DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines

Summary

We report a DNA-FISH metaphase spread protocol that visually detects locus copy number and location within the genome. This approach enables single-cell resolution of amplification states, specifically in cancer cell lines containing extrachromosomal DNA and homogeneously staining regions.

Abstract

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Introduction

The human genome is organized not only by its sequence, but also by its structure, and changes in either sequence or structure can lead to cancer^1,2,3,4. Oncogene amplification is present in approximately 40% of all tumors, where it contributes to tumor initiation, growth, metastasis, and drug resistance⁵. In some cases, amplified oncogenes are carried on extrachromosomal DNAs (ecDNAs, also called double minutes), which are circular, self-replicating, double-stranded DNA elements that reside outside of the linear chro....

Protocol

All human cell culture procedures were performed in compliance with institutional guidelines.

1. Arrest cells in metaphase using Colcemid (N-methyl-N-deacetyl-colchicine)

1. Begin with approximately 2.0 × 10⁶–2.0 × 10⁷ cancer cells or other cultured cells. Too few cells will yield fewer metaphase spreads, whereas too many cells may result in insufficient fixation.
2. Determine the appropriate Colcemid incubation time for the experimental system. Incubation time affects the number of metaphase spreads obtained and chromosome length, and optimal conditions vary among cell ....

Results

This protocol (Figure 1B) yields clean, well-dispersed, intact metaphase chromosomes suitable for cytogenetic analysis, with chromosomes and DNA-FISH signals clearly visible by fluorescence microscopy. Using a brightfield/phase-contrast microscope at low magnification (10× or 20×) is an important quality-control step to verify that nuclei have burst and chromosomes have dispersed (Figure 2). Successful fixation and tabletop dropping should yield well-dispersed m.......

Discussion

Despite their well-established advantages, DNA-FISH metaphase spreads are underused in the era of WGS. The physical organization of amplified DNA is functionally significant, as it can influence downstream biological behavior and therapeutic response^61,62,63,64. Focally amplified oncogenes can exist on ecDNA or within HSRs, and these two amplification states are not functionally equivalent

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15-mL conical centrifuge	any source	NA	Centrifugation.
15-mL conical tubes	any source	NA	Cell collection.
200 proof ethanol	any source	NA	Wash steps.
Acetic Acid, glacial, 99.7%	Millipore Sigma	695092	Carnoys fixative and slide wash steps.
Antifade Mounting Medium	FisherScientific	NC9265087	Slide mounting after completed slide preparation
Biological safety cabinet with vaccum outlet	any source	NA	Chemical storage.
Colcemid (N-methyl-N-deacetyl-colchicine)	Millipore Sigma	10295892001	Binds tubulin, arrests cells in metaphase.
Coplin jars	any source	NA	Wash steps.
DAPI (4′,6-diamidino-2-phenylindole)	Milipore Sigma	D8417-1MG	Fluorescent DNA stain.
EGFR/CON7 FISH probe	Empire Genomics	EGFR-CHR07	Locus-specific fluorescent probe.
Elmer's rubber cement	any source	NA	Keep coverslide in place during overnight probe hybridization.
Ethanol resistant permanent marker	any source	NA	Slide labeling.
Fluorescent microscope	any source	NA	Slide imaging.
Glass Cover Slip, 22 x 22mm	ThermoFisher	50-143-780	Probe hybridization on slide.
Glass Slide, precleaned	VWR	48311-703	To drop metaphase spreads and perform DNA-FISH.
Humidified CO2 Incubator	any source	NA	Live-cell incubation.
Hybridization Buffer	Empire Genomics	Hyb-Buffer	Buffer used with probe.
Ice bucket	any source	NA	Keep Carnoy's fixative cold.
KCl (2M)	ThermoFisher	AM9640G	Swell cells prior to fixation.
Methanol, 99.9%, HPLC grade	Millipore Sigma	1060351000	For Carnoys fixative.
Microcentrifuge	any source	NA	Centrifugation.
Microcentrifuge tubes	any source	NA	For cells during fixation, and for probe preparation.
MYC / SE 8 Probe	Leica	KI-10106	Locus-specific fluorescent probe.
PBS, pH 7.4	Gibco	10010-023	Wash steps.
Phase contrast microscope	any source	NA	Imaging of metaphase spreads immediately after dropping
Pipette tips	any source	NA	Pipetting.
Sodium chloride	Millipore Sigma	S9888	For SSC buffer.
Sodium citrate	Millipore Sigma	567446	For SSC buffer.
Sterile ultrapure water	any source	NA	Wash steps.
ThermoBrite programmable slide incubator	any source	NA	Slide incubation.
Tongs	any source	NA	For slide wash steps.
Water bath	any source	NA	For KCl incubation.