Cell labelling method from microscopy adapted for use in whole-body
imaging
Researchers develop imaging methods to examine bodily processes from the individual building blocks to the whole system

Date:
September 30, 2021
Source:
University of Mu"nster
Summary:
Scientist have utilized so-called SNAP-tag technology
to radioactively label cells in living organisms. In a
proof-of-principle study they developed a SNAP-tag substrate
equipped with the radioactive signal emitter fluorine-18 and
used it to make tumor cells in the bodies of mice visible in PET
images. The labeling method, already established in microscopy,
opens up the prospect of studying cells with different imaging
techniques and at different temporal stages -- for example, when
inflammation begins, continues and resolves again. This may help
reveal more about how the functions of individual cells and entire
organs are interconnected.



FULL STORY ========================================================================== Processes and structures within the body that are normally hidden from the
eye can be made visible through medical imaging. Scientists use imaging
to investigate the complex functions of cells and organs and search for
ways to better detect and treat diseases. In everyday medical practice,
images from the body help physicians diagnose diseases and monitor whether therapies are working. To be able to depict specific processes in the
body, researchers are developing new techniques for labelling cells or molecules so that they emit signals that can be detected outside the body
and converted into meaningful images. A research team at the University
of Mu"nster has now adapted a cell labelling strategy currently used in microscopy -- the so-called SNAP-tag technology -- for use in whole-body imaging with positron emission tomography (PET) for the first time.


==========================================================================
This method labels cells in two steps that work for completely different
cell types such as tumour and inflammatory cells. First, the cells are genetically modified to produce a so-called SNAP-tag enzyme on their
surface that is unique to the targeted cells. The enzyme is then brought
into contact with a suitable SNAP-tag substrate. The substrate is labelled
with a signal emitter and chemically structured so that it is recognised
and split by the enzyme allowing the signal emitter to be transferred to
the enzyme. In the process, the enzyme is modified so that it is no longer active and, as a result, the signal emitter remains tightly coupled to
it. "Through its biological activity, the SNAP-tag enzyme labels itself,
so to speak -- this happens very quickly and without disturbing the
natural processes in the organism," explains Dominic Depke, a biology
doctoral student and one of the lead authors of the new study.

In microscopy, fluorescent dyes are used to label cells, but they are
mostly not suitable for whole-body imaging because their signals are
scattered by thicker tissue layers with the result that they can no longer
be measured. To solve this problem, the scientists synthesised a new
SNAP-tag substrate using the radioactive signal emitter fluorine-18. The
team have successfully labelled tumour cells in mice by injecting this substrate into the organism via the bloodstream and were then able
to visualise the tumours using PET imaging. "The exciting thing for 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 temporal stages -- we call it multiscale imaging,"
explains nuclear medicine specialist Prof Michael Scha"fers.

"Radioactive signals from fluorine-18 remain stable for only a short
time," adds radiochemist Dr Christian Paul Konken, "but as we can repeat
the second labelling step, we can potentially visualise the same cells
again and again over 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 view provided by whole-body imaging
enables scientists to assess how these cells function as part of whole
organ systems. Time may reveal what role individual cell types play in inflammation, for example, as it begins, continues and resolves. "Only
by combining all this information can we understand how everything is
connected in the body," says Michael Scha"fers.

A small beginning with great potential "Our investigations are a very
first step, in which we have shown that labelling cells with SNAP-tags
works, in principle, in living organisms," emphasises biochemist
Prof Andrea Rentmeister. "What matters here is that the substrate is distributed rapidly in the organism and that it binds exclusively to the
cells to be studied." The next crucial steps will be to test how many
cells are needed to obtain a sufficiently strong signal and whether
the method can also be used to visualise cells that move within the
organism -- in particular immune system cells. 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 combat a specific disease. Such therapies are already being used for cancer treatment and
have the potential to help treat inflammatory diseases as well. Imaging
could help develop and improve such treatments.

When the scientists presented their results for the first time at a
scientific symposium, they were in for a surprise -- colleagues from
Tu"bingen presented a similar study there at the same time. Independently
of each other, both research teams had the same fundamental idea,
a SNAP-tag substrate labelled with fluorine-18. Chemically speaking,
they implemented the idea differently but they tested the resulting
substrates using the same biological model system and arrived at similar findings. "This shows how topical our question is and that our results are reproducible and really promising," says Michael Scha"fers. He adds that
the Tu"bingen team is developing new labelling methods to study immune
cells in cancer, while the team in Mu"nster is focusing on inflammatory diseases, so the research complements each other very well. The research
team from Mu"nster published their study in the scientific journal
"Chemical Communications," only a few days later the publication from
Tu"bingen was released in "Pharmaceuticals." Creating a new substrate
for the SNAP-tag Like all SNAP-tag substrates, the newly developed
molecule is based on benzylguanine to which the scientists attached the radioactive isotope fluorine-18, which is, in turn, ideally suited for
PET imaging. "Our goal was to design the synthesis in a few quick steps
so that we get as strong a signal as possible -- because fluorine-18 has
a short half-life, its radioactivity is reduced by half after every 110 minutes," explains Christian Paul Konken.

Initially, the scientists found that the fluorine-18 did not attach to
the desired position on the molecule. "The benzylguanine was apparently
too sensitive to be labelled directly with fluorine-18," says Lukas
Ro"sner, a biochemistry doctoral student, "so we first labelled a small molecule that is insensitive to the necessary chemical reactions -- the fluoroethylazide -- and then attached it to the benzylguanine using a
click reaction, which is very fast and selective." Tests in test tube,
cell cultures and the organism The scientists first checked whether the synthesised substrate remained stable when in contact with blood in the
test tube and then examined how the cells interacted with the substrate in
the first practical tests in cell cultures. In doing so, they compared
human tumour cells into which they had genetically incorporated the
SNAP-tag enzyme with those that did not produce the enzyme.

"We could see very clearly that the radioactivity was only taken up by the cells that produced the SNAP-tag enzyme," says Dominic Depke. Finally,
the team conducted targeted studies on individual mice. "This step
was decisive once again," explains Michael Scha"fers, "because how
a molecule behaves in the complex biological environment in a living
organism cannot be fully simulated in cell culture or with artificially produced organs." The scientists were able to show that once the substrate
is injected into the bloodstream it is distributed through the body very quickly. Additionally, they identified the pathways through which it
is excreted. They then compared how tumour cells with and without the
SNAP-tag enzyme reacted to the substrate in living organisms.

For this purpose, the tumour cells were injected under the skin of mice
and removed again after the examination in order to confirm the results
with autoradiography.

========================================================================== Story Source: Materials provided by University_of_Mu"nster. Original
written by Doris Niederhoff. Note: Content may be edited for style
and length.


========================================================================== Journal Reference:
1. Dominic Alexej Depke, Christian Paul Konken, Lukas Ro"sner,
Sven Hermann,
Michael Scha"fers, Andrea Rentmeister. A novel 18F-labeled clickable
substrate for targeted imaging of SNAP-tag expressing cells by
PET in vivo. Chemical Communications, 2021; 57 (77): 9850 DOI:
10.1039/ D1CC03871K ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/09/210930104837.htm

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