Mass cytometry offers a powerful approach for single-cell multiparametric analysis, enabling the simultaneous measurement of up to 50 proteins or protein modifications. This high-throughput capability has significantly advanced our understanding of cellular heterogeneity and complex biological processes. However, a major limitation of current mass cytometry techniques is their sensitivity. The detection threshold of the instrument typically requires hundreds of metal-tagged antibodies binding to each cellular epitope, hindering the analysis of low-abundance proteins, which play crucial roles in various biological processes and disease states. This limitation has restricted the application of mass cytometry to the study of many important components of the proteome, such as transcription factors, surface receptors, and intracellular phosphorylation sites. Existing signal amplification methods, such as tyramide signal amplification (TSA) and alkaline phosphatase-mediated amplification, suffer from high nonspecific signals and limited multiplexing capabilities. Rolling circle amplification (RCA) and hybridization chain reaction (HCR), while achieving high multiplexing in other contexts, encounter issues like nonspecific background binding and amplification efficiency limitations when applied to mass cytometry. Signal amplification by exchange reaction (SABER), although successful in imaging mass cytometry, presents challenges when used with suspension mass cytometry due to stringent washing requirements and DNA instability during the high-temperature single-cell droplet vaporization step. Therefore, a robust and highly sensitive signal amplification method is needed to address these limitations and unlock the full potential of mass cytometry.

The authors review existing signal amplification strategies for mass cytometry, highlighting their limitations. TSA and alkaline phosphatase methods are criticized for their high background noise and limited multiplexing ability. RCA, while effective in RNA quantification, proves problematic in antibody-based detection due to nonspecific binding and steric hindrance. Similarly, HCR's multiplexing limitations restrict its application in high-dimensional mass cytometry. Immuno-SABER, a previously developed method, while successful in imaging mass cytometry, faces challenges in suspension-based applications due to its sensitivity to stringent washing steps and heat instability of DNA duplexes during sample introduction. The authors position their novel method, ACE, as a superior alternative addressing these critical shortcomings.

The core of the ACE method involves the use of short DNA oligonucleotide initiators conjugated to antibodies targeting the proteins of interest. These antibodies are then applied to cell suspensions (or tissue sections for IMC). An extender oligonucleotide, complementary to the initiator, is introduced. Thermal cycling facilitates repeated in situ extension of the initiator, creating multiple copies of detector oligonucleotide binding sites. Crucially, the inclusion of 3-cyanovinylcarbazole phosphoramidite (CNVK) in the metal-conjugated detector oligonucleotide provides high thermal stability, preventing signal loss during the high-temperature vaporization step in mass cytometry. The authors optimize ACE using various parameters such as the number of thermal cycles (up to 500) and demonstrate the feasibility of branching amplification to further enhance the signal. Orthogonality is validated by testing a panel of 33 initiator strands, showing minimal crosstalk. The ACE protocol, including the thermal cycling steps, photocrosslinking, and detector hybridization, is described in detail. Different applications are investigated, including suspension mass cytometry and IMC. For single-cell suspension analysis, cells undergo mass-tag barcoding and are then subjected to the ACE amplification protocol. In IMC applications, the ACE amplification is performed directly on the tissue slide prior to imaging mass cytometry analysis. The authors extensively detail the cell culture methods (including EMT/MET induction), antibody conjugation and detection oligonucleotide conjugation protocols, and the data analysis pipeline involving UMAP and Scorpius for dimensionality reduction and trajectory inference. Statistical methods employed throughout the study, including Pearson correlation, BP-R², and area under the curve (AUC)/half-maximal point (HMP) calculations, are meticulously explained.

ACE achieves over 500-fold signal amplification with minimal crosstalk between channels (average 1.07% crosstalk). The method successfully quantifies low-abundance proteins in single cells, as demonstrated by the GFP transfection experiments. Applying a 32-parameter ACE panel to mouse Py2T cells undergoing EMT and MET revealed molecular signatures involving low-abundance transcription factors, such as Zeb1 and Snail/Slug. Analysis identified the expression ratio between Zeb1 and cyclin B1 as a hallmark for cells undergoing MET. A 30-parameter ACE panel was applied to profile TCR signaling networks in human T lymphocytes. ACE significantly enhanced signals from phosphorylation sites, enabling the detailed study of TCR network dynamics in response to patient postoperative drainage fluid (POF). Analysis revealed an immunosuppressive T cell signature caused by tissue injury. In IMC, ACE facilitated highly sensitive spatial analysis of human kidney tissues. The study successfully identified six main compartments in renal cortexes and revealed heterogeneous nestin expression levels in polycystic kidney disease, indicating potential mesangial expansion.

The successful application of ACE across various experimental systems highlights its versatility and robustness. The ability to amplify low-abundance proteins dramatically expands the scope of mass cytometry applications, addressing a significant limitation of previous techniques. The findings on EMT/MET transitions provide valuable insights into the regulatory mechanisms governing these processes. The analysis of TCR signaling networks demonstrates the potential of ACE to unravel complex signaling events in immune cells, offering new avenues for understanding immune regulation and responses to injury. The application of ACE to IMC provides high-resolution spatial information, revealing tissue heterogeneity and potential biomarkers for disease states. The superior sensitivity and multiplexing capability of ACE compared to existing methods significantly advances the field of mass cytometry and enables the study of previously inaccessible biological processes.

ACE presents a transformative signal amplification technology for mass cytometry, substantially improving sensitivity and enabling the analysis of low-abundance proteins. The study successfully demonstrated its application across diverse biological systems, from single-cell analysis of EMT/MET transitions and TCR signaling networks to high-resolution spatial profiling of kidney tissue. Future research could focus on expanding the range of compatible antibodies and optimizing ACE for even higher sensitivity and multiplexing. The development of automated workflows could further enhance the scalability and accessibility of this powerful technique.

The current ACE protocol requires the use of Triton X-100 for cell permeabilization, which may affect certain cell surface proteins. The compatibility of ACE with all antibodies needs further investigation. Poor antibody specificity, while not improved by ACE, may impact signal-to-noise ratio. The reliance on commercially available antibodies also limits the current scope of targetable low-abundance proteomes. The overall cost is also slightly increased by ~US$24 for a 30 target experiment.