Rapid uptake of glucose and lactate, and not hypoxia, induces apoptosis in three-dimensional tumor tissue culture

Abstract

The spatial arrangement of cellular metabolism in tumor tissue critically affects the treatment of cancer. However, little is known about how diffusion and cellular uptake relate to intracellular metabolism and cell death in three dimensions. To quantify these mechanisms, fluorescent microscopy and multicellular tumor cylindroids were used to measure pH and oxygen profiles, and quantify the distribution of viable, apoptotic and necrotic cells. Spheroid dissociation, enzymatic analysis, and mass spectrometry were used to measure concentration profiles of glucose, lactate and glutamine. A mathematical model was used to integrate these measurements and calculate metabolic rate parameters. It was found that large cylindroids, >500 μm in diameter, contained apoptotic and necrotic cells, whereas small cylindroids contained apoptotic but not necrotic cells. The center of cylindroids was found to be acidic but not hypoxic. From the edge to the center, concentrations of glucose, lactate and glutamine decreased rapidly. Throughout the cell masses lactate was consumed and not produced. These measurements indicate that apoptosis was the primary mechanism of cell death; acidity was not caused by lactic acid; and cell death was caused by depletion of carbon sources and not hypoxia. The mathematical model showed that the transporter enzymes for glucose and lactate were not saturated; oxygen uptake was limited by intracellular metabolism; and oxygen uptake was not limited by membrane-transport or diffusion. Unsaturated transmembrane uptake may be the cause of both proliferative and apoptotic regimes in cancer. These results suggest that transporter enzymes are excellent targets for treating well oxygenated tumors.

Figure 1. In vitro tumor-mimicking techniques: cylindroids and spheroid dissociation

A) Image of a multicellular tumor spheroid, with a transparent viable periphery and an opaque necrotic core. B) Tumor cylindroids are formed by constraining spheroids between the bottom of a well plate and a plug attached to the well-plate lid. Plugs are spaced 150 μm above the bottom of the plate. The wells are filled medium and interior regions of cylindroids can be observed from the underside by microscopy. C) The metabolic content of cells as a function of radius is determined by successive rounds of dissociation that isolated cells from concentric shells.

Figure 2. Distribution of cell viability and apoptosis in small and large cylindroids

A) Transmitted and fluorescence images of a small and large cylindroid. Fluorescence images show location of viable and dead cells using the Live/Dead cell viability assay. Scale bars are 200 μm. B) Transmitted and fluoresce images of a small and large cylindroids stained to identify activated caspase-3, an indicator of apoptosis. C, D) Radial profiles of viable cells, necrotic cells, and cells with activated caspase-3 in populations of small (C; diameter<500 μm; n=5 for Live/Dead; n=6 for apoptosis) and large (D; diameter>500 μm; n=5 for live/dead; n=3 for apoptosis) cylindroids. Some errors are small and the bars are obscured by data markers. E) Extent of viability, necrosis and apoptosis in the interior and peripheral 10% of the cylindroid populations. Cell viability was greater in the center of small compared to large cylindroids (*, P<0.001); cell death was greater in the center of large cylindroids (*, P<0.001); and apoptosis was greater in the center compared to the periphery for both small and large cylindroids (*, P<0.001). In large cylindroids, viability was greater in the periphery (*, P<0.001), and death was greater in the center (*, P<0.001).

Figure 3. Oxygen concentration and extracellular pH profiles in tumor cylindroids

A) Transmitted and fluorescence images of a cylindroid stained with ruthenium-tris(4,7-diphenyl-1,10-phenanthroline) dichloride. Brightness in the fluorescence image is inverse to oxygen concentration. Bright regions have lower oxygen concentrations and dark regions have more oxygen. B) Two-point calibration of Ru-dpp in DMEM in ambient air (159 mmHg O₂) and pure nitrogen (0 mmHg O₂). C) Oxygen partial-pressure (pO₂) radial profile of tumors cylindroids (n=14). Error bars represent standard error of the mean. The oxygen concentration at normalized radii less than 0.975 was lower than at the edge (*, P<0.01). D) Transmitted and fluorescence images of a cylindroid stained with BCECF. E) Radial pH profile in tumors cylindroids (n=54). The pH at normalized radii between 0 and 0.5 was lower than at 0.875 (*, P<0.005). Error bars represent standard error of the mean.

Figure 4. Concentration profiles of four intracellular metabolites in spheroids

Profiles of (A) glucose, (B) lactate, (C) glutamine and (D) glutamate as functions of normalized radius (n=3). The average spheroid radius was 287μm. Each point represents a single layer of cells removed from the outer edge of a group of spheroids. The concentrations of glucose and glutamine (A, C) in the inner three layers was lower than the outer edge (*, P<0.05). The concentration of lactate (B) was lower in the inner four layers than the edge (*, P<0.05). The concentration of glutamate (D) did not decrease with radius. Error bars represent standard error of mean.

Figure 5. Concentration profiles of intracellular amino acids in spheroids

A) Radial concentration profiles of three essential amino acids: valine, leucine, and isoleucine (n=3). Concentrations of all three were lower at a normalized radius of 0.70 than at the outer edge (*, P<0.05). B) Radial concentration profiles of five non-essential amino acids: alanine, glycine, proline, serine, and aspartate (n=3). Concentrations of all five were lower at a normalized radius of 0.70 than at the outer edge (*, P<0.05).

Figure 6. Computational analysis of metabolism and diffusion

A–C) Measured values and computational predictions of concentrations for (A) glucose, (B) lactate, and (C) oxygen. For both the measurements and the model, glucose and lactate were completely consumed at the edge and oxygen was present in the center. D) Uptake rates of oxygen, glucose, and lactate as a function of radius. Oxygen uptake was greater than glucose and lactate because of the stoichiometry of intracellular metabolism. E) Effect of hypothetically limiting cellular uptake of oxygen. Oxygen uptake would be reduced at the outer edge, and lactate would have been produced, rather than consumed. F) Effect of reducing the glucose rate constant (k) on uptake as a function of radius. At the edge, consumption would be reduced, but in the center it would be increased.