Hypoxia is thought to be a major cause of failure in
cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing
lactate dehydrogenase activity, endogenous
lactate and accumulation of neutral fat. An
ascites tumour (Yoshida
hepatoma) and a solid tumour (Ehrlich
carcinoma) were used as the experimental models. A gel film technique was used for visualizing
lactate dehydrogenase and "nothing
dehydrogenase" (or endogenous
lactate). The
fluorescent dyes Nile Red and
Acridine Orange were used to demonstrate
lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of
necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of
lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or
LDHk isoenzymes bound to membranous structures; 2) an intense granular positivity of "Nothing
Dehydrogenase" due to high concentrations of endogenous
lactate; 3) neutral lipid droplets emitting an intense yellow fluorescence in
Nile Red-stained preparations; 4) a yellow cytoplasmic fluorescence in
Acridine Orange-stained sections, attributable to a low cellular
RNA content. Electron microscopy revealed moderately osmiophilic
lipid globules in close association with damaged mitochondria. Better oxygenated cells showed: (a) a reddish-blue diffuse pattern of LDH, ascribed to moderately active soluble LDH
isoenzymes containing H subunits; (b) almost no "Nothing
Dehydrogenase" positivity; (c) no cytoplasmic lipid droplets; and (d) an intense orange-red fluorescence in the cytoplasm of
Acridine Orange-stained specimens, due to high concentrations of cellular
RNA.
Nile Red fluorescence showed that the
lipids of the solid tumour membranes were more hydrophobic than in the normal surrounding tissue. This suggests that there are abnormal domains of neutral
lipids in the tumour cell membranes. In solid tumours, cells with the characteristics attributable to
hypoxia were usually observed on the edge of
necrosis of cuff-like formations. In very advanced growth stages, however, they were also seen surrounding (and occasionally clogging) blood vessels, or in tentacular formations coming from a
necrosis border and polarized towards the vessels.
Lipid-loaded cells were also seen in blood vessels distant from the tumour. These observations point towards a chemotactic process of hypoxic cells towards better environments.