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 Massachusetts Institute of Technology (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,” said Scott Manalis, PhD, 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. Senior author Manalis, together with lead author, MIT researcher Weida (Richard) Wu, PhD, and colleagues, described their study in Nature Biomedical Engineering, in a paper titled, “High-throughput single-cell density measurements enable dynamic profiling of immune cell and drug response from patient samples.” In their paper, they concluded, “Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a biomarker for functional precision medicine.”
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. “Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and a fundamental determinant of cell state and function,” the authors wrote. “The coupling between crowding level and cell physiology makes cell density a key proxy for characterizing fundamental cellular processes such as proliferation, apoptosis, metabolic shifts, and differentiation, indicating its potential as a biomarker for cellular fitness and drug response. However, they pointed out, “… existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples.”
The new density measurement technique reported by Manalis et al. builds on work in the Manalis lab over the past two decades on technologies for making measurements of cells and tiny particles. In 2007, the 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. “… the throughput of this approach is also limited to a few hundred cells per experiment because it requires cells to be sequentially measured in two types of fluids,” the team noted.
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 explained. “From a cell’s mass and volume, we can then derive its density, without compromising the throughput or the precision.” The authors further explained, “Here, we present a fluorescence exclusion-coupled SMR (fxSMR) platform that simultaneously measures single-cell buoyant mass and volume, which allows us to profile cell density with a throughput of over 30,000 cells per hour and a precision of 0.03% (0.0003 g ml−1) for cells larger than 12 μm in diameter.”
The researchers used their new technique to track what happens to the density of T cells after they are activated by signaling molecules. “We performed daily measurements of CD8+ T cells from two human donors after anti-CD3 and anti-CD28 activation,” they noted. 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 g/mL to 1.06 g/mL. 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 commented. “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 that are expected to stimulate a strong antitumor 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 than using either one alone. “Both mass and density are revealing something about the overall competency of the immune cells,” Manalis said.
Another potential application for this approach is predicting how tumor cells will respond to different types of cancer drugs. In previous work, Manalis showed that tracking changes in cell mass after treatment can predict whether a tumor cell is undergoing drug-induced apoptosis. In the new study, the team found that density could also reveal these responses. “Since changes in cell density can reveal state transitions related to cell proliferation, we sought to determine whether it could be used as a biomarker for assessing the ex vivo treatment response of cancer cells,” they wrote.
In those experiments, the researchers treated pancreatic cancer cells with one drug that the cells are susceptible to, and a different drug to which they are resistant. The team 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 noted. “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 noted.
In their paper the team concluded, “In addition to its potential for exploration of the biology of density homeostasis, our approach may provide a much-needed method for functional precision medicine in patients … With our high-throughput approach, we believe that many drugs can now be more readily profiled on patient samples within a hyperacute time window, thereby enabling clinical studies for assessing the effectiveness of density response in guiding patient treatment.”
The post Rapid Method for Cell Density Measurement Could Help Predict Tumor Response to Treatment appeared first on GEN - Genetic Engineering and Biotechnology News.
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 Massachusetts Institute of Technology (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,” said Scott Manalis, PhD, 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. Senior author Manalis, together with lead author, MIT researcher Weida (Richard) Wu, PhD, and colleagues, described their study in Nature Biomedical Engineering, in a paper titled, “High-throughput single-cell density measurements enable dynamic profiling of immune cell and drug response from patient samples.” In their paper, they concluded, “Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a biomarker for functional precision medicine.”
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. “Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and a fundamental determinant of cell state and function,” the authors wrote. “The coupling between crowding level and cell physiology makes cell density a key proxy for characterizing fundamental cellular processes such as proliferation, apoptosis, metabolic shifts, and differentiation, indicating its potential as a biomarker for cellular fitness and drug response. However, they pointed out, “… existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples.”
The new density measurement technique reported by Manalis et al. builds on work in the Manalis lab over the past two decades on technologies for making measurements of cells and tiny particles. In 2007, the 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. “… the throughput of this approach is also limited to a few hundred cells per experiment because it requires cells to be sequentially measured in two types of fluids,” the team noted.
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 explained. “From a cell’s mass and volume, we can then derive its density, without compromising the throughput or the precision.” The authors further explained, “Here, we present a fluorescence exclusion-coupled SMR (fxSMR) platform that simultaneously measures single-cell buoyant mass and volume, which allows us to profile cell density with a throughput of over 30,000 cells per hour and a precision of 0.03% (0.0003 g ml−1) for cells larger than 12 μm in diameter.”
The researchers used their new technique to track what happens to the density of T cells after they are activated by signaling molecules. “We performed daily measurements of CD8+ T cells from two human donors after anti-CD3 and anti-CD28 activation,” they noted. 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 g/mL to 1.06 g/mL. 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 commented. “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 that are expected to stimulate a strong antitumor 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 than using either one alone. “Both mass and density are revealing something about the overall competency of the immune cells,” Manalis said.
Another potential application for this approach is predicting how tumor cells will respond to different types of cancer drugs. In previous work, Manalis showed that tracking changes in cell mass after treatment can predict whether a tumor cell is undergoing drug-induced apoptosis. In the new study, the team found that density could also reveal these responses. “Since changes in cell density can reveal state transitions related to cell proliferation, we sought to determine whether it could be used as a biomarker for assessing the ex vivo treatment response of cancer cells,” they wrote.
In those experiments, the researchers treated pancreatic cancer cells with one drug that the cells are susceptible to, and a different drug to which they are resistant. The team 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 noted. “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 noted.
In their paper the team concluded, “In addition to its potential for exploration of the biology of density homeostasis, our approach may provide a much-needed method for functional precision medicine in patients … With our high-throughput approach, we believe that many drugs can now be more readily profiled on patient samples within a hyperacute time window, thereby enabling clinical studies for assessing the effectiveness of density response in guiding patient treatment.”
The post Rapid Method for Cell Density Measurement Could Help Predict Tumor Response to Treatment appeared first on GEN - Genetic Engineering and Biotechnology News.