Steady progress in a variety of technologies, such as gene-expression profiling, proteomics, and next-generation sequencing, is helping to characterize tissues and cells and to catalog the differences between healthy and diseased tissues. The ability to go beyond these techniques and to analyze and monitor the status, activity, and features of individual cells is yielding information on why cells of the same type may respond differently to external stimuli or therapeutic compounds, how tumor heterogeneity may affect drug response and secondary drug resistance, and how surrounding microenvironments in vivo or in bioreactors can be manipulated to alter the growth and function of cells and microbes, for example.
A variety of tools and techniques are in use to enable single-cell analysis, including microfluidics and other methods to isolate single cells in controlled microenvironments, microfabrication techniques and nanoprobes to create cellular environments in vitro, and miniaturization technology to enable assays and molecular detection at the single-cell level.
At the upcoming Select Biosciences “Single Cell Analysis Europe” conference, Birgitta Knudsen, Ph.D., associate professor, department of molecular biology and genetics at the Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark, will describe a microfluidics-based molecular detection approach for measuring enzyme activity in single cells that can achieve multiplexed detection of individual enzymatic events.
The technology underlying this approach is isothermal rolling circle amplification (RCA), an established method for direct detection and quantification of nucleic acid sequences.
Dr. Knudsen’s group has developed a rolling-circle-enhanced enzyme activity detection (REEAD) assay. This assay is based on RCA technology that allows for detection and visualization of enzymatic DNA cleavage-ligation events using fluorescent probes and provides single-molecule sensitivity. REEAD can measure the activity of an enzyme and not merely its presence in a cell.
The initial application of this approach is to detect topoisomerase I (topI) activity in human cancer cells. TopI, a DNA-cleaving enzyme, is an important target for anticancer drugs used to treat ovarian, colon, and small-cell lung cancer, for example.
Approximately half of the patients do not respond to the treatment, however, and in more than 50% of patients secondary drug resistance develops after an initial response to chemotherapy. The reason for this is unclear, but it is known that average topI levels across a tumor cell population correlate to drug response. High topI activity is associated with a good drug response.
One of Dr. Knudsen’s research goals is to explore how individual cancer cells vary and how that variation affects cell response to chemotherapy. She then envisions being able to apply that knowledge to the analysis of tumor tissue from biopsy samples and to use markers such as topI activity to help clinicians predict drug response and guide drug selection.
Frederik Fritzsch, a researcher in the laboratory of chemical biotechnology at the department of biochemical and chemical engineering of the TU Dortmund University (Germany), will discuss two key terms: “controlled” and “contactless.” Why are these so important for single-cell analysis?
“The principal reasons for phenotypic diversity and heterogeneity in bioprocesses with isogenic populations are the varying microenvironments surrounding single cells,” says Fritzsch. The ability to control this microenvironment allows scientists to introduce systematic perturbations and perform time-resolved analyses to assess the effects of these perturbations on single cells.
The results can be used to decipher cellular mechanisms, biochemical pathways, and regulatory cascades, and may lead to the identification of biochemical targets for drug discovery. They can also facilitate metabolic engineering of hyper-producing microorganisms for industrial biotechnology applications.
The ability to control the microenvironment of a cell implies control over both physical (including temperature and mechanical impact such as pressure or shear stress) and chemical (by modifying the media and nutrient supply) parameters.
“Contactless” refers to the trapping of a single cell such that it does not come in contact with surfaces or other cells. Noncontact isolation of a cell is important because cells intrinsically interact with their surroundings, resulting in potential changes in their morphology, function, signaling pathways, and surface features.
Established lab-on-a-chip-based methods for performing contactless single-cell analysis typically use negative dielectrophoresis (nDEP) to trap cells. These approaches have limitations, including Joule heating; in addition, they are also not suitable for capturing cells <5 µm (such as platelets and many bacteria) in a continuous flow system, according to Fritzsch.