Spatial profiles of markers of glycolysis, mitochondria, and proton pumps in a rat glioma suggest coordinated programming for proliferation

Antibody labeling of GAPDH, Tom20 and V-ATPase was, on average, markedly increased
relative to labeling outside the C6 gliomas, an increased level extending from the
tumor rim to at least 2.5 mm into the tumor. This pattern is very different from that
shown previously for NHE1, which is upregulated in a peak at the rim, and for the
carbonic anhydrase, CAIX, which is not upregulated in this zone 19]. These radical differences appear to rule out the possibility that the observed profiles
of antibody labeling are the result of some artifactual tumor-specific increase in
the efficacy of antibody binding, or a reduction in the extracellular space fraction,
rather than a true increase in protein expression.

There was considerable variation in the measured intensity of the labeling (always
expressed relative to the intensity outside the glioma) from one profile to another
and one glioma to another. For example, when the mean value for each profile over
the distance 0.5–1 mm was calculated for the series of sections for which both GAPDH
and V-ATPase were labeled, the coefficients of variation (SD/mean) for the 9 pairs
of profiles were 0.229 for V-ATPase and 0.251 for GAPDH. A possible artifactual contributor
to this variance is tissue distortion caused by stretching or compression of parts
of sections. When the corresponding ratio V-ATPase/GAPDH was calculated, its coefficient
of variation was much less, being 0.103. i.e., the ratio of expression of V-ATPase
relative to expression of GAPDH is quite tightly controlled. Inspection of Figure 2b, c suggests that the same is true of Tom20 and GAPDH. These results strongly suggest
that the different components of cell activity are well-coordinated in C6 gliomas.
An unknown factor is how tightly the expression of GAPDH, Tom20 and V-ATPase are regulated
in normal brain.

Upregulation of Tom20

The production of lactate by tumors has sometimes obscured the fact that oxygen consumption
is, in most tumors, greater than in normal tissue 34], 63]. In rat gliomas, the oxygen saturation in much (or all) of the volume is as high
as in normal brain tissue 64], although zones of hypoxic necrosis can develop 65]. In keeping with this expected availability of oxygen in C6 gliomas, expression of
CAIX, which is upregulated by Hypoxia Inducible Factor 1 alpha (HIF1alpha; 66]) has been found not to be upregulated over at least 2 mm in from the rim 19]. It is therefore unsurprising that Tom20, a component of mitochondria, should be
present, as we find.

Comparison of Tom20 and GAPDH

The metabolic pathways associated with glycolysis and with mitochondria can be programmed
in either of two directions, to produce a maximum of ATP, or to produce precursor
molecules for cell growth and proliferation, the latter function becoming predominant
in proliferating cells 28], 67]. That both Tom20 and GAPDH were upregulated with a ratio close to 1 is reminiscent
of the finding by Gullino et al. 34], in various types of tumor, that “glucose consumption and lactate elimination were
in direct proportion to the oxygen utilized and a lack of oxygen blocked both of them”.

Although the upregulation of Tom20 compared to upregulation of GAPDH was greatest
near the rim, Tom20 upregulation was still significantly higher than GAPDH deeper
(1 mm) into the tumor, although only by 10% (Figure 2b). Since C6 gliomas release lactate, there is presumably less pyruvate available
to feed into the TCA cycle. Instead, part of the upregulation of Tom20 may be necessary
to metabolize glutamine, supplied by the blood and a major entrant of the TCA cycle
in cancer cells 27], 68]–70].

Closer to the rim (0.8 mm), on sections in which Tom20 was compared directly with
GAPDH, GAPDH expression was somewhat reduced so that there was a marked peak in the
ratio Tom20/GAPDH. The peripheral location of MCT1 in cervix squamous carcinoma has
led to the hypothesis that there can be net production of lactate within a tumor and
that some of this is taken up close to the rim as a fuel for oxidative phosphorylation
35], 36]. A peak in MCT1 near the rim has also been reported in C6 gliomas 19]. Transfer of lactate, or other metabolic fuel, from one cell to another, is well
known as a physiological phenomenon in muscle and nervous tissue 71]–74]. The observed excess of Tom20 over GAPDH near the rim seems to fit well with the
hypothesis of lactate transfer in C6 gliomas (Figure 4).

Figure 4. Scheme summarizing the results and discussion. Compared to non-tumoral tissue, the
tumor takes up more glucose (yellow arrows) and oxygen (blue arrows). Part of the mitochondrial metabolism in the rim is fuelled by lactate. NHE1 has
a specific role in migrating cells. V-ATPase is present both on plasma membranes and
intracellular vacuoles (pink). EMP Embden-Meyerhof Pathway.

V-ATPase

The presence of V-ATPase has been reported in several types of tumor where it subserves
the handling of molecules for cell growth in intracellular vacuoles, and also contributes
to the export of protons across the plasma membrane 40], 43], 44], 51], 52]. For the C6 gliomas of Figure 3a, the mean upregulation of V-ATPase expression was about 1.7 compared to normal brain,
and it was tightly regulated against GAPDH (Figure 3b). In these sections, expression of GAPDH was upregulated more than V-ATPase. A possible
explanation is that upregulation of glycolysis in C6 gliomas serves two functions:
to support an increase in production of biomass (which requires V-ATPase) and also
to produce pyruvate for ATP production (which does not require V-ATPase).