ACE technology enhances single-cell protein detection through advanced signal amplification.

Since the 1950s, researchers have used a well-known method invented by Wallace Coulter called “flow cytometry” to characterize different types of immune cells in research studies and human blood samples. This has led to a better understanding of immune cell development and has also led to new ways to assess human health and diagnose various blood cancers. Flow cytometry has since been applied to other cell types.

In traditional flow cytometry, cell surface and intracellular proteins are detected with antibody molecules linked to fluorescent probes. However, although this method offers single-cell sensitivity, it is limited in its ability to detect multiple proteins by the number of fluorophores that can be clearly distinguished within the entire fluorescence spectrum. The advent of “mass cytometry” in 2009 enabled the simultaneous quantification of 50 different proteins in single cells, providing a finer analysis of the cell’s identity and physiological state. In mass cytometry, antibodies are linked to non-radioactive isotopes of metallic elements. These isotopes can be quantified in different channels of the mass cytometer instrument based on their mass. However, mass cytometry and its cousin “image mass cytometry” (IMC), used to visualize cellular proteins in intact tissue sections, suffer from lower sensitivity compared to flow cytometry and fluorescence microscopy.

Now, another 15 years later, a collaboration led by the Wyss Institute at Harvard University, and involving researchers from MIT and the University of Toronto, has developed a method to significantly improve the sensitivity of mass cytometry and IMC using DNA nanotechnology. By applying a new signal amplification technique called “Amplification by Cyclic Extension (ACE)” to DNA barcodes linked to antibodies, the protein signal generated by metal isotopes attached to the antibodies was amplified by more than 500-fold, allowing for the simultaneous and highly sensitive detection of more than 30 proteins. This new method allowed for the quantitative detection of rare proteins, the investigation of changes in complex biological tissues, and the study of how the entire network of interconnected proteins that control immune cell function responds to stimuli and pathological conditions. ACE applied to IMC also enabled the identification of cell types and tissue compartments in tissue sections, as well as changes in tissue organization associated with polycystic kidney disease pathology. The results showed that Nature Biotechnology.

“ACE helps fill a critical gap in cytometric analysis. By increasing the sensitivity of mass cytometry, we enable a single-cell analysis platform that is simultaneously highly sensitive, highly multiplexed, and high-throughput. It opens the opportunity to interrogate single cells in suspension and intact tissues in a highly multiplexed and sensitive approach, providing a deeper understanding of normal and pathological biological processes,” said Peng Yin, PhD, Wyss Institute Core Faculty Member who led the study. Dr. Yin is also a professor in the Department of Systems Biology at Harvard Medical School (HMS).

More DNA means more metal isotopes, which increases sensitivity

To date, Yen and his group at the Wyss Institute have developed several DNA-driven imaging techniques that can reveal the inner workings of cells at ultra-high resolution at the single-molecule level, as well as visualize many different RNA and protein molecules within a single tissue section. However, the DNA structures created using these methods are not durable enough to withstand the relatively harsh conditions used in mass cytometry.

“ACE solves the current sensitivity issue of mass cytometry by allowing us to associate antibody molecules with a significantly increased number of metal isotopes compared to traditional mass cytometry, which greatly facilitates quantification of a wide range of low-abundance proteins that have been difficult to quantify with single-cell approaches to date,” said co-first author Xiao-Kang Lun, PhD, a postdoctoral researcher in Yin’s group. Dr. Lun worked on the project in collaboration with co-first author Kuanwei Sheng, PhD, who initially developed ACE for other applications such as multiplexed imaging and works with Dr. Yin. “Inspired by our previous work on primer extension reactions to create linear DNA concatamers (multiple copies of the same DNA sequence linked in series) and the PCR reaction, which achieves amplification by synchronized thermal cycling, we invented ACE to synthesize linear concatamers. On-site This is achieved through thermal cycling in a controllable way,” Shen said.

ACE creates a scaffold with multiple binding sites for short “detection strands” carrying metal isotopes. Moreover, by branching the synthesis of the scaffold strands, the researchers were able to further increase the sensitivity of this method for the detection of rare proteins. While linear ACE provides a 13-fold signal amplification on average, branched ACE increases the initially unamplified signal by more than 500-fold. To stabilize the entire ACE array complex and keep it intact during mass cytometry analysis, the short duplexes formed between the scaffold and the added detection strands were crosslinked with a chemical crosslinker. “Following this recipe, we designed a panel containing 33 identifiable (orthologous) ACE arrays whose synthesis does not interfere with each other and applied it to three completely different types of analysis,” says Sheng, who is also a postdoctoral researcher in Yin’s team.

ACE’s Work

The team first used ACE to investigate the transition from epithelial to mesenchymal cells and back again. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) occur during embryonic development, but the former is also recapitulated when tumors become invasive and metastatic. By profiling a total of 32 epithelial and mesenchymal markers, signaling molecules, and rare transcription factors in a single mouse breast cancer cell multiple times during its 28-day transition from epithelial to mesenchymal state and back, and then computationally analyzing the results, they were able to shed new light on the two processes.

“ACE enabled us to profile the levels of low-abundance transcription factors in parallel with markers reflecting cell physiological and signaling states in single cells. This enabled us to gain a more detailed understanding of how the molecular programs of EMT and MET are driven by increased or decreased abundance of key transcription factors such as Zeb-1 and Snail/Slug.”

Dr. Quan-Wei Shen, first author

In the second example, the researchers focused on the inner workings of a single T cell. When surface T cell receptor (TCR) molecules are stimulated, a complex network of intracellular signaling proteins is activated. Analyzing these signaling responses at single-cell resolution has been difficult, in part because of the small size of T cells. Individual proteins in this network are activated by phosphate residues bound by other network proteins, commonly known as kinases. Many of these activated network proteins then phosphorylate other proteins in the network. This ultimately leads to changes in the behavior of the T cell, for example, against pathogens or cancer cells. The researchers applied ACE to a panel of 30 antibodies that specifically bind to phosphorylation motifs in TCR network proteins that function in stress, inflammation, cell proliferation, and other responses. “Using ACE-enhanced mass cytometry analysis, we obtained a quantitative snapshot of the dynamically changing TCR network within individual primary human T cells. This allowed us to study single-cell variations in the timing and duration of specific T cell activation events and reveal how the network is activated from a basal state by extracellular signals,” Lun said.

The research team used the same ACE-enhanced antibody panel to investigate a phenomenon they call “injury-induced T cell paralysis.” T cells that experience injury in their environment, such as tissue damage from major surgery, often become immunosuppressed. To understand how the TCR network causes this, Yin’s group collaborated with co-author Michael Yaffe, M.D., Ph.D., the David H. Koch Professor of Science and Professor of Biology and Bioengineering at MIT, who has a strong interest in how the microenvironment around sites of tissue injury suppresses the immune system. Yaffe provided the research team with samples of “post-operative effluent” (POF) taken from patients who had undergone surgery. By stimulating T cells with POF and their TCRs, the researchers were able to isolate distinct network changes that cause single T cells to stop dividing and become exhausted.

Finally, the researchers also investigated the utility of ACE in the spatial analysis of proteins in tissue sections using IMC, focusing on the human kidney. Kidney tissue is difficult to analyze with a fluorescent microscope due to its strong autofluorescence, and even with conventional IMC due to a lack of sensitivity. The researchers developed a panel of 20 ACE-enhanced antibodies against various kidney markers and used them to examine sections of renal cortex taken from patients with polycystic kidney disease. In collaboration with co-author Hartland Jackson, PhD, a professor at the University of Toronto in Canada and an expert in multiplex imaging, this approach allowed them to identify different cell types and their organization within the kidney’s proximal and distal tubules, collecting ducts, and blood-filtering glomeruli. “We discovered new disease-specific signatures of cell and tissue organization, and found that the stem cell marker nestin, which is also associated with kidney disease, is expressed very heterogeneously throughout the glomerulus,” Lun said. “This could mean that different parts of the tissue may be undergoing different pathological stages at the same time.”

“This new mass cytometry method developed by the Peng Yin team and collaborators once again demonstrates the power of leveraging DNA nanotechnology to accelerate an existing technique that is highly relevant to clinical care, taking its sensitivity and specificity to much higher levels. This relatively simple method will lead to entirely new insights into the function of cells, tissues and organs both in health and disease,” said co-senior author and Wyeth Founding Director Donald Ingber, M.D., Ph.D., who provided key expertise on T cell stimulation. He also Judah Folkman Professor of Vascular Biology HMS and Boston Children’s Hospital, Hansjörg Wyss Professor of Bio-Inspired Engineering at the John A. Paulson School of Engineering and Applied Sciences at Harvard University.

Other authors on the study are Xueyang Yu, Ching Yeung Lam, Gokul Gowri, Matthew Serrata, Yunhao Zhai, Hanquan Su, Jingyi Luan and Youngeun Kim. This research was supported by grants from the National Institutes of Health (grant numbers ES028374, CA226898, UG3HL145600, UH3CA255133, DP1GM133052, R01GM124401, RF1MH124606 and RF1MH128861) and the Ontario Cancer Institute.