Imaging extrachromosomal DNA (ecDNA) in cancer

Abstract

Extrachromosomal DNA (ecDNA) are circular regions of DNA that are found in many cancers. They are an important means of oncogene amplification, and correlate with treatment resistance and poor prognosis. Consequently, there is great interest in exploring and targeting ecDNA vulnerabilities as potential new therapeutic targets for cancer treatment. However, the biological significance of ecDNA and their associated regulatory control remains unclear. Light microscopy has been a central tool in the identification and characterisation of ecDNA. In this review we describe the different cellular models available to study ecDNA, and the imaging tools used to characterise ecDNA and their regulation. The insights gained from quantitative imaging are discussed in comparison with genome sequencing and computational approaches. We suggest that there is a crucial need for ongoing innovation using imaging if we are to achieve a full understanding of the dynamic regulation and organisation of ecDNA and their role in tumourigenesis.

Keywords: Double-minute; Fluorescence in situ hybridisation; Homogeneously staining regions; Oncogene; Transcription hubs.

Fig. 1

ecDNA at metaphase. A 4′,6-Diamidino-2-phenylindole (DAPI) stained metaphase spreads from a recurrent glioblastoma cell line E37. ecDNA appear as small DAPI-stained dots (arrowed). Scale bar: 10 µm. B Violin plot of number of ecDNA per metaphase spread in E37 cells, median and quartiles are shown. Number of metaphase spreads = 53

Fig. 2

Detection of oncogenes on ecDNA by DNA-FISH. Left: DAPI stained metaphase spreads from a recurrent glioblastoma cell line E37. Right: DNA-FISH with a probe (red) detecting c-MYC. Arrows indicate examples of DNA-FISH signal on ecDNA for orientation. Scale bar: 10 µm

Fig. 3

Left: hypothesis for ecDNA transcription driven by ecDNA–ecDNA and ecDNA–RNA Pol II hubs. Right: hypothesis that ecDNA (red and green) localise in the nucleus independent of each other and RNA Pol II hubs

Fig. 4

Schematic of nuclei showing clustered (top) and random (bottom) distribution of DNA-FISH signals detecting ecDNA, with overlay of increasing radii (r) to indicate Ripley’s K. The graph shows how this is plotted per nucleus, with the expected values ± confidence interval shown to represent the null distribution, i.e. random distribution. Observed values within this null distribution would be considered randomly distributed. The lines indicate observed values as they would plot if foci were clustered (burgundy) or dispersed (blue). Adapted from Purshouse et al.