The analysis of individual cells is now a reality. Single-cell signatures may tell us a lot about the tissue microenvironment, differentiation, aging, or regeneration. For instance, tumor microenvironment contains multiple cell types including immune cells, stroma, cells of blood vessels, and all their chemical signals.
Communication of tumor cells with their microenvironment helps drive tumor progression. Single-cell analysis can explain this communication in great detail, potentially yielding new therapeutic approaches for targeting these signals.
Until recently, the sensitivity of most of the “omics” techniques (genomics, proteomics) was not sufficient enough to trace the contribution of individual components. Many current molecular biology techniques require multiple cells. This inevitably blends cell populations and, therefore, reports the average values.
“This is rapidly changing,” comments Daojing Wang, Ph.D., life sciences division, Lawrence Berkeley National Laboratory. “Single-cell analysis is a new frontier in omics. It will enable systems biology at the level of individual cells.” Single-cell technologies will help to understand the molecular stasis of a particular cell or cell type in a specific biological environment, its interconnections with the surrounding environment, and the roles that individual phenotypes play in healthy and diseased tissue function.
Key cells may be present in small numbers and produce infrequent and weak signals that get diluted and lost during averaging. For example, circulating cancer cells are rare but important, since they play a key role in cancer metastasis. Measuring gene expression within individual circulating cancer cells will hopefully shed light on cancer dissemination and metastasis.
“Single-cell mRNA and genomic analysis have already became a reality,” adds Dr. Wang, who will be speaking at Select Bioscience’s “Single Cell Analysis Congress” in May. “Proteomics and metabolomics experienced more challenges, which may be resolved by combining extremely efficient sample manipulation and highly sensitive detection. In that regard, micro-/nanofluidics interfaced with mass spectrometry (MS) could be a promising combination for single-cell proteomic analysis.”
Dr. Wang and colleagues have developed special multinozzle emitters for MS. Each emitter consists of a parallel silica nozzle array. These emitters could be linked to microfluidic circuits on one side and MS on the other side, thus forming an integrated lab-on-a-chip system with potential for future single-cell proteomics and metabolomics.
qPCR from Single Cells
“A comprehensive understanding of life requires combining molecular data from individual cells and the macro data from the organ/organism,” comments Hideki Kambara, Ph.D., Hitachi. Dr. Kambara’s group participated in a three-year multiprong effort called the “Life Surveyor”. Over 70 scientists from multiple research entities formed workgroups aimed at developing better tools for single-cell analysis.
“While tissues may seem uniform on the macro level, each cell may be individually different,” continues Dr. Kambara. “Our group focused on quantitative PCR as the most accurate method of detecting these differences. We adapted qPCR for single-cell analysis in a cost-efficient, amplification-independent fashion.”
The concept of this method is not new, as it involves using capture of mRNA on magnetic beads followed by reverse transcription into cDNA. However, the adaptation to a single cell required multiple modifications, such as introduction of a special polymer to prevent DNA and RNA adsorbtion to the surfaces.
“Our goal was to reuse this cDNA library multiple times, but we observed that 90 percent of DNA was stripping from beads after only 10 rounds of qPCR,” says Dr. Kambara. “This desorption was caused by thermal decomposition of the bead’s polymer coating. We had to develop a low-temperature PCR reaction, which decreased the desorption rate to only 20 percent per 10 rounds.”
This technology was used to discern whether the lineage of mesenchymal stem cells is predetermined a priori, or if the differentiation is induced by the addition of a chemical agent.
While this research is still ongoing, the preliminary data seems to suggest that the fate of undifferentiated cells is already predetermined. “Our ultimate goal is to develop tools for multiple gene expression at single-cell resolution while preserving the spatial information on cells within the tissues,” says Dr. Kambara. “Such measurements will have a profound impact on understanding the effect of each cell on a tissue or organ.”