Single-cell analysis of replicative fate in vivo by heavy water labelling of DNA and mass spectrometry

New methods will be developed for quantifying cell division in living organisms. This is of central concern in cancer, the immune system, ageing, and understanding normal tissue maintenance in the body. Cell division is difficult to measure in living organisms, especially in adults, because cells often divide to replace dying cells, so their total number may not change. "Tracer" substances are therefore used to identify cells that have divided recently. One reason for the use of animals in this research is that the tracers used to study cell division in animals are too hazardous (radioactive, cancerpromoting, or toxic) to use in humans. Non-radioactive, non-toxic "tracers" are now available for measuring cell division, e.g., water (H2O) with hydrogen atoms that are twice as heavy as those of normal water (called "deuterium"; water made with deuterium is "heavy water"). When new cells are made in a patient drinking heavy water (which is perfectly safe for the patient), a new copy of their DNA is made, which is slightly heavier because it was made with deuterium replacing some of its hydrogen atoms. The weight difference can be measured with "mass spectrometers" (instruments for weighing molecules), and this reveals how many cells have divided during the time a person has been drinking heavy water. Unfortunately, current techniques permit this analysis to be performed only on large collections of cells (thousands, at least). In many cases, however, it is important to know exactly which cells have divided, which requires the analysis of DNA from single cells. We have shown recently that it is possible to improve sensitivity of the technique to the point where single cells can be analysed. Thus far, we have already managed to reduce sample requirements to less than 100 cells, and shown by computer simulation that single-cell analysis will indeed provide adequate counting of the number of times a cell has divided in the presence of heavy water. We have also shown that this provides more information on the mechanisms of cell maintenance than determining population averages, as is done with current techniques. The studentship will be devoted to developing the single-cell analytical technology. A student with a background in analytical chemistry, biomedical engineering, or other relevant discipline will be recruited and placed in an interdisciplinary teaching and research environment (analytical chemists, biochemists, cell biologists, immunologists, oncologists, engineers) to support this effort. The relevant Departments at the University of Cambridge have a world-leading track record and offer excellent support to PhD students. The first aim of the studentship is to achieve single-cell sensitivity in practice, and to demonstrate the ability to measure deuterium incorporation into DNA accurately in single cells. The second aim will involve the design of a "lab on a chip" that can perform this analysis on hundreds of single cells. Thirdly, the student will explore model settings where cells are known to divide at varying rates, (e.g. immune cells after immunization) to demonstrate the capabilities of the method. Lastly, the student will collaborate within and beyond the supervisor's lab to address fundamental unsolved problems in cell dynamics, focusing on immunology and cancer biology in the first place. Collectively, these experiments will improve the design of studies of cell division in animals, reducing animal use by more efficient use of the samples generated and eliminating toxicity inherent in the current use of DNA labels for measuring cell proliferation. Some animal use will be replaced entirely by studies of cell proliferation in patients. We estimate that, in all, perhaps 40% of the use of animals in studies of cell division/life span/death might be avoided by widespread adoption of this approach. This studentship will create the technological basis for these advances and provide proof of feasibility.