New Single-Cell Measurement Techniques Reveal Significant Functional Heterogeneity
As analytical technologies have improved over the past decade, it has become clear that cells within the same tissue can differ greatly in how they are behaving at any given moment. Now, two teams of collaborators have developed technologies that can characterize various aspects of this phenotypic heterogeneity in thousands of cells simultaneously. These high-throughput techniques will not only shed new light on a variety of biological processes, but could also provide important diagnostic information that could improve the treatment of cancer and other diseases.
In a paper published in the journal Integrative Biology, Denis Wirtz, principal investigator of the Johns Hopkins University Physical Sciences-Oncology Center, and his collaborators describe a high-throughput microscopy technique that can measure simultaneously both the state of the cell cycle and a number of parameters of cellular morphology. Then, in a paper published in the journal Analytical Chemistry, Rong Fan, head of the Single Cell Profiling Core facility at the Dana Farber Cancer Institute Physical Sciences – Oncology Center, working with Dr. Wirtz’s group, describe a simple-to-use high-throughput method for simultaneously measuring a wide range of secreted proteins from more than one thousand individual cells.
Maintaining a properly regulated cell cycle, the highly coordinated process of cell division, is essential to the growth of cells and organisms and when the cell cycle goes awry, the results can be devastating, leading to diseases such as cancer. A limitation in many studies of how various cellular properties relate to the cell cycle is the use of chemical methods for cell cycle synchronization or from measurements averaged over many cells in various stages of the cell cycle.
Dr. Wirtz and colleagues designed their method to interrogate the cell cycle by analyzing morphometric properties in live cells in an attempt to tease out some of the subtleties of this key process. Using this technique, which relies on automated image analysis software, the team were able to accurately infer the cell-cycle stage of up to 2000 cells at a time from fluorescent images. The researchers demonstrated the effectiveness of their method in nine different cell types. These included normal and diseased cells, human and rodent cells, as well as cells harvested from primary tumors and metastatic lesions from cancer patients.
The Johns Hopkins team was able to pinpoint a number of key changes in cell properties that are tied to distinct phases of the cell cycle, and were even able to formulate a simple equation that defines how the physical properties of a cell are affected by cell-cycle changes and genetic manipulations of the cell. They also used this technique to identify new regulators of cell cycle distribution.
Dr. Fan’s team, meanwhile, was interested in quantifying the various proteins that individual cells secrete as a function of their health or disease status. Their method relies on two components – one that encodes a high-density antibody barcode array deposited on a glass substrate, the other consists of an array of 5440 subnanoliter microchambers etched into the biocompatible polymer PDMS for capturing single cells. Once a suspension of cells is deposited on the microchamber plate, the barcode plate is aligned over the array so that each microchamber interacts with a complete set of barcodes. After a suitable incubation time, the two components are separated, processed, imaged, and analyzed. The entire process can be completed in as little as a few hours, and the technique can detect as few as 160 molecules of a given protein secreted from a single cell.
Using this method, Dr. Fan’s group, working with Dr. Wirtz’s laboratory, was able to measure the levels of 14 different proteins secreted by over a thousand single cells isolated from human lung tumor samples simultaneously. Because the technique does not harm the cells, they were also able to measure the migratory behavior of the cells and correlated the expression of specific proteins to this behavior. These findings could provide new insights into metastasis.
The investigators note that their technique “has a number of clinician-friendly features such as ease of operation, low sample consumption, and standardized data analysis.” They also state that it represents “a potentially transformative tool for informative monitoring of cellular function and immunity in patients.”
The studies reported were supported in part by the National Cancer Institute's Physical Sciences in Oncology initiative, a program that aims to foster the development of innovative ideas and new fields of study based on knowledge of the biological and physical laws and principles that define both normal and tumor systems. The microscopy work was detailed in a paper titled, “Functional interplay between the cell cycle and cell phenotypes.” An abstract of this paper is available at the journal’s Web site. View abstract.
The microchamber array device is detailed in a paper titled, “High-throughput secrotomic analysis of single cells to assess functional cellular heterogeneity.” An abstract of this paper is available at the journal’s Web site. View abstract