A dash of DNA improves protein identification in single cells

Many diseases and treatments rely on knowing not only what proteins are behaving abnormally, but also where in the cell they are acting. Without this information, developing new therapeutics to tackle problems at their roots can be difficult — or even impossible.

Current methods for discerning the levels and locations of proteins in the cell often require users to choose between sensitivity and scale. Flow cytometry, which offers single-cell resolution, is limited in the number of parameters it can examine simultaneously; mass cytometry expands those parameters, but the process takes longer and can’t effectively detect low-abundance proteins (1). Now, researchers have developed a new technique that combines the sensitivity of flow cytometry with the wide net cast by mass cytometry to yield new insights into disease and treatment (2).

“Mass cytometry is a really versatile technology, but sensitivity is always a problem,” study coauthor Xiao-Kang Lun, a postdoctoral scholar at the Wyss Institute at Harvard University, explained. “A lot of the markers we’re interested in, especially rare posttranslational modifications, are not abundant enough to be detectable, but they’re critical to answering biological questions.”

For that reason, Lun and his colleagues decided to experiment with ways of increasing the sensitivity of mass cytometry. Inspired by primer exchange and polymerase chain reactions (PCR), which allow predesigned DNA sequences to form long repeating chains, the researchers developed a technique called amplification by cyclic extension (ACE). In this technique, an antibody attached to a short DNA sequence binds to the target protein of interest. A PCR-like process then extends that DNA, ultimately yielding hundreds of copies of the short sequence that each bind to a metal ion detected by the mass cytometer. In conventional mass cytometry, a single protein molecule is labeled with just one metal ion, meaning that the signals from low-abundance proteins would be too weak to detect, but attaching each protein to hundreds of ions amplifies the signal and makes the proteins visible.

The first time the researchers tried the technique, however, they saw very little signal and a lot of background noise. Realizing that the mass cytometry process was heating their extended DNA sequence up to the point where it denatured and the metal ions detached, they added a photo-crosslinking step to stabilize the DNA and make it “heat-proof.”

Lun and his colleagues envision a wide range of applications for ACE mass cytometry. One crucial opportunity includes examining previously challenging aspects of the tumor microenvironment, such as posttranslational modifications on T cell markers. Because T cells have limited cytosolic material, many of these modifications are too scarce to be detected by conventional techniques, but a better understanding of their role in cancer could support the development of new therapeutics.

A lot of the markers we’re interested in, especially rare posttranslational modifications, are not abundant enough to be detectable, but they’re critical to answering biological questions. 
- Xiao-Kang Lun, Wyss Institute at Harvard University

To validate ACE in T cell signaling, the researchers created a 30-antibody ACE panel that allowed them to examine phosphorylated proteins within a single T cell. Without ACE, quantifying the levels of these phosphoproteins was difficult or even impossible. With ACE, however, the signal was amplified an average of 17-fold, making it easy to detect, compare, and describe relationships between proteins in the T cell signaling network. For further reinforcement, the researchers used their panel to gain insights into the immunosuppressive effects of tissue injury. By examining T cell signaling in samples of human postoperative drainage fluid, they determined which signaling molecules were suppressed, knowledge that could one day lead to therapeutic interventions.

Imaging mass cytometry can also benefit from ACE’s added sensitivity. “Low-abundance proteins are sometimes very important in regulating cells,” said Yetrib Hathout, a pharmaceutical researcher at Binghamton University who was not involved in the study. “Transcription factors, for instance, are always low-abundance and difficult to detect or quantify by conventional methods.” By applying ACE to imaging mass cytometry, these targets could be visualized and localized, potentially leading to advancements from intraoperative tumor detection to analyzing and quantifying treatment responses.

In fact, Lun and his colleagues investigated transcription factors, developing a 32-antibody ACE panel to gain insight into how cells transition between epithelial and mesenchymal states, a process that underlies wound healing, organ fibrosis, cancer, and much more. Performing ACE mass cytometry on samples from 11 timepoints throughout the transition revealed the behaviors of several low-abundance transcription factors whose changing levels over time had not previously been observed. Factors like Zeb1, which helps regulate the epithelial-to-mesenchymal transition in cancer progression, are of particular interest because drugs targeting these factors may help prevent metastasis and treatment resistance (3).

Lun and his colleagues are currently working to improve ACE further, for example, by developing protocols that don’t require a permeabilizing detergent, which can damage cell surface markers of interest. They are also working on ways to reduce or avoid the time-consuming antibody conjugation process. The need for highly specific, validated antibodies also presents a challenge; currently, ACE mass cytometry can’t be used for discovery or to examine markers that lack such antibodies.

Nonetheless, the future of ACE is promising. “This is a good technique for understanding the mechanisms of diseases and identifying better therapeutic targets,” said Hathout. “Right now, we just know when a biomarker increases or decreases, but what’s the function of that protein? Where is it in the cell? This breakthrough can tell us.”

References

1. Maecker, H.T. & Harari, A. Immune monitoring technology primer: flow and mass cytometry. J Immunother Cancer  3, 44 (2015).
2. Lun, X.K. et al. Signal amplification by cyclic extension enables high-sensitivity single-cell mass cytometry. Nat Biotechnol (2024).
3. Zhang, P., Sun, Y. & Ma, L. ZEB1: at the crossroads of epithelial-mesenchymal transition, metastasis and therapy resistance. Cell Cycle  14, 481487 (2015).