Mass Cytometry for Comprehensive Single-Cell Proteomics

Mass cytometry, which enables high-dimensional measurement of numerous proteins across millions of individual cells, has driven significant advances in single-cell protein analysis. This technology offers a detailed view of cellular diversity and functional states within complex tissues and cell types. Investigating this diversity is essential for uncovering predictive biomarkers that enable improved therapeutic development and understanding of disease mechanisms. In this article, we review the use of mass cytometry for single-cell protein analysis, covering its benefits, capabilities, applications, and future.

Basics of mass cytometry

Cytometric methods measure various cell characteristics, including cell markers, size, and morphology. Among these methods, mass cytometry, also known as cytometry by time-of-flight (CyTOF^®), differs significantly from traditional approaches. “Mass cytometry is a cytometric technique that measures metal-conjugated antibodies bound to cell antigens,” explained Jennifer Ellis, Director of Scientific Content at Standard BioTools. After ionization, a mass spectrometer detects the metal tags, enabling highly multiplexed protein measurement in single cells. “CyTOF technology gives researchers the ability to interrogate more than 50 surface and functional markers in one experiment to reveal critical biomarkers of predictive response, mechanism of action, and patient stratification in translational and clinical research,” added Ellis.

Key advantages and capabilities

Mass cytometry overcomes many of the limitations experienced with similar technologies. Ellis explained that CyTOF solutions "capture highly multiplexed surface and functional markers simultaneously through the ability to precisely and accurately distinguish between reporter molecules." When compared with flow cytometry, she emphasized that the CyTOF platform has shown lower coefficients of variation, and the resulting data is more dependable for broad, longitudinal studies, capturing details from cell types and low-abundance intracellular markers. CyTOF technology also has much higher parameter capabilities to detect all markers at once compared to spectral flow cytometry, and researchers can easily use barcoding to multiplex samples in a single tube, further improving reproducibility.

The high-parameter capabilities of mass cytometry are particularly valuable because they allow researchers to measure many different features of cells at once and study complex cell interactions as a unified system. “Scalable throughput is essential for thorough characterization of this type of complexity, where vital rare cell populations might otherwise be missed,” Ellis stated. “This utility extends to further investigating cellular metabolism, including protein levels, posttranslational modifications, and proteolysis products that can all be quantified from a single experiment.”

Integration and analysis

Mass cytometry is often integrated with other technologies to gain more comprehensive insights. Frequently used together, CyTOF is an ideal complement to fluorescent technologies given its unique rapid and scalable immune profiling capabilities across sites or timepoints and ability to capture a wide range of functional markers in a single panel. Leveraging CyTOF can be an important difference in capturing new or unexpected targets. Ellis noted that if researchers do not have local access to instrumentation or expertise, samples can be easily shipped to service labs. Mass cytometry can also be used to validate single-cell RNA sequencing (scRNA-seq) results, or these technologies can be combined to provide a deeper level of understanding of the phenotypic and functional diversity of cell populations within a single sample. This multimodal approach produces vital data for studying complex microenvironments, like those in tumors or areas of inflammation, helping to assess therapeutic effects, find new targets, and link immune responses to treatment.

A common challenge associated with mass cytometry is analyzing the large datasets that are produced. To address this issue, Ellis recommends using the Maxpar^® Pathsetter™ software. This tool fully automates reporting and analysis, identifying 37 immune cell types in FCS files derived from samples processed through the Maxpar Direct Immune Profiling Assay. Since these files follow the same format as fluorescence flow cytometry FCS files, researchers can apply any analysis software or workflow to CyTOF data as they would with fluorescence data. Researchers can also build their own panels with ready-to-go pre-validated CyTOF products, which overcome common challenges, complexity, and labor involved with fluorescence flow cytometry panel design.

Applications in immunology and oncology

In clinical and translational research, mass cytometry has become an important tool for immune profiling and biomarker discovery. “CyTOF technology provides the widest coverage available for comprehensive screening, capturing the most relevant immune metrics,” stated Ellis. Applications of this technology have been demonstrated in numerous studies. Recently, a pre-print described how researchers applied mass cytometry to precisely profile different tissue components located near a pancreatic lesion.¹ Mass cytometry provided a high-dimensional view of immune cell compositions around these pancreatic precancers, identifying distinct immune microenvironments critical to understanding pancreatic cancer progression. Another study showcased how mass cytometry, combined with amplification by cyclic extension (ACE), allowed for precise quantification of low-abundance proteins at the single-cell level.² This approach improved sensitivity over 500-fold through DNA crosslinking and thermal cycling, enhancing both suspension and Imaging Mass Cytometry.

Ellis highlighted additional applications of mass cytometry through several notable publications. For example, CyTOF data have identified activated tissue-resident T cells associated with non-progressive disease.³ In another study, CyTOF analysis provided a baseline immune peripheral score indicative of objective response, progression-free survival, and overall survival.⁴ This technology was also applied to identify distinct subsets of CD8+ T cells and CD8+ TCM cells strongly linked to a complete or near-complete pathologic response⁵ and a mechanism of persistent NKG2D-CAR T cell response.⁶

Future directions

Mass cytometry has become essential for high-dimensional single-cell analysis, especially in immunology and precision medicine research. The technology’s ability to provide high-level protein data on individual cells offers important advantages against other cytometric approaches. This is further complemented by emerging computational tools that simplify data processing. Ellis shared that her group is excited about the future of CyTOF due to its broad coverage of surface and functional markers, providing consistent, high-content data across sites. “All of this is critical to improving immunotherapies by mitigating risks of off-target effects and understanding patient stratification and immune response biomarkers,” she concluded.

References

1. Kiemen AL, Almagro-Pérez C, Matos V, et al. 3D histology reveals that immune response to pancreatic precancers is heterogeneous and depends on global pancreas structure. bioRxiv. Published online January 1, 2024:2024.08.03.606493.

2. Lun X, Sheng K, Yu X, et al. Signal amplification by cyclic extension enables high-sensitivity single-cell mass cytometry. Nature Biotechnology. Published online 2024.

3. Lheureux S, Matei DE, Konstantinopoulos PA, et al. Translational randomized phase II trial of cabozantinib in combination with nivolumab in advanced, recurrent, or metastatic endometrial cancer. J Immunother Cancer. 2022;10(3):e004233.

4. Rochigneux P, Lisberg AE, Garcia A, et al. Mass cytometry reveals classical monocytes, NK cells and ICOS+ CD4+ T cells associated with pembrolizumab efficacy in lung cancer patients. Clinical Cancer Research. Published 2022. 

5. Hieken TJ, Nelson GD, Flotte TJ, et al. Neoadjuvant cobimetinib and atezolizumab with or without vemurafenib for high-risk operable Stage III melanoma: the Phase II NeoACTIVATE trial. Nature Communications. 2024;15(1):1430.

6. Driouk L, Gicobi JK, Kamihara Y, et al. Chimeric Antigen Receptor T Cells Targeting NKG2D-Ligands Show Robust Efficacy Against Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia. Frontiers in Immunology. 2019;11.