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

Project Information

3D-SpatiOmics

Grant agreement ID: 101189497

DOI

10.3030/101189497

Project closed

EC signature date 12 December 2024

Start date 1 January 2025

End date 30 June 2026

Funded under

• European Research Council (ERC)

Total cost

No data

EU contribution

€ 150 000,00

Coordinated by

CHARITE - UNIVERSITAETSMEDIZIN BERLIN
Germany

Periodic Reporting for period 1 - 3D-SpatiOmics (Development of a nanobody-based, slide-free approach for 3D-Histological analysis of the spatial tumor microenvironment using lightsheet imaging)

Reporting period: 2025-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

Accurate histopathological analysis of tumour tissue is essential for diagnosis and treatment decisions, yet conventional histopathology relies on thin, two-dimensional tissue sections. This 2D approach cannot fully represent the three-dimensional spatial organisation of a tumour and its microenvironment, the distribution of immune cells, blood vessels and tumour cells within intact tissue, which limits precise diagnosis and patient stratification. The 3D-SpatiOmics proof-of-concept project set out to demonstrate the feasibility of a nanobody-based, slide-free three-dimensional histopathology approach using non-destructive volumetric light-sheet microscopy. The central idea is to stain entire tissue specimens with small antibody fragments (nanobodies), image them in three dimensions without physically sectioning the tissue, and to repeat this staining for many markers on the very same specimen. Because the method is non-destructive, the sample remains available afterwards for further molecular analysis. The overall objective is to provide a richer, spatial characterisation of the tumour microenvironment and thereby to improve tumour diagnosis and the stratification of patients for treatment.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project pursued three technical lines of work. First, new nanobodies were generated and validated for immune-system and tumour-relevant markers to enable whole-mount staining of intact tissue. Fully validated nanobodies were obtained for the clinically important immune-checkpoint markers such as PD-1 and PD-L1, performing reliably in both two- and three-dimensional staining. Further markers reached different stages of validation, and for several technically challenging targets alternative production strategies were initiated. Second, a fluorescent labelling method was established that attaches dyes to nanobodies in a controlled, directional manner and allows the dye to be removed again, so that a specimen can be stained, imaged, cleared and re-stained for the next set of markers. This removable labelling was demonstrated on thin tissue. In thicker specimens the removal step still requires optimisation. Third, the full workflow was validated on tumour tissue: sequential multiplex staining with intermediate removal and re-staining of the same specimen was demonstrated, and the same sample was afterwards successfully analysed by molecular diagnostics, confirming that the non-destructive imaging does not compromise downstream analysis. Together, these achievements demonstrate the feasibility of the proposed integrated pipeline.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The key advance is the combination of three properties in a single workflow: three-dimensional, slide-free imaging of intact tumour specimens, sequential multiplex staining of many markers on the same sample using removable fluorescent labels, and full compatibility with subsequent molecular diagnostics on the identical specimen ("no touch, no waste"). Conventional 2D histopathology offers none of these in combination. This enables a more complete spatial characterisation of the tumour microenvironment and more informative patient stratification. The main next steps are completion of the full marker panel, optimisation of the labelling chemistry for thick tissue, and validation on larger, clinically annotated sample sets. Building on these results, exploitation is pursued through a spin-off company and industrial partnerships, and a dedicated spatial diagnostics platform for oncology has been established to support translation towards clinical and commercial use.