A boost for cellular profiling

Sep. 22, 2013 — A team of researchers affiliated with Ludwig Cancer Research and the Karolinska Institutet in Sweden report in the current issue of Nature Methods a dramatically improved technique for analyzing the genes expressed within a single cell -- a capability of relevance to everything from basic research to future cancer diagnostics.

"There are cells in tumors and in healthy tissues that are not present in sufficient numbers to permit analysis using anything but single-cell methods," explains senior author, Rickard Sandberg, PhD. "This method allows us to identify rare and important subpopulations of cells in all sorts of tissues. We can also use it to tease apart, more rigorously than ever before, how the expression of unique suites of genes transform cells from one state to another as, say, an embryo develops into an organism, or a tumor becomes metastatic."

Traditional approaches, which depend on the collective analysis of gene expression in millions of cells at once, tend to obscure biologically significant differences in the genes expressed by specialized cells within a particular kind of tissue. Single-cell analysis of gene expression overcomes this limitation. The leading method for such analysis -- Smart-seq -- was developed in 2012 by the biotechnology firm Illumina, together with Sandberg's laboratory.

To develop the new technique, named Smart-seq2, Sandberg's team conducted more than 450 experiments to improve upon their initial method. The new procedure consistently captures three to four times as many RNA molecules, which often translates into 2,000 more genes per cell than current methods allow. It also captures far more full-length gene sequences, a steep challenge in such studies, which often capture only partial sequences of expressed genes. This will permit researchers to conduct a more granular analysis of how subtle differences between the same genes in different people -- known as single nucleotide polymorphisms (or SNPs) -- contribute to differences in biology and disease.

The new method is likely to be of great value to cancer research. Identifying rare sub-populations of cells in tumors and understanding their role in the survival and progression of cancers can provide invaluable information for the development of diagnostics and targeted therapies. A study recently published by Ludwig researchers described, for example, how certain subpopulations of cells in melanomas can be pushed into a drug-susceptible state and then destroyed by chemotherapy. More such strategies might be devised as researchers get a better handle on the cellular species found in different types of tumors, and the patterns of gene expression that define them.

Because Smart-seq2 relies on off-the-shelf reagents, it costs roughly a twentieth as much as the commercialized kit, which should allow researchers to conduct sophisticated analyses of single cells on a much larger scale. It can also be improved further by the scientific community, since its constituent components and rationale are both open to the public.

Armed with the more effective and affordable Smart-seq2, Sandberg's lab is now moving ahead on projects that require a large-scale, single-cell gene expression analysis. "Now all researchers can do their own single-cell gene expression analysis by buying the components of the process described in this paper and assembling their own kits," says Sandberg.

View the original article here

Protein identified that regulates cellular trafficking, potential for anti-cancer therapy

Sep. 22, 2013 — Molecular microbiologists at the University of Southern California (USC) have uncovered intricate regulatory mechanisms within the cell that could lead to novel therapeutics for the treatment of cancer and other diseases. Their findings, which have long-standing significance in the basic understanding of cell biology, appear in the journal Nature Cell Biology.

"Our research reveals a new regulatory mechanism that coordinates two distinct intracellular processes that are critical to cellular homeostasis and disease development," said Chengyu Liang, M.D., Ph.D., a member of the USC Norris Comprehensive Cancer Center and principal investigator of the study.

The endoplasmic reticulum (ER) and Golgi apparatus are cellular organelles in eurkaryotic organisms where proteins are synthesized and packaged for secretion through the body. The trafficking of proteins between the ER and Golgi must be tightly modulated to maintain the health of the cell and prevent diseases like cancer from taking hold.

"Interest in the role of ER-Golgi network during cancer cell death has been gaining momentum," said Shanshan He, Ph.D., research associate at the Keck School of Medicine of USC and one of the study's first authors. "In this study, we identified a novel regulatory factor for the Golgi-ER retrograde transport and a new mechanistic connection between the physiological trafficking and the autophagic transportation of cellular material."

The researchers discovered that the UV irradiation resistance-associated gene protein (UVRAG), which has been implicated in the suppression of colon and breast cancer, coordinates trafficking of proteins between the ER and Golgi apparatus and also autophagy, the natural process of breaking down cellular components.

"Given that the ER-Golgi network is often dismantled in malignant conditions and that UVRAG is intensively involved in different types of human cancers, this study gives us a new avenue to investigate anti-cancer agents that target UVRAG and/or the ER-Golgi pathway in cancer and other relevant diseases," Liang said.

View the original article here

Protein identified that regulates cellular trafficking, potential for anti-cancer therapy

Sep. 22, 2013 — Molecular microbiologists at the University of Southern California (USC) have uncovered intricate regulatory mechanisms within the cell that could lead to novel therapeutics for the treatment of cancer and other diseases. Their findings, which have long-standing significance in the basic understanding of cell biology, appear in the journal Nature Cell Biology.

"Our research reveals a new regulatory mechanism that coordinates two distinct intracellular processes that are critical to cellular homeostasis and disease development," said Chengyu Liang, M.D., Ph.D., a member of the USC Norris Comprehensive Cancer Center and principal investigator of the study.

The endoplasmic reticulum (ER) and Golgi apparatus are cellular organelles in eurkaryotic organisms where proteins are synthesized and packaged for secretion through the body. The trafficking of proteins between the ER and Golgi must be tightly modulated to maintain the health of the cell and prevent diseases like cancer from taking hold.

"Interest in the role of ER-Golgi network during cancer cell death has been gaining momentum," said Shanshan He, Ph.D., research associate at the Keck School of Medicine of USC and one of the study's first authors. "In this study, we identified a novel regulatory factor for the Golgi-ER retrograde transport and a new mechanistic connection between the physiological trafficking and the autophagic transportation of cellular material."

The researchers discovered that the UV irradiation resistance-associated gene protein (UVRAG), which has been implicated in the suppression of colon and breast cancer, coordinates trafficking of proteins between the ER and Golgi apparatus and also autophagy, the natural process of breaking down cellular components.

"Given that the ER-Golgi network is often dismantled in malignant conditions and that UVRAG is intensively involved in different types of human cancers, this study gives us a new avenue to investigate anti-cancer agents that target UVRAG and/or the ER-Golgi pathway in cancer and other relevant diseases," Liang said.

View the original article here

A boost for cellular profiling

Sep. 22, 2013 — A team of researchers affiliated with Ludwig Cancer Research and the Karolinska Institutet in Sweden report in the current issue of Nature Methods a dramatically improved technique for analyzing the genes expressed within a single cell -- a capability of relevance to everything from basic research to future cancer diagnostics.

"There are cells in tumors and in healthy tissues that are not present in sufficient numbers to permit analysis using anything but single-cell methods," explains senior author, Rickard Sandberg, PhD. "This method allows us to identify rare and important subpopulations of cells in all sorts of tissues. We can also use it to tease apart, more rigorously than ever before, how the expression of unique suites of genes transform cells from one state to another as, say, an embryo develops into an organism, or a tumor becomes metastatic."

Traditional approaches, which depend on the collective analysis of gene expression in millions of cells at once, tend to obscure biologically significant differences in the genes expressed by specialized cells within a particular kind of tissue. Single-cell analysis of gene expression overcomes this limitation. The leading method for such analysis -- Smart-seq -- was developed in 2012 by the biotechnology firm Illumina, together with Sandberg's laboratory.

To develop the new technique, named Smart-seq2, Sandberg's team conducted more than 450 experiments to improve upon their initial method. The new procedure consistently captures three to four times as many RNA molecules, which often translates into 2,000 more genes per cell than current methods allow. It also captures far more full-length gene sequences, a steep challenge in such studies, which often capture only partial sequences of expressed genes. This will permit researchers to conduct a more granular analysis of how subtle differences between the same genes in different people -- known as single nucleotide polymorphisms (or SNPs) -- contribute to differences in biology and disease.

The new method is likely to be of great value to cancer research. Identifying rare sub-populations of cells in tumors and understanding their role in the survival and progression of cancers can provide invaluable information for the development of diagnostics and targeted therapies. A study recently published by Ludwig researchers described, for example, how certain subpopulations of cells in melanomas can be pushed into a drug-susceptible state and then destroyed by chemotherapy. More such strategies might be devised as researchers get a better handle on the cellular species found in different types of tumors, and the patterns of gene expression that define them.

Because Smart-seq2 relies on off-the-shelf reagents, it costs roughly a twentieth as much as the commercialized kit, which should allow researchers to conduct sophisticated analyses of single cells on a much larger scale. It can also be improved further by the scientific community, since its constituent components and rationale are both open to the public.

Armed with the more effective and affordable Smart-seq2, Sandberg's lab is now moving ahead on projects that require a large-scale, single-cell gene expression analysis. "Now all researchers can do their own single-cell gene expression analysis by buying the components of the process described in this paper and assembling their own kits," says Sandberg.

View the original article here