By Peter Van Loo
It’s always fascinated me how cancers can develop from normal cells. And how that process – unfortunately – occurs so frequently that it affects one in every three of us. Cancer development is, in essence, a process of Darwinian evolution of cells, very much like how species develop.
Because changes to our DNA drive cancer development, using large-scale, rapid sequencing techniques seem like the ideal technology to study it. The Wellcome Trust Sanger Institute has developed quite some expertise in using massively parallel sequencing to understand cancers over the years, so I came to the Institute to be a part of that.
In the two and a half years I’ve now been here, I’ve seen the field mature. We’ve become very good at sequencing the genomes of cancers, and we have certainly learnt a lot.
The trouble is that we are always looking at DNA extracted from millions of cells collected from a single cancer. To disentangle the DNA sequences of the different cell populations within the sample, we have needed to develop complicated methods to analyse the data we gathered.
Imagine if, instead of collecting sequences from many genomes at once and only getting an overview of the cancer’s genetic makeup, we were able to study the genomes of every single cell in a tumour individually. We would then be able to look directly at how that cancer evolved, instead of needing to infer it.
To make this idea a reality all we need to do is to isolate single cells from a sample, extract the cell’s DNA and sequence it. It sounds simple, but is surprisingly hard to do.
The trouble is that a cell contains only a very small amount of DNA (about 6 picograms). So first we need to amplify the DNA to have enough for our techniques to work. To do this, we can use either methods based on PCR or isothermal multiple displacement amplification (MDA). Both approaches work quite well, but they do make the occasional mistake (and sometimes, not so occasional). Since we want to know the precise DNA sequence for the genome of a single cell to follow a cancer’s evolution, we need to know which of the changes we find are real (and may contribute to the cancer) and which are not (and are an artefact of our amplification technique).
In mid-2010, Dr Thierry Voet (assistant professor at KU Leuven, Belgium) came to the Sanger Institute to try to find a solution to this problem. He tested and optimized both PCR-based techniques and MDA, and compared their strengths and weaknesses. Finally, Thierry performed single-cell sequencing using either MDA or PCR-based methods on a series of cancer cells.
He studied several pairs of sister cells, related by only one cell cycle: a single cell was isolated, allowed to divide and both daughter cells were single-cell sequenced separately. We then developed sophisticated analysis methods to separate true DNA changes from amplification artefacts by comparing the DNA changes we found in one cell to those found in its sister, to other single cells, and to a multi-cell reference.
Using the techniques we developed, we are able to uncover:
- copy number changes, where a gene or genetic region is deleted or duplicated
- genomic rearrangements, where different chromosomes break apart or join together, or parts of chromosomes are spliced and rejoined
- changes of a single base in the genome (under certain conditions).
Our studies led to some surprising observations. Sometimes, we saw two sister cells that shared an aberration that was not found in other cells. This change must have occurred recently because it was present in the two cells’ ‘mother’ cell, but it was not found in other cells from the same sample.
Even more interestingly, we occasionally observed events only in one of the two sister cells. For example, in some cases, a fragment was missing from one cell but appeared as an extra copy in its sister. Something had gone wrong during the most recent cell division. This discovery elegantly showed that we could follow tumour evolution in real time (one cell cycle at a time) by using single-cell sequencing.
We can also use single-cell sequencing to study the earliest stages of human life: the very first cycles of cell division after in vitro fertilisation. We discovered that even the time directly after conception, when original cell divides rapidly to produce a cluster of smaller cells from which the embryo will grow, can be prone to genomic instability. Recent results suggest that these signatures of chromosome instability also occur in in vivo conceptions, suggesting that this phenomenon may lead to a spectrum of conditions, including not only loss of conception, but also genetic mosaics, genomic disorders and genetic variation development.
I’m looking forward to what single-cell sequencing will tell us next. No doubt, it will shed considerable light onto how cancers develop and evolve, and how other (normal) cell types maintain stable genomes. It will be interesting to compare the genetic data from single cells with our previous efforts to disentangle the different cell populations in cancer, and explore how we can combine both sets of information to understand more about rare cancer cell populations.
Techniques such as single-cell sequencing have enormous potential to help us better understand cancer’s growth and evolution. Hopefully, as we understand more about the genetic changes that drive cancer development, someday we will be able to conquer it.
Voet T, Kumar P, Van Loo P et al. Single-cell paired-end genome sequencing reveals structural variation per cell cycle. Nucleic Acids Res. 2013 Apr 29
Doi: 10.1093/nar/gkt345 – http://dx.doi.org/10.1093/nar/gkt345
The Cancer Genome Project (CGP) – http://www.sanger.ac.uk/research/projects/cancergenome/
Single-cell genomics – http://www.sanger.ac.uk/research/projects/singlecellgenomics/
Laboratory of Reproductive Genomics – http://med.kuleuven.be/cme/subpage.html?section=laboratories&subsection=laboratory-of-reproductive-genomics&en
Theirry Voet – http://www.sanger.ac.uk/research/faculty/tvoet/
Nik-Zainal S, Van Loo P, Wedge DC et al. The life history of 21 breast cancers. Cell 2012 May 25. http://www.sciencedirect.com/science/article/pii/S0092867412005272
Vanneste E, Voet T et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med. 2009 April 26. http://www.nature.com/nm/journal/v15/n5/full/nm.1924.html