Appropriate cell labeling method by microscopy

image: PET imaging of tumors (dotted circles) in the body of a mouse (right in section). The researchers used a newly developed radioactive substrate to label tumor cells in a living organism. Cells genetically modified to produce an enzyme called SNAP-tag have absorbed the radioactive marker (orange), unlike cells without this enzyme.
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Credit: Depke D et al.

The processes and structures in the body that are normally hidden from the eye can be made visible through medical imaging. Scientists use imaging to study the complex functions of cells and organs and to find ways to better detect and treat disease. In everyday medical practice, images of the body help doctors diagnose illnesses and monitor whether therapies are working. In order to be able to describe specific processes in the body, researchers are developing new techniques for labeling cells or molecules so that they emit signals that can be detected outside the body and converted into meaningful images. A research team from the University of Münster has adapted for the first time a cell labeling strategy currently used in microscopy – the so-called SNAP-tag technology – for use in whole-body imaging with positron emission tomography (PET).

This method labels cells in two steps that work for completely different cell types such as tumor and inflammatory cells. First, the cells are genetically engineered to produce an enzyme called SNAP-tag on their surface which is unique to the targeted cells. The enzyme is then contacted with an appropriate SNAP-tag substrate. The substrate is labeled with a signal emitter and chemically structured so that it is recognized and divided by the enzyme allowing the signal emitter to be transferred to the enzyme. In the process, the enzyme is changed so that it is no longer active and therefore the signal emitter remains tightly coupled to it. “Thanks to its biological activity, the SNAP-tag enzyme self-labels itself, so to speak – this happens very quickly and without disturbing the body’s natural processes,” says Dominic Depke, doctoral student in biology and the one of the main authors of the new study.

In microscopy, fluorescent dyes are used to label cells, but they are generally not suitable for whole-body imaging because their signals are scattered through layers of thicker tissue, so they can no longer be measured. To solve this problem, the scientists synthesized a new SNAP-tag substrate using the fluorine-18 radioactive signal transmitter. The team succeeded in labeling tumor cells in mice by injecting this substrate into the body via the bloodstream and then were able to visualize the tumors using PET imaging. “What interests us about SNAP-tag technology is that it opens up the prospect of visualizing genetically encoded cells in the body with different imaging modalities and at different time stages – we call it multiscale imaging. », Explains Professor Michael Schäfers, specialist in nuclear medicine. . “The radioactive signals of fluorine-18 only remain stable for a short time,” adds radiochemist Dr Christian Paul Konken, “but because we can repeat the second labeling step, we can potentially visualize the same cells over and over for long periods of time. days and weeks. ” The high level of detail provided by microscopy makes it possible to study how individual cells communicate with each other. The big picture provided by whole body imaging allows scientists to assess how these cells function as part of whole organ systems. Time can reveal the role that individual cell types play in inflammation, for example, when it begins, continues, and resolves. “It is only by combining all this information that we can understand how everything is connected in the body,” explains Michael Schäfers.

A small start with great potential

“Our investigations are a very first step, in which we have shown that the labeling of cells with SNAP-tags works, in principle, in living organisms”, emphasizes biochemist Prof Andrea Rentmeister. “What matters here is that the substrate is distributed quickly in the organism and that it attaches itself exclusively to the cells to be studied. The next crucial steps will be to test how many cells are needed to get a strong enough signal and whether the method can also be used to visualize cells moving around the body – especially cells of the immune system. If the approach continues to prove successful, the technique may become important for future research into immunotherapies in which the body’s own immune cells are genetically modified in the laboratory so that they can fight a specific disease. Such therapies are already being used for the treatment of cancer and can also help treat inflammatory diseases. Imaging could help develop and improve such treatments.

When scientists first presented their results at a science symposium, they were surprised – colleagues from Tübingen presented a similar study at the same time. Independently of one another, the two research teams came up with the same basic idea, a SNAP-tag substrate labeled with fluorine-18. Chemically speaking, they implemented the idea differently, but they tested the resulting substrates using the same biological model system and came up with similar results. “It shows how topical our question is and how reproducible and really promising our results are,” says Michael Schäfers. He adds that the Tübingen team is developing new labeling methods to study immune cells in cancer, while the Münster team is focusing on inflammatory diseases, so the research complements each other very well. The Münster research team published their study in the scientific journal “Chemical Communications”, a few days later, the Tübingen publication was published in “Pharmaceuticals”.

Creation of a new substrate for the SNAP-tag

Like all SNAP-tag substrates, the newly developed molecule is based on benzylguanine to which scientists have attached the radioactive isotope fluor-18, which is, in turn, perfectly suited for PET imaging. “Our goal was to design the synthesis in a few quick steps in order to get as strong a signal as possible – because fluorine-18 has a short half-life, its radioactivity is halved every 110 minutes”, explains Christian Paul . Konken. Initially, scientists discovered that fluorine-18 did not attach itself to the desired position on the molecule. “Benzylguanine was apparently too sensitive to be labeled directly with fluorine-18,” explains Lukas Rösner, PhD student in biochemistry, “so we first labeled a small molecule insensitive to necessary chemical reactions – fluoroethylazide – and then we labeled it. have attached to benzylguanine using a click reaction, which is very fast and selective.

Test tube, cell cultures and organism

Scientists first checked whether the synthesized substrate remained stable on contact with blood in the test tube, and then looked at how cells interacted with the substrate during the first practical tests in cell cultures. In doing so, they compared human tumor cells in which they had genetically incorporated the SNAP-tag enzyme with those which did not produce the enzyme. “We could see very clearly that the radioactivity was only taken up by the cells which produced the SNAP-tag enzyme,” explains Dominic Depke. Finally, the team conducted targeted studies on individual mice. “This step was once again decisive,” explains Michael Schäfers, “because the behavior of a molecule in the complex biological environment of a living organism cannot be fully simulated in cell culture or with artificially produced organs. . ” Scientists have been able to show that once the substrate is injected into the bloodstream, it is distributed very quickly in the body. In addition, they have identified the routes by which it is excreted. They then compared how tumor cells with and without the SNAP-tag enzyme reacted to the substrate in living organisms. For this purpose, the tumor cells were injected under the skin of mice and taken again after the examination in order to confirm the results by autoradiography.

Author: Doris Niederhoff

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