Development of a nanobody-based, slide-free approach for 3D-Histological analysis of the spatial tumor microenvironment using lightsheet imaging

For histopathological analysis and subsequent diagnosis of diseased tissues a rapid, accurate histopathological and genetic analysis of biopsied tissues are essential.

However, traditional 2-dimensional slide-based histopathological analysis does not represent spatial tissue structures and molecular targets sufficiently, which aggravates an accurate and spatial analysis of tissue, a detailed description of the tumor microenvironment as well as patient stratification.

To overcome these limitations, I propose an innovative, nanobody-based 3D-histopathology approach using non-destructive volumetric lightsheet-microscopy. This approach bears the potential to revolutionize the way pathological analysis of tumor samples are done.

In comparison to conventional 2D-pathology, non-destructive 3D-histopathology allows rapid slide-free histological imaging of an entire tissue sample, volumetric analysis of diagnostic relevant structures such as immune cells, vessels and an improved spatial analysis of cell distribution relevant to study e.g. the microenvironment.

To achieve this, we will generate nanobodies for improved wholemount tissue staining of the immune system as well as tumor-relevant markers.

By labelling nanobodies with cleavable fluorescent dyes using click chemistry and integration of linkers for stoichiometric and directed labelling, we will be able to perform several rounds of multiplex staining of the identical sample and therefore provide cutting-edge diagnostic phenotyping of the specimen.

Due to non-destructive nature of the optical-sectioning methodology, the sample is available for downstream applications such as molecular diagnostics of the previously imaged specimen.

In summary, this innovative project holds the great potential to revolutionize cancer histology by providing a spatial and detailed description of the tumor microenvironment, improving diagnosis of tumor samples and consequently patient stratification and outcome.