Evidence for hypoxia increasing the tempo of evolution in glioblastoma

Abstract

Background: Tumour hypoxia is associated with metastatic disease, and while there have been many mechanisms proposed for why tumour hypoxia is associated with metastatic disease, it remains unclear whether one precise mechanism is the key reason or several in concert. Somatic evolution drives cancer progression and treatment resistance, fuelled not only by genetic and epigenetic mutation but also by selection from interactions between tumour cells, normal cells and physical micro-environment. Ecological habitats influence evolutionary dynamics, but the impact on tempo of evolution is less clear.

Methods: We explored this complex dialogue with a combined clinical-theoretical approach by simulating a proliferative hierarchy under heterogeneous oxygen availability with an agent-based model. Predictions were compared against histology samples taken from glioblastoma patients, stained to elucidate areas of necrosis and TP53 expression heterogeneity.

Results: Results indicate that cell division in hypoxic environments is effectively upregulated, with low-oxygen niches providing avenues for tumour cells to spread. Analysis of human data indicates that cell division is not decreased under hypoxia, consistent with our results.

Conclusions: Our results suggest that hypoxia could be a crucible that effectively warps evolutionary velocity, making key mutations more likely. Thus, key tumour ecological niches such as hypoxic regions may alter the evolutionary tempo, driving mutations fuelling tumour heterogeneity.

Fig. 1. Model schematic and example oxygen maps.

a Cell fate decisions per each cellular update are determined by the flow charts displayed. Note that the non-italicised p in this schematic refers to probability rather than oxygen tension. Example static heterogeneous oxygen maps with b 1, c 15 and d 357 vessels.

Fig. 2. Co-registration and cell detection analysis.

A necrotic boundary is marked on the H&E slide by the pathologist (marked here by the green line). On the Ki-67 stain, cells that meet the threshold for Ki-67 positive are marked by red dots and those below the threshold by blue dots. Finally, p53-positive cells are marked by red (+) symbols on the final stain. The region shown above encompasses an area of 87.52 mm² (15.67 mm × 5.58 mm).

Fig. 3. Simulation results of hypoxia effects.

Clonogenic division in a low-density oxygen map and b high-density oxygen map. Colour bars indicate oxygen partial pressure, and vertical bars indicate divisions of clonogenic cells, normalised to the maximum number of divisions in the simulation. c, d show respective clonogenic cell divisions higher under hypoxia. In e, f, clonogenic cells are shown in green and TACs in red, with β = 15 after 10,000 time-steps. e depicts cells initially seeded in a high oxygen environment and f initial cell seeded on the hypoxic niche. In e the firewall effect of long-lived TACs can be clearly seen, whereas in f clonogenic cells proliferate along the anoxic ridge, yielding an ‘edge creep’ effect, allowing invasion invade along the hypoxic ridge.

Fig. 4. Physiological evidence of hypoxia effects in glioblastoma samples.

Pooled data from 23 regions of 9 patient glioblastoma samples after image analysis depicting (a) distribution of p53-stained cells versus Ki-67-stained cells relative to known necrotic borders. b Probability distributions for stained cells close to necrosis. c An example from a patient glioblastoma histologic section. Physiological p53 stress detected by image analysis is illustrated by blue dots overlaid on the histology section. Green lines depict pathologist-marked necrosis; contour lines with red opacity show the probability density of p53-positive cells (calculated from a Sheather–Jones smoothing-kernel distribution function). Near necrotic regions, the probability of finding stress markers increases relative to non-necrotic zones. The mitotic rate appears constant throughout the tissue, suggesting that these regions are more likely to give rise to mutations. Physiological stress indicates potential topography of evolutionary velocity. A decoupled version of the figure is available in Supplementary Material S1.