Flow cytometry, invented in the 1950s, uses antibodies linked to fluorescent probes to detect cell surface and intracellular proteins. Although able to achieve single-cell sensitivity, the method is limited by the number of fluorophores that can be distinguished within the spectrum of fluorescent light. The newer approach of mass cytometry allows the simultaneous quantification of 50 proteins in single cells through the use of antibodies linked to non-radioactive isotopes of metal elements, but has reduced sensitivity compared to flow cytometry and fluorescence microscopy.
Now, a research collaboration led by the Wyss Institute at Harvard University has developed a method to significantly enhance the sensitivity of mass cytometry and image mass cytometry (IMC) using DNA nanotechnology. Applying a new signal amplification technology called “Amplification by Cyclic Extension” (ACE) to DNA barcodes linked to antibodies, they were able to amplify protein signals produced by antibody-bound metal isotopes more than 500-fold, and to simultaneously (and with high sensitivity) detect more than 30 different proteins. More specifically, ACE implements “thermal-cycling-based DNA in situ concatenation in combination with 3-cyanovinylcarbazole phosphoramidite-based DNA crosslinking to enable signal amplification simultaneously on >30 protein epitopes.”
The new method enables the quantitive detection of rare proteins, investigation of complex biological tissue changes, and the study of how entire networks of interconnected proteins that regulate immune cell functions respond to stimulation and pathological conditions. Applied to IMC, ACE also allowed the identification of cell types and tissue compartments in histological sections, and changes in tissue organization related to the pathology of polycystic kidney disease.
This work is published in Nature Biotechnology in the paper, “Signal amplification by cyclic extension enables high-sensitivity single-cell mass cytometry.”
“ACE helps to close a crucial gap in cytometric analysis: by enhancing the sensitivity of mass cytometry, it enables a single cell analysis platform that simultaneously achieves high sensitivity, high multiplexing, and high throughput,” noted Peng Yin, PhD, professor at Harvard Medical School Department of Systems Biology and Wyss Institute Core Faculty member. “The opportunities it opens for investigating single cells in suspension and intact tissues with highly multiplexed and sensitive approaches can provide a much deeper understanding of normal and pathological biological processes.”
“ACE solves current sensitivity problems of mass cytometry by allowing researchers to associate antibody molecules with substantially increased numbers of metal isotopes compared to conventional mass cytometry. This significantly facilitates the quantification of a broad range of low-abundance proteins, which has been challenging using previous single-cell approaches,” said Xiao-Kang Lun, PhD, a postdoctoral fellow in Yin’s group.
ACE creates a scaffold with multiple binding sites for short metal isotope-carrying “detector strands.” By branching the synthesis of the scaffold strand, the researchers could further increase the method’s sensitivity for the detection of rare proteins. Linear ACE on average provides a 13-fold signal amplification while branching ACE allows an initially unamplified signal to be increased more than 500-fold. To stabilize the entire ACE sequence complex and keep it intact during mass cytometry analysis, they crosslinked the short double strands formed between the scaffold and the added detector strands with a chemical crosslinker.
“Following this recipe, we designed a panel with 33 distinguishable (orthologous) ACE sequences whose synthesis doesn’t interfere with one another, and applied it to three entirely different types of analysis,” said Kuanwei Sheng, PhD, a postdoctoral fellow on Yin’s team who had initially developed ACE for other applications, including multiplexed imaging.
The team first used ACE to investigate the transitions of epithelial cells into mesenchymal cells and back into epithelial cells again. Epithelial-mesenchymal transitions (EMTs) and mesenchymal-epithelial transitions (METs) occur during embryonic development but the former in particular is also re-enacted when tumors become invasive and metastatic. By profiling 32 epithelial and mesenchymal markers, signaling molecules, and rare transcription factors in single mouse breast cancer cells multiple times during their 28-day transition from an epithelial to a mesenchymal state and back, and computationally parsing the results, they were able to shed new light on the two processes. “ACE allowed us to profile levels of low-abundance transcription factors simultaneously with markers reflecting cellular physiological and signaling states in single cells. This led to a more refined picture of how molecular programs in EMT and MET are driven by increasing and decreasing amounts of key transcription factors, including Zeb-1 and Snail/Slug,” said Sheng.
The team also applied ACE to a panel of 30 antibodies that specifically bound to phosphorylated motifs in T-cell receptor (TCR)-network proteins. “Using ACE-enhanced mass cytometry analysis, we captured quantitative snapshots of the dynamically changing TCR network in individual primary human T cells. This allowed us to study the single-cell variations in the timing and duration of specific T-cell activation events and to reveal how the network is activated from its ground state by extracellular cues,” said Lun.
The team used the same ACE-enhanced antibody panel to investigate “injury-induced T-cell paralysis.” T cells experiencing injury in their environment, such as tissue injuries caused in major surgical procedures, often become immunosuppressive. To understand how the TCR network causes this, the team analyzed samples of “postoperative drainage fluid” (POF) that were obtained from patients undergoing surgery. Stimulating T cells with POFs as well as their TCRs enabled the researchers to isolate distinct network changes that cause single T cells to stop dividing and become exhausted.
Finally, they investigated the utility of ACE for spatial analysis of proteins in tissue sections using IMC. The researchers developed a panel of 20 ACE-enhanced antibodies for various kidney markers and used it to examine sections of the renal cortex derived from a patient with polycystic kidney disease. This approach allowed them to identify the 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 features of cell and tissue organization and found that the stem cell marker Nestin, which is also associated with kidney disorders, was expressed very heterogeneously across glomeruli,” said Lun. “This could mean that different parts of the tissue could be simultaneously going through different pathological stages.”
“This new mass cytometry approach developed by Peng Yin’s team and their collaborators once again shows the power of leveraging DNA nanotechnology to turbocharge an existing technique that is highly relevant for clinical care, and to bring it to a much higher level of sensitivity and specificity,” said Donald Ingber, MD, PhD, Wyss founding director whose group provided critical expertise on stimulating T cells. “This relatively simple method will lead to entirely new insights into cell, tissue, and organ function, both during health and disease.”
