introduction
In modern life science research, mass cytometry is capable of simultaneously measuring approximately 50 proteins or protein modifications in millions of single cells by using metal isotope-labeled antibodies to label targets of interest. However, current mass spectrometry cytometry is limited in terms of sensitivity, often requiring hundreds of metal-labeled antibodies to bind to each cell epitope to reach the instrument's detection threshold. This limits the analysis of low-abundance protein fractions, including many transcription factors, surface receptor proteins, and intracellular phosphorylation sites, which play an important role in health and disease. On July 29, Nature Biotechnology reported "Signal amplification by cyclic extension enables high-sensitivity single-cell mass cytometry", introducing a method called Amplification by Cyclic Extension (ACE) Signal amplification is achieved by thermally cycling DNA in situ ligation and DNA cross-linking of 3-cyanovinylcarbazole phosphoramide. ACE technology enables simultaneous signal amplification on more than 30 protein epitopes, significantly increasing the detection sensitivity of low-abundance proteins. The research team demonstrated the application of ACE technology in the characterization of molecular reprogramming during epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) by suspension mass spectrometry cytometry. At the same time, ACE technology also demonstrated its ability to quantify the response dynamics of human T lymphocyte signaling networks. In addition, by incorporating imaging mass cytometry (IMC), ACE technology enables multiparametric tissue imaging to identify tissue compartments and analyze spatial features associated with pathological status. This study demonstrates that ACE technology not only solves the sensitivity challenges of mass cytometry, but also enables the analysis of low-abundance protein fractions at the single-cell level, expanding the application potential of mass cytometry in life science research. Through IMC analysis of human kidney tissue, ACE technology revealed six major compartments in the renal cortex and demonstrated heterogeneity in the expression levels of the stem cell marker nestin in polycystic kidney disease tissues. Overall, ACE technology provides a highly sensitive method for mass spectrometry cytometry analysis, enabling the profiling of low-abundance protein fractions at the single-cell level, and providing important technical support for the research and diagnosis of related diseases.
Mass cytometry is a technique that enables the simultaneous measurement of approximately 50 proteins or protein modifications in millions of cells at the single-cell level by using metal isotope-labeled antibodies to label targets of interest. However, current mass spectrometry cytometry is limited in terms of sensitivity, often requiring hundreds of metal-labeled antibodies to bind to each cell epitope to reach the instrument's detection threshold. This limitation hinders the analysis of low-abundance protein fractions, including many transcription factors, surface receptor proteins, and intracellular phosphorylation sites that play important roles in health and disease.
为了克服质谱细胞术的灵敏度限制，研究团队开发了一种新型的信号放大技术，称为循环扩增放大(Amplification by Cyclic Extension, ACE)。 该技术通过热循环DNA原位连接和3-氰基乙烯基咔唑(3-cyanovinylcarbazole, CNVK)磷酰胺的DNA交联来实现信号放大。
研究中使用了小鼠乳腺癌Py2T细胞，首先用4ng/ml的转化生长因子β1(TGFβ1)处理14天以诱导间充质转化(epithelial-to-mesenchymal transition, EMT)，然后去除TGFβ1，使细胞在接下来的14天内回复到上皮状态(mesenchymal-to-epithelial transition, MET)。 在这一过程中，在不同的时间点(0、1、2、3、6、9、14、17、21、24、28天)收集细胞样本。
循环扩增放大(Amplification by Cyclic Extension, ACE)技术及其应用的示意图（Credit: Nature Biotechnology）
ACE Method Overview: Figure a details how the ACE method works, including steps such as initial labeling, primer extension, thermal cycling, and metal probe hybridization. Demonstrates how primer chains can be amplified through multiple thermal cycles, significantly amplifying the metal signal on each antibody. Applications in Suspension Mass Cytometry: Figure b illustrates the application of ACE in suspension mass cytometry, illustrating that ACE technology can amplify the signal, enabling the detection and quantification of low-abundance markers in mass cytometry. Applications in Imaging Mass Cytometry: Figure c illustrates the application of ACE in combination with Imaging Mass Cytometry (IMC). The figure shows the highly sensitive multiparametric spatial analysis of ACE technology in tissue samples, enabling the identification of cells and tissue compartments in healthy and diseased tissue samples.
Cycle amplification amplifies initial labeling: The antibody targeting the protein is first bound to a short DNA oligonucleotide primer (TT-a, 11-mer). Labeling applications: Primer-bound antibodies are applied to cell suspensions for cell surface or intracellular labeling. Primer extension: Primer extension of the primer strand is mediated by Bst polymerase by introducing an elongated oligonucleotide (a*-T-a*, 19-mer) that is complementary to the primer, hybridized at the appropriate temperature. Thermal cycling: Through multiple thermal cycling (1 minute per cycle), the primers are progressively extended to form hundreds of repeats. Metal probe hybridization: Finally, the probe containing the metal ions is hybridized to the extended primers, which significantly amplifies the metal signal on each antibody.
ACE技术在质谱细胞术中的验证和信号放大的定量结果（Credit: Nature Biotechnology）
HEK293T cell validation: Figure a illustrates the experimental design of HEK293T cells transiently transfected with green fluorescent protein (GFP) to validate the ACE scale-up method. The specificity and amplification efficiency of ACE were verified by ACE amplification of GFP-expressing cells and compared with conventional fluorescently labeled antibodies and immunosmune-labeled SABER (immunorolling circle amplification). Signal correlation: Figure b illustrates the signal amplification of ACE amplification through 1 to 500 thermal cycles and compares to conventional metal-labeled secondary antibodies. The results showed that the Pearson correlation coefficient between the ACE-amplified GFP signal and the secondary antibody signal was high under all conditions, which verified the specificity of ACE in intracellular epitope staining. Signal Enhancement and Specificity: Figures c and D illustrate the effect of signal enhancement through different thermal cycles. The data are divided into 10 equally wide intervals, showing the median of each interval at different thermal cycles. The results show that the signal amplification efficiency is highest in the first 100 cycles (about 2 hours), and then the amplification efficiency gradually decreases. Amplification Intensity and Signal-to-Noise Ratio: Figure e shows the signal strength and signal-to-noise ratio over 500 cycles of amplification. The results showed that 500 cycles of amplification could achieve 13-fold amplification intensity and 6-fold signal-to-noise ratio enhancement compared to the unamplified control. Branching amplification: Figure F illustrates further signal enhancement by introducing branching primers (a*-T-a*-b). With additional thermal cycling, the number of probe sites is significantly increased, with a further 9-fold signal enhancement achieved by 50 thermal cycling and an additional 5-fold signal increase by secondary branching compared to linear amplification, for a total of 500-fold initial signal amplification. Orthogonality verification: Figures g and h show the orthogonality verification of 33 primer sequences in the ACE amplification system. The results showed that the average crosstalk signal between the 33 primer sequences was only 1.02%, validating the high specificity and orthogonality of the ACE primers and detectors after individual staining, barcoding and pooling of GFP antibody-labeled HEK293T cells.
Application in EMT and MET processes Using ACE technology to analyze mouse breast cancer Py2T cell models, the research team was able to detect molecular reprogramming that occurs during EMT and MET with high sensitivity. Changes in Zeb1 and Snail/Slug expression: During EMT, the expression of Zeb1 increased dramatically after day 6, while the level of Snail/Slug slowly decreased during the first 3 days and showed a secondary peak on day 6. Phenotypic changes: During TGFβ1 treatment, the expression of epithelial markers E-cadherin, CK14, EpCAM, and β-catenin decreased, and the expression of mesenchymal markers vimentin and CD44 increased. In contrast, the expression of these markers is reversed after TGFβ1 removal.
Analysis of molecular regulation caused by different transcription factor expression levels during epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) by multiplex ACE technology (Credit: Nature Biotechnology)
Experimental Flow: Figure A illustrates the overall design and flow of the experiment. Mouse breast cancer Py2T cells are treated with transforming growth factor β1 (TGFβ1) for 14 days to induce EMT, and then TGFβ1 is removed for a 14-day MET reversal process. Cells were collected at different time points (0, 1, 2, 3, 6, 9, 14, 17, 21, 24, 28 days) for ACE labeling and mass spectrometry analysis. Dimensionality Reduction Analysis: Figures b and c illustrate the results of dimensionality reduction analysis of data using the Uniform Manifold Approximation and Projection (UMAP) method. Panel B color-codes cells based on treatment time points, and panel c color-codes cells based on the abundance of measurement markers. These graphs show changes in cell phenotype and molecular signatures during EMT and MET. Pseudo-time analysis: Figure d shows the results of the reconstruction of the EMT-MET process using the Scorpius pseudo-time analysis method. Comparing the pseudo-time with the actual time, it was found that the molecularly defined mesenchymal phenotype began to emerge on day 6 and disappeared completely 9 days after TGFβ1 stimulation. During MET, the cell population with epithelial molecular properties gradually expands over a 14-day time series, while most of the cells maintain their mesenchymal state. Molecular Regulatory Trajectories: Figure e illustrates the molecular regulatory trajectories of markers measured by Scorpius analysis during EMT and MET. The results showed that the increase in Zeb1 expression occurred in the late phase of EMT, accompanied by the downregulation of CK14. During reverse MET, a decrease in Zeb1 expression was associated with a sharp decrease in vimentin expression, while an increase in E-cadherin level occurred earlier. Cell population characteristics: Figure F shows the biaxial plot of cell populations with high Zeb1 expression and low cyclin B1 expression and cell populations with low Zeb1 expression and high cyclin B1 expression at different time points during the MET process. The results showed that the cell population with low Zeb1 expression and high cyclin B1 expression gradually increased during the MET process. Marker Expression Levels: Figure g shows the expression levels of vimentin, E-cadherin, and CK14 in cell populations with high Zeb1 expression and low cyclin B1 expression and cell populations with low Zeb1 expression and high cyclin B1 expression. The results showed that the expression levels of Zeb1 and cyclin B1 were closely related to the phenotypic characteristics of the cells.
Analysis of the Human T Lymphocyte Signaling NetworkThrough ACE technology, the research team was able to analyze the dynamic changes of the human T lymphocyte signaling network with high sensitivity: TCR signaling: Signaling at key phosphorylation sites in the TCR signaling pathway (e.g., p-CD3ζ, p-ZAP70, p-SLP76, p-ERK1/2) was significantly enhanced by anti-CD3/anti-CD28 stimulation, showing a strong signaling response. Immunosuppressive analysis: Co-culture with postoperative drainage fluid (POF) found that POF1 and POF2 samples resulted in a reduced and shorter TCR signal response, suggesting that these samples were immunosuppressive.
Multiparametric Tissue Imaging ACE Techniques Combined with Imaging Mass Spectrometry Cytometry (IMC), multiparametric tissue imaging analysis is possible: Renal Tissue Analysis: When analyzing renal cortical tissue in patients with polycystic kidney disease, ACE technology reveals six major compartments in the renal cortex and demonstrates the heterogeneous expression of the stem cell marker nestin in these compartments. Tissue compartment identification: IMC analysis enables the identification and differentiation of different types of tubular, glomerular, and vascular compartments, providing detailed tissue structure information.
ACE technology provides a powerful tool for the analysis of low-abundance protein fractions at the single-cell level by significantly increasing the sensitivity of mass spectrometry cytometry. This not only expands the application potential of mass spectrometry cytometry in life science research, but also provides important technical support for the research and diagnosis of related diseases. Through highly sensitive multiparameter analysis, ACE technology makes it possible to map low-abundance protein fractions at the single-cell level, providing new perspectives and methods for scientific research.
bibliography
Lun XK, Sheng K, Yu X, Lam CY, Gowri G, Serrata M, Zhai Y, Su H, Luan J, Kim Y, Ingber DE, Jackson HW, Yaffe MB, Yin P. Signal amplification by cyclic extension enables high-sensitivity single-cell mass cytometry. Nat Biotechnol. 2024 Jul 29. doi: 10.1038/s41587-024-02316-x. Epub ahead of print. PMID: 39075149.https://www.nature.com/articles/s41587-024-02316-x
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