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Using super-resolution shadow imaging (SUSHI) to quantify the extracellular space structure in tissue sections

English title Using super-resolution shadow imaging (SUSHI) to quantify the extracellular space structure in tissue sections
Applicant Stein Jens Volker
Number 190484
Funding scheme Spark
Research institution Microbiologie Département de Médecine Université de Fribourg
Institution of higher education University of Fribourg - FR
Main discipline Immunology, Immunopathology
Start/End 01.01.2020 - 31.03.2021
Approved amount 96'400.00
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All Disciplines (2)

Discipline
Immunology, Immunopathology
Cellular Biology, Cytology

Keywords (4)

Immune surveillance; Tissue architecture; Super-resolution microscopy; Extracellular space

Lay Summary (German)

Lead
Kartierung des extrazellulären Raums
Lay summary

Alle Zellen des Körpers werden vom extrazellulären Raum (EZR) umspült, der oft nur wenige Nanometer breit ist. Der EZR dient zum Transport von extrazellulären Boten-und Nährstoffen. Ausserdem ist es erwiesen, dass die dreidimensionale (3D) Struktur des EZR von Immunzellen als «Pfad» benutzt wird um entzündete Gewebe zu infiltrieren. Umgekehrt dient der EZR zum Auswandern von metastasierenden Krebszellen aus primären Tumoren. Die tatsächliche 3D Feinstruktur des EZR ist allerdings nicht genau bekannt, da bisher dafür geeignete bildgebende Verfahren gefehlt haben. In diesem Projekt wenden wir eine neuartige hochauflösende Mikroskopie-Methode an, SUSHI (für «super-resolution shadow imaging»), welche es erlaubt, die genaue 3D Struktur des EZR in verschiedenen Geweben herauszuarbeiten. Basierend auf diesen Daten erstellen wir «EZR-Karten», welche die organspezifische Verteilung von Immunzellen und Krebszellen mit dem vorhandenem EZR abgleichen und Einblicke geben in den Prozess der Immunantwort und Metastasierung.

Direct link to Lay Summary Last update: 16.12.2019

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Abstract

All cells of the body are surrounded by extracellular space (ECS) with sizes varying between 30 to several 1000 nm. ECS serves to transport extracellular fluids, nutrients and metabolites, and is central for ion homeostasis in the central nervous system (CNS). Furthermore, the 3D structure of the ECS is thought to determine the pathway along which immune cells and metastazing cancer cells infiltrate tissue. Yet, while the molecular composition of intercellular junctions and the cell anchorage to extracellular matrix (ECM) via cadherins, claudins, integrins and other adhesion molecules are well characterized, the tissue-specific 3D structure of the ECS has remained elusive. Using a newly developed microscopy method called super-resolution shadow imaging (SUSHI), the quantification of the intricate ECS structure of the CNS in cultured mouse brain slices has been recently published. In brief, brain slices are overlaid with a cell-impermeable dye and imaged using 3D stimulated emission depletion microscopy (STED) to record the ECS structure with tens of nm resolution. By subjecting slices to a hyperosmotic challenge, intercellular adhesions and cell attachment to ECM become clearly visible through dynamic changes in ECS structure, thus highlighting intrinsic physical connectivity within tissue in unprecedented detail. In collaboration with the developers of SUSHI, we have obtained promising preliminary high-resolution images of the ECS structure of sectioned mouse salivary glands. This unpublished proof-of-concept experiments constitute the first successful attempt to apply SUSHI to freshly sectioned tissue. Importantly, our findings shed light on potential surveillance pathways of protective CD8+ T lymphocytes during antiviral immune reponses and have the potential to explore a completely new venue of tissue analysis. Yet, the applicability of SUSHI to additional mouse (or human) organs remains to be examined, since every organ displays distinct biochemical and physical properties. This in turn may impact on their suitability for SUSHI-driven analysis and requires careful adaptation of sample preparation, image acquisition and analysis. Given the very recent development of SUSHI, it is currently impossible to predict whether this imaging approach is amenable to map the complex ECS structure of various organs.Here, we propose to examine the suitability of SUSHI to produce high-resolution maps of the 3D ECS spatial organization of multiple mouse organs, including lymph nodes, spleen, small intestine, kidney and bone marrow. To investigate intercellular attachment, we will challenge tissue sections with hyperosmotic solutions to induce cell shrinkage and uncover tight versus loose connections between different cell types, using changes in ECS structure as readout. While this constitutes a highly ambitious approach, we are confident from preliminary results obtained with salivary glands that at least some organs are amenable to this pioneering analysis. In case of success, our data will serve as a blueprint for a comprehensive analysis of ECS architecture on tumor slices and inflamed tissue in future experiments. We anticipate that this unconventional approach serves to close a gap between detailed knowledge of cellular adhesion derived from in vitro experiments on one end of the spectrum and conventional immunohistology of fixed tissue on the other end of the spectrum. This will pave the way for a more contextualized understanding of organ-specific immune surveillance by leukocyte subsets and of pathways taken by disseminating tumor cells during metastasis formation.
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