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Harvard Team Develops Tissue-Based Protein Imaging System


NEW YORK – A team led by researchers at Harvard University's Wyss Institute for Biologically Inspired Engineering has developed an imaging technique that allows for multiplex protein analysis in tissue samples.

Described in a study published this week in Nature Biotechnology, the approach, called Immuno-SABER (immunostaining with signal amplification by exchange reaction) combines DNA-barcoded antibodies with DNA concatemer-based signal amplification to allow for multiplexed protein imaging with subcellular resolution and at relatively high throughput.

The method is one of a number of tissue-based protein imaging technologies to emerge in recent years. According to Sinem Saka, first author on the paper and a post-doc in the lab of Harvard professor Peng Yin, the paper's senior author, the Immuno-SABER approach is distinguished from other imaging methods primarily by its signal amplification step, which its developers expect will improve sensitivity and throughput.

Researchers have long used antibody-based methods to measure the expression of proteins in tissue, but these approaches were quite limited in terms of the number of proteins they could look at in a sample. That has changed in recent years as academic researchers and industry have developed a number of proteomic platforms capable of simultaneously imaging dozens of analytes. Systems like Fluidigm's mass cytometry-based Hyperion and IONPath's multiplexed ion beam imaging instrument as well as Akoya Biosciences' CODEX system have made it possible for researchers to multiplex measurements of large numbers of proteins at resolutions down to the single-cell level.

As the Harvard researchers noted, instruments like Hyperion and the IONPath system typically scan small portions of tissue at a time, limiting their throughput with larger tissue sections. An alternative approach that provides for higher throughput is that used by the Akoya CODEX system — labeling protein targets of interest with DNA-barcoded antibodies.

Samples are then stained with fluorescing reagents that bind not the antibody but the DNA barcode, making them detectable by microscope. By analyzing the sample iteratively — exciting a set of oligos, denaturing them and stripping them from the sample, then repeating the processes with the next set — researchers can collect data on large numbers of proteins across fairly large tissue sections.

The Immuno-SABER approach also uses DNA-barcoded antibodies for protein detection, but it includes a signal amplification step that Saka said both improves sensitivity and throughput.

"We think [signal amplification] will be important especially where you have tissue sections with high levels of autofluorescence or light scattering, which can cause signal to be lost," she said, adding that it could also improve measurement of less abundantly expressed proteins that might otherwise go undetected.

This sensitivity improvement should also lead to higher throughput by reducing the exposure times required to collect enough signal during the imaging process, Saka said.

"That is going to be important when we image large volumes of tissues [like whole organs], because imaging times will be really important when [an experiment] takes days and days of imaging," she added, noting that she and her colleagues believed the technique could prove very useful for this sort of large-scale tissue mapping work.

The Immuno-SABER approach uses DNA concatemers generated using primer exchange reactions. These concatemers are added to the samples after they are stained with the DNA-barcoded antibodies and bind to their corresponding DNA barcodes. DNA imaging strands containing fluorophores are then added. These attach to the bound concatemers creating the desired sample amplification.

Because the concatemer binding can be controlled to form secondary concatemer structures containing additional binding sites for the fluorophore-containing imaging strands, the technique can provide different levels of signal amplification for different proteins, allowing researchers to account to an extent for the high dynamic range of protein expression. In the Nature Biotech study, the researchers managed signal amplification ranging from five- to 180-fold while multiplexing up to ten proteins.

Saka said that, in theory, the technique could multiplex many more proteins, adding that she and her colleagues are currently working to validate additional antibody and DNA reagents with the aim of increasing their multiplexing. Akoya's CODEX system typically multiplexes between 25 and 50 proteins, though academic experiments exploring the platform's capabilities have multiplexed as many as 120.

Saka said the Immuno-SABER system currently could complete a multiplexed analysis of six proteins in around six hours.

The approach can achieve subcellular resolution in standard tissue samples, and in the Nature Biotech paper, the researchers combined the method with expansion microscopy to enable super-resolution analysis, imaging six proteins in an expanded section of mouse retina tissue.

Saka said that she and her colleagues are currently using the technique as part of tissue mapping studies.

"Currently, there is a big interest in molecularly mapping human tissues, to determine different cell types, to identify different markers for these cell types across large areas of tissue so that we have a reference map for how these tissues are organized," she said. "We have a lot of information from techniques like RNA sequence, but that comes usually after dissociation of the tissues, so we are losing spatial information and it is mostly at the RNA level. So, we're trying to apply our technology first to these kinds of large-scale mapping investigations."

Saka said that there was commercial interest in the system, though she referred questions on that subject to the Wyss Institute's communications department, which declined to comment beyond noting that the institute is "pursuing a commercialization path" for the technology.

Co-author Peng Yin is a co-founder and director at tissue imaging company Ultivue, which provides reagents for multiplexed tissue imaging as well as services using those reagents.