Measuring the density of a cell can reveal a great deal about the cell's state. As cells proliferate, differentiate, or undergo cell death, they may gain or lose water and other molecules, which is revealed by changes in density.
Tracking these tiny changes in cells' physical state is difficult to do at a large scale, especially with single-cell resolution, but a team of MIT researchers has now found a way to measure cell density quickly and accurately - measuring up to 30,000 cells in a single hour.
The researchers also showed that density changes could be used to make valuable predictions, including whether immune cells such as T cells have become activated to kill tumors, or whether tumor cells are susceptible to a specific drug.
"These predictions are all based on looking at very small changes in the physical properties of cells, which can tell you how they're going to respond," says Scott Manalis, the David H. Koch Professor of Engineering in the departments of Biological Engineering and Mechanical Engineering, and a member of the Koch Institute for Integrative Cancer Research.
Manalis is the senior author of the new study, which appears today in Nature Biomedical Engineering . The paper's lead author is MIT Research Scientist Weida (Richard) Wu.
Measuring density
As cells enter new states, their molecular contents, including lipids, proteins, and nucleic acids, can become more or less crowded. Measuring the density of a cell offers an indirect view of this crowding.
The new density measurement technique reported in this study builds on work that Manalis' lab has done over the past two decades on technologies for making measurements of cells and tiny particles. In 2007, his lab developed a microfluidic device known as a suspended microchannel resonator (SMR), which consists of a microchannel across a tiny silicon cantilever that vibrates at a specific frequency. As a cell passes through the channel, the frequency of the vibration changes slightly, and the magnitude of that change can be used to calculate the cell's mass.
In 2011, the researchers adapted the technique to measure the density of cells. To achieve that, cells are sent through the device twice, suspended in two liquids of different densities. A cell's buoyant mass (its mass as it floats in fluid) depends on its absolute mass and volume, so by measuring two different buoyant masses for a cell, its mass, volume, and density can be calculated.
That technique works well, but swapping fluids and flowing cells through each one is time-consuming, so it can only be used to measure a few hundred cells at a time.
To create a faster, more streamlined system, the researchers combined their SMR device with a fluorescent microscope, which enables measurements of cell volume. The microscope is positioned at the entrance to the resonator, and cells flow through the device while floating in a fluorescent dye that can't be absorbed by cells. When cells pass by the microscope, the dip in the fluorescent signal can be used to determine the volume of the cell.
After that volume measurement is taken, the cells flow into the resonator, which measures their mass. This process, which allows for rapid calculation of density, can be used to measure up to 30,000 cells in an hour.
"Instead of trying to flow the cells back and forth at least twice through the cantilever to get cell density, we wanted to try to create a method to do a streamlined measurement, so the cells only need to pass through the cantilever once," Wu says. "From a cell's mass and volume, we can then derive its density, without compromising the throughput or the precision."
Evaluating T cells
The researchers used their new technique to track what happens to the density of T cells after they are activated by signaling molecules.
As T cells transition from a quiescent state to an active state, they gain new molecules, as well as water, the researchers found. From their pre-activation state to the first day of activation, the densities of the cells dropped from an average of 1.08 grams per milliliter to 1.06 grams per milliliter. This means that the cells are becoming less crowded, as they gain water faster than they gain other molecules.
"This is suggesting that cell density is very likely reflecting an increase in cellular water content as the cells transit from a quiescent, non-proliferative state to a high-growth state," Wu says. "These data are pointing to the notion that cell density is an interesting biomarker that is changing during T-cell activation and may have functional relevance to how well the T cells could proliferate."
Travera, a clinical-stage company co-founded by Manalis, is working on using the SMR mass measurements to predict whether individual cancer patients' T cells will respond to drugs meant to stimulate a strong anti-tumor immune response. The company has also begun using the density measurement technique, and preliminary studies have found that using mass and density measurements together gives a much more accurate prediction that using either one alone.
"Both mass and density are revealing something about the overall competency of the immune cells," Manalis says.
Using physical measurements of cells to monitor their immune activation "is very exciting and may offer a new way of evaluating and measuring changes in immune cells in circulation," says Genevieve Boland, an associate professor of surgery at Harvard Medical School and vice chair of research for the Integrated Department of Surgery at Mass General Brigham, who was not involved in the study.
"This is a complementary, but very different, method than those currently used for immune assessments in cancer and other diseases, potentially offering a novel tool to assist in clinical decision-making regarding the need for and the choice of a specific cancer therapy, allow monitoring of response to therapy, and/or in early detection of side effects of immune-based therapies," she says.
Making predictions
Another potential application for this approach is predicting how tumor cells will respond to different types of cancer drugs. In previous work, Manalis has shown that tracking changes in cell mass after treatment can predict whether a tumor cell is undergoing drug-induced apoptosis. In the new study, he found that density could also reveal these responses.
In those experiments, the researchers treated pancreatic cancer cells with one of two different drugs - one that the cells are susceptible to, and one they are resistant to. They found that density changes after treatment accurately reflected the cells' known responses to treatment.
"We capture something about the cells that is highly predictive within the first couple of days after they get taken out from the tumor," Wu says. "Cell density is a rapid biomarker to predict in vivo drug response in a very timely manner."
Manalis' lab is now working on using measurements of cell mass and density as a way to evaluate the fitness of cells used to synthesize complex proteins such as therapeutic antibodies.
"As cells are producing these proteins, we can learn from these markers of cell fitness and metabolic state to try to make predictions about how well these cells can produce these proteins, and hopefully in the future also guide design and control strategies to even further improve the yield of these complex proteins," Wu says.
The research was funded by the Paul G. Allen Frontiers Group, the Virginia and Daniel K. Ludwig Fund for Cancer Research, the MIT Center for Precision Cancer Medicine, the Stand up to Cancer Convergence Program, Bristol Myers Squibb, and the Koch Institute Support (core) Grant from the National Cancer Institute.
