Helen Albert Writer GEN

Narrowing Focus to The Single-Cell Genomics Level Targets Cancer Complexity on Its Own Terms

The potential of genomic sequencing technology to help in the fight against cancer was recognized at an early stage, with the first report of cancer genome sequencing in breast and colorectal tumors appearing in 2006, just five years after completion of the Human Genome Project.

Since then, efforts such as the Cancer Genome Project in the U.K. and the Cancer Genome Atlas in the U.S., have collected genetic information about many different cancer types, helping to significantly increase knowledge of cancer genomics. The increased efficiency and rapid cost reduction of sequencing technology and analysis techniques over the same period has also contributed to the fast pace of development in the field. 

Cancer sequencing efforts to date have consistently demonstrated how genetically diverse tumors can be. This heterogeneity can make it difficult to discover which mutations trigger tumor development, complicating efforts to develop therapies that target specific mutations and to better understand drug resistance.

Today, researchers are quickly adopting a relatively new method of cancer researcher, that has the promise to unlock the riddle of tumor heterogeneity—single-cell sequencing. Nicholas Navin, Ph.D., pioneered single-cell cancer genome sequencing while working on breast cancer cells in 2010. This work, published in Nature in 2011, transformed the field of cancer genomics. This approach allowed researchers to zoom in on individual cells within a given tumor and sequence their DNA.

Paul Robson, Ph.D., director of Single Cell Genomics at the Jackson Laboratory for Genomic Medicine in Farmington, CT explained the value of these techniques to Clinical Omics: “Single cell genomics…allows you to study the heterogeneity of cancer mutations and how they might evolve over time or in response to therapy.

“Cells with different sets of mutations may respond differently to cancer drugs or may be more likely to give rise to metastatic cells. Understanding mutational features at single-cell resolution and how they behave in response to therapy may provide better opportunities for creating combinatorial therapies.”

Around the same time Dr. Navin was working on single-cell genome sequencing, other researchers were also working on developing techniques for sequencing RNA from single cells (transcriptomics).

By sequencing the RNA (RNA-seq) present in single cancer cells, it is possible to find out what type of cell it is or what signals it is producing or receiving from its neighbors. “This provides a whole new level of understanding of the biology of a tumor,” Dr. Robson said.

Dr. Robson and many other cancer researchers are now using these techniques to investigate tumor heterogeneity and are moving closer to developing better diagnostic and prognostic tools, as well as more targeted therapies, for use in the clinic.

left: Human colon cancer cells with the cell nuclei stained red and the protein E-cadherin stained green created by Urbain Weyemi, Christophe E. Redon and William M. Bonner; middle: A cluster of slow-cycling (AKT-low/Hes 1-high) breast cancer cells (red) within a human ER+ primary breast tumor (cell nuclei in blue; rapidly cycling, AKT-high, cancer cells in green), created by Sheheryar Kabraji and Sridhar Ramaswamy; right: A single gene called Pem (purple) has been localized using fluorescence in situ hybridization. DNA is stained blue; the cell cytoplasm is stained green, created by Tom Misteli. [NCI]

Little Is More

Single-cell sequencing has a number of attractions for cancer researchers over more traditional “bulk” sequencing techniques. 

 “Each tumor is composed of a very diverse set of cells, including both cancer cells with distinct genetic and phenotypic features, and various noncancer cells, such as immune and stromal cells,” said Itay Tirosh, Ph.D., a postdoctoral fellow at the Broad Institute in Cambridge, MA, who specializes in single-cell analysis.

“Without single cell methods, we are averaging across all of their cells, but single-cell methods allow us to accurately profile the tumor and distinguish its various components,” he explained.

“If you look at RNA expression, many genes are expressed in almost every cancer cell. So if the level is high in one and low in the other you would never know from the bulk RNA sequencing,” commented Nir Hacohen, Ph.D., director of the Massachusetts General Hospital Center for Cancer Immunotherapy.

“With bulk DNA sequencing we have some chance because there are typically only two copies of the gene, so you can make assumptions about the number of copies, but with RNA we have no idea, it’s a random number.”

Another advantage of single-cell sequencing is that it can help identify the sometimes crucial influence of small subpopulations of cells that might otherwise be missed using bulk techniques.

“For example, tumors may respond to a drug treatment but contain a rare subpopulation of drug resistant cells that underlie tumor relapse. Similarly, metastasis may be driven by very few cells that have the ability to invade other tissues and adapt to those niches,” explained Dr. Tirosh.

Elise Courtois, Ph.D., of the Genome Institute of Singapore, worked with Dr. Robson on a study using single cell RNA-seq on cells from 11 colorectal tumors that was recently published in Nature Genetics.

Discussing their work, Dr. Courtois said: “If we compare this to previous studies based on bulk transcriptomics, we were able to use this single cell signature to subdivide the different types of patients and identify important cell types for the survival of the patient, or a worse prognosis.” 

Transitioning from Bench to Bedside

Single-cell cancer genomics is still in the early stages of being developed and has yet to reach the clinic in a meaningful way. However, the consensus of researchers in the field seems to be that this goal is firmly on the horizon.

Thierry Voet, Ph.D., is a founding member of the Sanger-EBI Single-Cell Genomics Centre in the U.K., as well as an associate professor at the University of Leuven. Speaking about how he thought single cell research will positively impact patients he said: “I think what technology will bring in the first instance is a better understanding of tumor biology. Based on that, if we understand the cellular heterogeneity that arises within the tumor, we can devise better anticancer treatments.”

Dr. Voet highlighted the problems with cancer treatment failure caused by drug resistance. “There may be a variety of drug resistance mechanisms that can arise within a tumor,” he explained. “Understanding how these are acquired within the tumor, or within a cancer, will be of real importance to design better anticancer treatments in the future.”

Dr. Courtois believes her recent findings in colorectal cancer could lead to direct patient benefits. “Single cell transcriptomics can allow us to identify new biomarkers that we can use for future clinical use,” she said. “For example, we identified different types of fibroblasts in our study and we have specific markers for each type. Maybe in the future we can develop a panel that can screen for both different types of fibroblasts present in the tumor and have a prognostic effect.”

Dr. Tirosh and his colleagues recently discovered that gliomas with a mutation in the IDH1 or IDH2 genes are made up of three different populations of cancer cells. Two of these types do not proliferate, but the third population of cells was linked to tumor growth and aggressiveness.

“This latter subpopulation, which is consistent with the cancer stem cell model, is the least frequent and was never previously described, but it is the one that we need to target in order to eradicate glioma,” he emphasized.

“In future work, we will study the regulation of this subpopulation with the goal of developing new treatments that directly target those glioma stem cells. Another future direction involves longitudinal studies in which we would … follow the evolution of the tumor over time and how it changes following treatments and development of drug resistance.”

While single-cell genomics and transcriptomics have advanced enormously in the last 5 years, both techniques still require refinement. Dr. Hacohen cautioned: “I think that it will take a while, like all things when a new technology develops, for the discoveries to become robust enough that you can say this is going to be what we do for this patient.”

In order to successfully sequence RNA from a single cell, researchers must convert it into a copy DNA library that can then be sequenced. Dr. Hacohen explained that the efficiency of such conversion is still very low at around 5% to 10%. He added that the problem is even worse for DNA as, while there are multiple copies of RNAs in a single cell, there are only two copies of the DNA.

“Via the transcriptome, at least there are multiple copies of the RNA so if there are 10 copies of the RNA maybe you’ll see one or if there are 100 copies you’ll see 10 of them. That’s pretty good, but with DNA if your efficiency is low you are simply not going to see certain events. So, I think sensitivity is probably the biggest issue right now, I would say. The sensitivity is not high enough.”

Future Directions

Single-cell cancer genomics has developed rapidly over the last five years and is likely to continue to do so over the next decade. Based on recent research, it seems likely that it will continue to expand our understanding of tumor heterogeneity and allow rare, potentially disease-causing or resistance-associated subpopulations of cells to be identified more easily. This improved understanding of the cell biology of tumors will also lead to better design of targeted cancer drugs and provide insight into the mechanisms of drug response to current therapies.

If the current upwards trajectory of technology development continues, such techniques will become more accurate, efficient and affordable. “I believe we will arrive at higher accuracy DNA and RNA sequencing together with methodologies where we are able to do this on the same single cell,” predicted Dr. Voet.

Additional ways in which single-cell techniques are forecast to develop include the ability to image genes and their activity during analysis.

“Imagine taking a section of the tumor and imaging thousands of genes and their activity in every cell. So, you can see not only what state are the cells in, but also where they are,” commented Dr. Hacohen.

“Are they next to the tumor? Are they far away from the tumor? Are two cell types that interact next to each other or far away from each other? These are things that we haven’t been able to do very well by the current methods where we dissociate the cells and study them individually, and so I think it’s going to be important to link spatial analysis under single-cell technologies.”

Dr. Voet agrees. “These technologies will be brought to a spatial context so that we can interrogate tissue slices from a single cell perspective and we can learn about the gene expression profiles in a spatial context.”

While improvements in amplification efficiency and sensitivity are still needed to help the techniques reach the clinic, it seems certain that single-cell cancer genomics is here to stay.

“There is really a technological movement right now that is unprecedented in biology,” emphasized Dr. Hacohen. “It will bring us to a deeper understanding of cancer that will both lead to greater discovery and therapeutic targets, but also indirectly affect patient care as well.”

This article was originally published in the May/June 2017 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to www.clinicalomics.com.

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