Mass Cytometry for Clinical Research

Mass cytometry™ is a single-cell proteomics technology. It uses metal-tagged antibodies to measure dozens of different protein levels in about 1,000 individual cells per second. It is similar to flow cytometry but can read out the relative expression of more protein targets at once.¹ Mass cytometry’s cost is also comparable to flow cytometry, making the technology a natural choice for research labs studying everything from infectious diseases like COVID-19 to immuno-oncology.

Larger panels for deeper profiling

Mass cytometry machines, sometimes referred to as a CyTOF^®, are made by a single company: Fluidigm. The technology is being used in more than 100 FDA-described clinical trials, ranging from Phase I through Phase IV.²

“It's a single-cell technology, just like flow cytometry or RNA-seq, and we can measure 50 or so parameters per cell,” says Marie Iannone, Director of the University of North Carolina at Chapel Hill’s Mass Cytometry Core Facility. Mass cytometry can not only detect specific proteins, but also measure their relative abundance in each cell.

“The CyTOF’s main use is deep phenotyping of cell populations, but its real strength is when you overlay function with that phenotype to ask questions about which cells are signaling, which cells are producing cytokines, which cells are showing certain characteristics of the cell cycle or apoptosis,” says Iannone. “All of those things are brought to bear when you look at these complex and heterogeneous cell populations.”

Fluidigm sells panels, or collections of optimized, metal-tagged antibodies, for mass cytometry experiments. These panels are usually designed to target the proteins involved in a specific cell function, like cytokine signaling or cell death. Additional antibodies can also be tagged with metals and added to commercial panels, according to Iannone.

As for cost, Iannone estimates that a mass cytometry experiment is roughly on par with flow cytometry. “A vial of antibodies costs around $400, and you could probably run 100 tests with a vial,” she says. “So that’s about $4 per marker and, for 50 markers, $200 per sample.” A 10-tube experiment costs about $2,000.

The mass cytometer has specific advantages compared to single-cell genomics and flow cytometry. “For single-cell sequencing, like with 10x Genomics, you dissociate small amounts of tissue and then do deep profiling on a very small number of cells. The CyTOF is able to do many more cells,” says Karen McKinnon, Assistant Professor and Director of the Immune Monitoring and Genomics Facility at the University of North Carolina at Chapel Hill.

And while the CyTOF is usually used to measure cells in a liquid suspension, there’s a commercially available module that can be attached to the machine to look at intact tissues slices.

“Tissue sections are stained with the metal-labeled antibodies and a precisely directed laser beam focused at 1 micron collects the selected tissue sample from a region of interest. The discrete signals from each ionized metal tag enable a high number of markers to be detected in a single scan using CyTOF^® technology,” says Clare Rogers, Marketing Director for Mass Cytometry at Fluidigm.

Metal-tagged antibodies are also more stable than fluorescently labeled antibodies; they’re largely impervious to changes in light or temperature. That means patient samples can be labeled with antibodies, frozen, and then shipped around the world to track infectious disease outbreaks or to analyze cancer biopsies in nationwide clinical trials, according to Rogers.

Tracking tumor cell populations over time

Mass cytometry is ideal for immuno-oncology studies; researchers are using the technique to see how deeply drugs penetrate in tumors and how immune cell populations shift over time.

“We’re using it to find out how tumors suppress the immune system, or how they cause the T cell response to be exhausted, or the B cell response,” says McKinnon.

And in patients with solid tumors, researchers are trying to understand why checkpoint inhibitors often fail. “Checkpoint inhibitor medications are supposed to turn on the immune system to infiltrate the tumor and attack the tumor cells,” says Rogers. “Unfortunately, for many types of cancer, this approach doesn't work.”

Since the mass cytometry can look at tissue samples, researchers use the technology to study the spatial positions of cells. In the case of checkpoint inhibitors, researchers take a little bit of cancerous tissue and analyze the tumor microenvironment at the single-cell level to “see whether the immune cells have infiltrated the tumor,” says Rogers. “In a person whose tumor is not responding, the immune cells are just kind of scattered around the outer periphery of the tumor. For whatever reason, they can't get inside the tumor to really start attacking the tumor cells. By analyzing the expression of key markers in those ineffective immune cells, investigators are starting to understand why those cells can’t infiltrate the tumor.”

A number of clinical trials are using mass cytometry to track immune cells in patients receiving CAR-T therapy.³ “Suspension mass cytometry is an excellent way to get a detailed look at how circulating immune cells are responding to immuno-therapy. Another recent development in this area is use of CyTOF in CAR-T cell development and testing. Several recent publications have shown how the sensitivity of mass cytometry allows researchers to identify CAR cells in circulation after infusion and over long time periods,” says Rogers.⁴ “But not only that, you can also see if those CAR cells that you've put back into the patient are all jazzed up and ready to attack, or if they are exhausted,” using mass cytometry.

In other words, “using mass cytometry, you can see both ends of the spectrum—what’s going on in the circulation and in the tumor,” says Rogers. “That's really powerful.”

Mass cytometry for infectious disease research

In a nationwide COVID-19 trial, called IMPACC, researchers at 20 different U.S. hospitals are using mass cytometry to analyze dozens of parameters in circulating immune cells collected from patients with COVID-19.⁵ They first stain whole blood samples from the patients using a 30-marker panel, then freeze the samples and ship them to a central mass cytometry facility. That panel was “created in a specialized format,” says Rogers. “The antibodies are in a dry-format, all in a single tube, so that all the person has to do at the bench is pipette in whole blood or peripheral blood mononuclear cells [PBMCs], follow the protocol, and within a few hours the sample is ready to go right on the mass cytometer.”

Elsewhere, researchers are using mass cytometry to study how immune cells, such as CD4 T cells, respond to HIV infection.⁶ Nadia Roan, Associate Professor of Urology at UCSF and associate investigator at Gladstone Institutes, uses the CyTOF to look at HIV-infected gut cells, lymph node cells, and reproductive tract cells.

“Most of the field has primarily analyzed blood specimens because that’s the most accessible source of cells, but infected cells in tissues are probably the more relevant ones to analyze,” says Roan. The gut harbors one of the highest frequencies of HIV-infected cells in people living with HIV.⁷

Roan uses mass cytometry to look at “what human proteins are present on the cells that HIV preferentially infects, and how HIV modifies these proteins in order to promote its own replication.” Over the last few years, her laboratory has used mass cytometry to track various proteins in cells obtained from people living with HIV fully suppressed on antiretroviral therapy. “We are doing these longitudinal analyses on these specimens to better understand how HIV-infected cells persist and can change over time.”

Back at the University of North Carolina, Avinash Kollipara, Assistant Professor of Pediatric Infectious Diseases, uses mass cytometry for “hypothesis generation.”

“One mass cytometry experiment will lead to multiple research avenues,” says Kollipara, who studies how chlamydia infections alter host immune responses in PBMCs and in the genital tract.⁸ “We started with a 34-marker panel that looks at T cells, B cells, monocytes, NK cells and dendritic cells, including each of their subsets, to comprehensively analyze a broad population of cells,” he says. Before, in the chlamydia research field, “people were looking at the tail of the elephant, or the ear of the elephant. Now we’re looking at the whole elephant using a systems immunology approach.”

References

1. Bendall, S.C., Nolan, G.P., Roederer, M. and Chattopadhyay, P.K. A Deep Profiler’s Guide to Cytometry. Trends Immunol 33(7), 323-332 (2012).

2. List of 100-plus National Clinical Trials. Fluidigm.com (2021).

3. Corneau, A., Parizot, C., Cherai, M. et al. Mass Cytometry: a robust platform for the comprehensive immunomonitoring of CAR-T-cell therapies. Br. J. Haematol. 194(4), 788-792 (2021).

4. Melenhorst, J.J. et al. Decade-long remissions of leukemia sustained by the persistence of activated CD4+ CAR T-cells. bioRxiv 2021.05.07.443194;  https://doi.org/10.1101/2021.05.07.443194

5. IMPACC Manuscript Writing Team. Immunophenotyping assessment in a COVID-19 cohort (IMPACC): A prospective longitudinal study. Sci Immunol. 6(62), eabf3733 (2021).

6. Cavrois, M., Banerjee, T., Mukherjee, G. et al. Mass Cytometric Analysis of HIV Entry, Replication, and Remodeling in Tissue CD4+ T Cells. Cell Rep 20(4), 984-998 (2017).

7. Monaco, C.L., Gootenberg, D.B., Zhao, G. et al. Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome. Cell Host Microbe 19(3), 311-322 (2016).

8. Kollipara, A., Iannone, M.A., Poston, T. et al. Multidimensional phenotypic immune profiling of Chlamydia trachomatis infected women using mass cytometry platform. J. Immunol. 200(1), 166.16 (2018).

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