 profiles within spheroids are governed by spheroid geometry and by the ratio of volume-related oxygen consumption rate (Statistical Analysis of Biphasic Dependencies
As stated in Statistical Analysis, the dependence of some of the assessed parameters appeared to be biphasic, i.e., below and above a common limiting diameter two distinctly different linear regression lines seemed to be necessary for an optimal fit. On the other hand, whenever two independent linear least-squares fits were used for the ranges of small and large spheroid diameters, a discontinuity at the limiting diameter was almost certain to be present, which one would not expect to exist in the functions relating these parameters to spheroid diameter, neither from the biological background nor from obvious evidence. Therefore, in selecting a type of expression function that was better suited to fit the data, a polygon was chosen that consisted of two straight-line segments that intersected at the limiting diameter. Such a polygon is uniquely defined by the limiting diameter and three additional parameters specifying slopes and intercepts of the polygon segments. For the polygonal expression functions, the sums of squares of weighted deviations from the measured data were minimized by determining one optimal limiting diameter as well as three more parameters for each of the polygons approximating TLI,Evidently, different diameter dependencies of a number of parameters in small and large spheroids may be reflections of differences between biological properties in each of the two size ranges. However, for any physiological interpretation of this phenomenon to be feasible, it first needs to be shown that the described biphasic relations actually do exist and are more than merely subjective impressions. This is performed according to Motulsky and Ransnas (28) by statistically comparing the goodness of the approximation of the data by straight lines or by polygons using an F-test analysis. As approximations based on a larger number of variable parameters obviously tend to yield better results than fits based on fewer ones (no matter if they really furnish a better description of the actual biological dependence or not), the different numbers of fit parameters (6 for 3 straight-line fits in the first case and 9 for the 3 polygon fits plus 1 limiting diameter in the 2nd) and, accordingly, the degrees of freedom need to be considered to make a valid comparison.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Simm from the Institute of Physiological Chemistry II, University of Wuerzburg, Wuerzburg, Germany, for providing oncogene-transfected rat fibroblast cell lines and Dr. J. P. Freyer from the Los Alamos National Laboratory, Los Alamos, NM, for critically reviewing this manuscript.
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FOOTNOTES |
---|
This work was supported by Deutsche Forschungsgemeinschaft Grants Mu 576/2-4 and Mu 576/4-1.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1
Viable cell rim oxygen consumption
O2 × (viable volume)/(total spheroid volume) =
O2 × [1
(d
29 µm)3/d3] for a spheroid of
diameter d and viable rim thickness of 29 µm (cf.
Spheroid and Cell Characteristics).
2
It should be noted that, in large MR1
spheroids, overall mean cellular oxygen consumption was observed to
rise. This average has been taken over the whole 304-µm-thick and
partly anoxic viable cell layer rather than the "respiring" cell
rim that measured only 225 µm and consisted of 10-15% fewer
cells (cf. Oxygen Consumption). Hence the rise in
cO2 of
well-oxygenated MR1 cells is even bigger.
3 Our findings indicate that energy metabolism during growth is not affected by glucose depletion. Glucose concentration was held constantly high at 25 mM in the medium and was found in the center of the viable cell rim to drop during the observation period of spheroid growth from ~20 mM to values not less than 13 mM. Moreover, even in the spheroid center, glucose concentrations were >2.5 mM in Rat1-T1 and MR1 and >13 mM in Rat1 and M1 spheroids (data not shown). Physiological glucose levels, on the other hand, are 2.5-6 mM in the plasma and considerably less next to tissue cells located remote from capillaries.
4 Comparisons on day 4 of monolayer culture ("late exponential growth") are less meaningful because at that time growth in the ras transfectants is still clearly exponential, whereas Rat1 and M1 have already come very close to their plateaus.
Address for reprint requests and other correspondence: L. Kunz-Schughart, Institute of Pathology, Univ. of Regensburg, 93042 Regensburg, Germany (E-mail: leoni.kunz-schughart{at}klinik.uni-regensburg.de).
Received 12 May 1999; accepted in final form 15 October 1999.
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