INSIHGT: A scalable and homogeneous 3D multiplexed molecular mapping platform for spatial biology and precision medicine.

Abstract

e15180 Background: The dynamic interplay between cancer cells and their microenvironment is a determinant of disease evolution and response to treatment. However, the current state of three-dimensional (3D) tumor microenvironment analysis is hindered by inadequate penetration of antibodies into thick tissue sections, leading to a substantial gap in acquiring unbiased, precise 3D multiplexed immunohistochemistry (IHC) images. Methods: We developed INSIHGT to surmount these limitations by enabling multi-round, multiplexed 3D IHC in standard formalin-fixed tissues. This fully automatable, fast-track method requires a simple series of chemical cocktail exchanges, translating to a tissue-to-image time of 6 days for 1 cm³ samples and as little as 6 hours for biopsy specimens. INSIHGT offers a scalable and efficient solution for detailed 3D spatial mapping of cancerous cells and their interactions within their native context. Results: 3D IHC with INSIHGT shows ideal penetration homogeneity, which quantitatively matches reference signals in 1cm3 samples. We further validated INSIHGT with 309 commercially available primary antibodies and is compatible with simultaneous fluorescent in situ hybridization (FISH) to detect RNA transcripts or DNA chromosomal abnormalities. Notably, the quality of post-INSIHGT traditional histological analyses, such as H&E and standard IHC, was not altered. We demonstrated a 3D mapping of over 20 markers in a 1 mm-thick tumor sample across seven INSIHGT cycles, and an atlas of multiple tumour samples with 3D IHC & H&E. The large datasets seamlessly integrates with single-cell segmentation and analysis workflows, yielding comprehensive maps that elucidate protein expression, cell morphology, spatial positioning, and potential cell-cell interactions. Conclusions: INSIHGT provides an unparalleled level of visualization and analytical detail concerning the spatial dynamics within the tumor microenvironment at a single-cell resolution. As a transformative next generation histopathology platform, it can uncover new structural and molecular details within clinical specimens, accelerating biomarker discovery and novel diagnostic assays development, thereby enriching the field of tumor biology and propelling the progress of personalized oncology treatments.