In what could possibly speed up cancer research and drug development dramatically, Stanford scientists have found a way to modify pairs of cancer-related genes in the lungs of mice and then precisely track individual cells of the resulting tumour. The work could finally allow scientists to mimic and then study genetic diversity of cells found in tumours outside of the lab.
“Human cancers don’t have only one tumour-suppression mutation. They have combinations. The question is as how do different mutated genes cooperate or not with one another?” said geneticist Monte Winslow at Stanford’s School of Medicine and a senior author of the study.
A few years ago, such a mapping study would have been a monumental and would have required breeding several lineages of genetically modified mice, each with a different pair of inactivated tumour suppressor genes. In order to explore all possible combinations, hundreds or thousands of mice would have been needed. In contrast, the experiments involved fewer than two dozen mice. “We’ve analysed more genotypes of lung cancer tumours than the whole field has in 15 years,” Winslow said.
This result was achieved by using CRISPR-Cas9, a powerful gene-editing tool that can easily replace, modify, or delete genetic sequences inside organisms, to create multiple, genetically distinct tumours in the lungs of individual animals. “We can induce thousands of clonal tumours in a single mouse,” Winslow said.
To draw useful conclusions about the combinatory effects of different gene mutations, scientists required a precise way to label and track the growth of different tumours. Conventional techniques, which involved trying to excise and compare the sizes of individual tumours, were insufficient.
Solution came from evolutionary biologist at Stanford, Dmitri Petrov, who had been working with physicist Daniel Fisher and geneticist Gavin Sherlock to develop DNA barcoding as a way of investigating rapid evolution in yeast. When Petrov learned about the experiments in Winslow’s group, he thought the technique might also work in mice.
Petrov’s idea was to attach short, unique sequences of DNA to individual tumour cells inside mice lungs. Each sequence functions as a heritable genetic barcode, and as each cancer seed cell divides, growing into a tumour, the number of barcodes also multiplies.
Instead of having to painstakingly cut out individual tumours, the scientists could take an entire cancerous lung, grind it up and then use high throughput DNA sequencing and computational analysis to very precisely determine how big a tumour is by counting how often its barcodes pop up. By tallying different barcodes, scientists can compare tumour sizes much more quantitatively than was previously possible.
“This is 10 steps forward in our ability to model human cancer,” said Petrov, adding, “We can generate a large number of tumours with specific genetic signatures in the same mouse and follow growth individually at scale and with high precision. The previous methods were both orders of magnitude slower and much less quantitative.”
The combination of CRISPR-Cas9 and DNA barcoding could allow scientists to replicate in the lab the kind of genetic diversity observed in cancer patients. “It gets around this fear of the complexity of cancer,” Winters said. “We’ve known for decades that human tumours are extremely complex and different from patient to patient, but how do you actually recreate that so you can study it? It’s not by doing it one at a time. Now, we can model 30 different genetic variations of a cancer simultaneously.”
Another finding is that many tumour suppressor genes are context dependent ie they only affect cancer growth in the presence or absence of another gene. The team’s hybrid technique could prove valuable for cancer drug testing. Pharmaceutical companies could test a drug on thousands of tumour variations simultaneously to see which ones respond to treatment and, equally important, which ones don’t.
“We can help understand why targeted therapies and immunotherapies sometimes work amazingly well in patients and sometimes fail,” Petrov said, adding, “We hypothesise the genetic identity of tumours might be partially responsible, and we finally have a good way to test this.”