NEW YORK – Researchers from Harvard University and their collaborators have developed a new signal amplification method for mass cytometry that promises to enable the simultaneous detection of dozens of rare protein targets at the single-cell level.
Described in a recent Nature Biotechnology study, the method, dubbed Amplification by Cyclic Extension (ACE), offers researchers a tool to address sensitivity challenges in suspension or imaging mass cytometry, and potentially beyond.
"Mass cytometry is a really versatile method, but it has a sensitivity problem," said Xiao-Kang Lun, a postdoctoral researcher at Harvard University and one of the lead authors of the study. "There's really high demand to apply any signal amplification approach to mass cytometry."
Currently, mass cytometry can quantitatively detect about 50 protein targets at once at the single-cell level. Because the technology typically requires hundreds of metal-tagged antibodies to bind to each type of epitope in order to reach the instrument's detection threshold, many low-abundance targets remain undetected, said Lun, who works in the lab of Peng Yin, the corresponding author of the study.
Previously, Yin's team had developed a signal amplification method called immunostaining with signal amplification by exchange reaction (Immuno-SABER), which deploys DNA-barcoded antibodies and presynthesized DNA concatemers generated by the so-called primer exchange reaction (PER) to help boost the signals of low-abundance proteins.
While this method has been utilized for imaging mass cytometry, it is still technically challenging to be adopted for suspension mass cytometry since the stringent washing conditions required in the protocol cannot be easily implemented for cells in suspension, said Kuanwei Sheng, also a postdoctoral researcher in Yin's lab and the other lead author of the study.
To address the problem, the Harvard team developed ACE, a signal amplification strategy that uses thermal-cycling-based DNA in situ concatenation as well as 3-cyanovinylcarbazole phosphoramidite-based DNA crosslinking.
As part of the ACE workflow, antibodies targeting the protein analytes are first conjugated to short DNA oligonucleotide initiators and subsequently applied to cell suspensions for cell surface or intracellular marker staining.
After that, an extender oligonucleotide containing two sequence repeats complementary to the initiator is introduced to the stained cells, where the extender and initiator sequences hybridize and the initiator strand is extended by a polymerase. After denaturing the initiator–extender pair, the initiator strand undergoes multiple thermal cycling reactions to create hundreds of repeats on each antibody conjugation site. The extended initiator then binds to hundreds of metal-conjugated detectors, thus dramatically enhancing the metal signal on the antibody for the downstream.
Lun said one challenge of applying ACE in mass cytometry is that the heat-mediated single-cell droplet vaporization step involved can denature DNA double strands, unwinding the hybridization between metal-conjugated detectors and initiator DNA and, thus, diminishing the amplified signal.
To solve this issue, the team further introduced a photo-crosslinking step into ACE. After the detector has hybridized to the initiator, the photo-crosslinker is activated by short UV light, forming a covalent bond between the detector and the initiator DNA strand, preventing them from coming apart.
Based on their study results, the Harvard researchers concluded that ACE can help achieve over 500-fold signal increase for a target with "uncompromised signal-to-noise ratios." They also validated 33 orthogonal ACE initiators with a low chance of crosstalk, indicating the method's multiplexing capability while ensuring that most of these initiator sequences could only be extended and detected by their corresponding extenders and detectors.
Additionally, the researchers showed that ACE can help profile low-abundance proteomic markers during the epithelial-to-mesenchymal transition (EMT) of mouse cells and the reverse mesenchymal-to-epithelial transition (MET). Moreover, they were able to analyze T-cell receptor (TCR) signaling networks at single-cell resolution in the human T cells.
Beyond suspension mass cytometry, the researchers applied ACE to imaging mass cytometry, where they were able to identify tissue compartments and profile pathological states in polycystic human kidney tissues where autofluorescence limits the use of conventional fluorescence-based spatial imaging.
Sheng said the method has not reached its peak multiplex capability yet and that there is room to accommodate more targets.
The method is "an elegant twist" to the conventional mass cytometry workflow, said Jared Burks, codirector of the flow cytometry and cellular imaging core facility at MD Anderson Cancer Center.
A peer reviewer of the study, Burks noted that one unique design of the ACE approach is the UV crosslinking step that tethers the metal-isotope probe to the oligo. In addition, he applauded the method for being streamlined, being easy to use, and adding little cost to the existing mass cytometry workflow.
"I think this [method] is going to be very exciting," said Burks, who is also a consultant for mass cytometry platform maker Standard BioTools (formerly known as Fluidigm). "In all honesty, I would expect that this is likely going to get commercialized at some point because of the low cost and the relatively rapid pace that this could be executed," he noted.
According to the study authors, the ACE protocol adds about $24 in cost to a 30-target conventional mass cytometry experiment. Sheng said the ACE workflow can be carried out in two days and does not require an elaborate experiment setup, other than reagents and a thermal cycler.
The Harvard researchers have filed patent applications pertaining to the ACE method, and Lun said the team is interested in talking with companies to help commercialize the technology.
Despite ACE's promises, the method currently has some limitations. For one, Lun said the chemistry is developed to automatically permeabilize the cells, which can be an issue when researchers only want to stain and study cell surface markers. In addition, the effectiveness of ACE hinges upon antibody availability and their affinity to their corresponding targets. Furthermore, the team is trying to standardize the antibody-oligo conjugation protocol, hoping to make it more accessible to researchers who might not be experts in the technique.
Encouraged by the proof of concept, the Harvard team plans to further harness ACE to help boost molecular detection in applications beyond mass cytometry. In their paper, for instance, the study authors noted that they have demonstrated using the method's framework to enhance signals during fluorescence microscopy imaging, though their data has not yet been published.
"ACE is indeed a platform technology that can be used in many application scenarios," one being mass cytometry, Sheng said.