NEW YORK – Swedish startup Pixelgen Technologies aims to enable a new dimension of single-cell protein analysis as it launches sales of its new spatial proteomics tool.
The company’s Single Cell Spatial Proteomics (SCSP) Kit allows researchers to determine both the quantity and location of 76 protein targets on the surfaces of single cells. According to early-access user Petter Brodin, a professor of immunology at Karolinska Institute and Imperial College London, the Pixelgen technology enables the study of cell surface protein localization and clustering with levels of multiplexing and throughput not previously possible.
The SCSP Kit uses a variation on the proximity extension assay (PEA) technology developed by proteomics firm Olink, where Pixelgen Cofounder and CEO Simon Fredriksson was previously cofounder, CEO, and CSO.
PEA uses pairs of antibodies linked to DNA strands that are brought into proximity when the antibodies bind to their target. The DNA is then extended by a polymerase that creates a new sequence that can be used as a surrogate marker for the target protein.
Pixelgen likewise uses antibodies tagged with DNA to detect and quantify target proteins but uses elongation of those DNA strands to localize those proteins on the cell surface. The process starts with incubation of fixed single cells with nucleic acid-tagged antibodies to cell surface proteins of interest. Users then treat the cell with pieces of DNA containing sequences designed to fuse with the DNA tag on the antibody. The final step of the assay uses elongation to link all of these DNA pixels to each other, forming a grid of DNA tags that allows researchers to identify both which proteins are present on the cell surface and where on the cell they are located. Readout of this grid is done using next-generation sequencing on Illumina instruments.
Scientists have long known that the spatial organization of proteins on a cell surface is important to cell function, but studying this spatial dimension has been challenging, Brodin said.
“There is a lot of literature showing that these types of adaptations — the spatial localization of proteins, the polarization of proteins towards a target cell or surface the cell is moving on, segregation of different proteins at different ends of a cell — all of these things happen for a reason,” he said.
Typically, researchers have explored this spatial information using fluorescent imaging approaches like imaging cytometry, in which cell surface proteins of interest are tagged with fluorescent-labeled antibodies and then photographed as they are run through a flow cytometer. However, such experiments are typically limited to 10 or fewer protein targets, Brodin said.
Pixelgen’s technology has far greater multiplexing capacity. While the initial SCSP Kit targets 76 proteins, Brodin said that targeting on the order of hundreds of proteins was feasible.
Pixelgen’s initial SCSP Kit release is its Immunology Panel 1, Human, which, as the name suggests, targets human proteins of particular interest to immunology researchers. Fredriksson said that this initial panel of 76 targets aims primarily to cover important proteins on T cells, B cells, NK cells, and macrophages.
“We will be expanding from here,” he said, adding that on the surface of immune cells, “there are probably around 500 to 600 very interesting proteins that you would like to study.”
Fredriksson said the SCSP Kits are meant to run around 1,000 cells per experiment. Throughput will largely be a matter of the user’s sequencing capacity, he said.
In a BioRxiv preprint published this month, Pixelgen researchers along with Brodin, who is a member of the company’s scientific advisory board, used the technology to study protein localization patterns in T cells stimulated by chemokines. They looked specifically at the formation of uropods, a protein formation that they noted is “critical for cytotoxic T-cells to infiltrate tumors” and “is associated with immune checkpoint inhibition efficacy and overall cancer survival.”
The SCSP technology returns results along three dimensions: protein abundance; protein polarization, meaning whether a given protein is clustered at particular locations on the cell; and protein colocalization, meaning how different proteins cluster together on the cell. The researchers identified 12 proteins with different abundances in stimulated cells versus controls, 15 with different polarization scores, and 15 with differences in colocalization. These proteins included a number of proteins, including CD2, CD44, CD50, and CD162, known to be involved in the formation of T-cell uropods.
Brodin said that his lab is now applying a variety of stimuli to different types of immune cells and using the technology to observe how proteins change in abundance or localization in response. He said that such phenomena remain little understood due to the lack of tools for exploring them.
“In my opinion, this is wide open territory,” he said. “We believe that there will be conditions where this will add information that we have previously missed and that it will allow us to better understand mechanistically what is happening.”
He suggested a number of areas where the SCSP Kits could prove useful, for instance, in the study of the anti-tumor responses of immune cells and engineered immune cells like CAR T cells or how immune cells adapt and rearrange their protein architectures over time.
“I think there is an enormous amount of information to glean from this,” Brodin said.
He added that he saw the technology as an interesting new complement to other single-cell protein analysis tools like mass cytometry and CITE-Seq that his lab uses. He suggested that these latter tools had begun to reach the point of diminishing returns in terms of protein multiplexing.
“In our experience, having 100 or 150 or 200 proteins doesn’t really make a huge difference,” he said. “You are kind of saturating where you have reached a really granular view of the different [cell] populations that are present. I think you can probably do with 50 [proteins], to be honest. What we are doing with this technology is actually adding new dimensions we haven’t had before.”
Fredriksson said that Pixelgen’s early-access users are primarily using the technology for basic research into immune cell responses to stimuli, as Brodin’s group has done, and for drug development work studying drug mode of action on immune cells.
Pixelgen has begun taking orders for the SCSP Kits and plans to begin shipping them in September or October of this year.
The company, which currently has 25 employees, raised roughly $6 million in seed funding last year. Fredriksson declined to say if it planned to raise additional funds to support the SCSP Kit launch.