As the importance of a few selected cells is increasingly recognized as driving tumor development, metastatic dissemination, and resistance to therapy, single-cell genomics emerges as a promising tool to understand tumor heterogeneity.
“Obtaining the whole transcriptome from single cells is invaluable, because every cancer cell is different, every cell is at a different developmental stage, and to truly understand carcinogenesis you really need to do it at a single-cell level,” states Kaiqin Lao, Ph.D., principal scientist in the molecular cell biology division at Applied Biosystems, a division of Life Technologies.
In collaboration with a group from the University of Cambridge, Dr. Lao used Applied Biosystems’ SOLiD system to perform digital expression profiling of a single mouse blastomere and identified 75% more genes than by microarray methodologies.
This approach unveiled 1,753 previously unknown splice junctions and, for the first time, unambiguously confirmed that 8–19% of the genes with multiple known transcript isoforms, expressed at least two isoforms within the same blastomere or oocyte. This finding will provide insight into biological processes relying on specific isoforms and facilitate their selective therapeutic manipulation.
“This is really intriguing and important,” states Dr. Lao. “With conventional transcriptome assays it was not possible to ascertain if the multiple transcript isoforms found in a tissue or organ coexist in the same cell, or if they are just expressed in different cell types or at different cell cycle stages of the same cell type. This knowledge is crucial to provide a real understanding of the transcriptome complexity within individual cells. Such single-cell technology would be very important to gain a greater understanding of the behavior cancer stem cells and solid tumors, such as breast cancer, exhibit.”
Measurements relying on genetic material extracted from entire organs or tissues do not reveal the heterogeneity of genomic information. “The ability to truly perform single-cell nucleic acid measurements, which retain the true, unbiased information coming from cells, is the gold mine in genome biology,” believes Patrice Milos, Ph.D., vp and CSO at Helicos BioSciences.
A recent innovative approach developed at Helicos relies on sequencing RNA from small numbers of cells by capturing the polyA tails of the cellular transcripts through hybridizing to an oligo-dT surface. The RNA-DNA hybrids formed as a consequence of this strong and stable interaction are then subjected to sequencing-by-synthesis by using Helicos’ True Single Molecule Sequencing (tSMS)™ technology. This approach does not require ligation or PCR amplification steps and thus offers an unbiased view of events at what will become single-cell nucleic acid levels, according to Dr. Milos.
Increasingly, new experimental tools become informative about discreet features that set individual cells apart and reveal their contribution to the population as a whole. In this context, Richard Feynman’s words, so eloquently illustrating the need to explore individual components that shape entire systems, become very relevant: “Nature uses only the longest threads to weave her patterns, so each small piece of her fabric reveals the organization of the entire tapestry.”