New article: 3D OTLS reveals interconnected immune structures in melanoma

A new preprint, “Spatially Resolved Multimodal Hallmarks of Response to Neoadjuvant Immunotherapies in the Melanoma Ecosystem in 2D and 3D,” combines spatial multi-omics, computational pathology, and 3D imaging to study response and resistance to neoadjuvant immune checkpoint blockade in melanoma.

The study analyzed 91 FFPE specimens from 87 patients with stage III melanoma across three immune checkpoint blockade regimens and treatment-naïve controls. Across the cohort, the authors identified mature tertiary lymphoid structures, germinal centers, TCF7+ stem-like T cells and distinct immune and stromal neighborhoods associated with response. They also developed computational tools to quantify cell-cell signaling, delineate TLS and germinal centers, and detect these structures directly from routine H&E images.

As part of the study, open-top light-sheet microscopy was used to examine the 3D architecture of selected tumor beds. Germinal centers that appeared separate in individual 2D sections could be followed through hundreds of microns of tissue and were shown to form interconnected structures in 3D. CODA-based reconstruction from serial H&E sections independently supported the same observation, showing that immune structures that appear isolated in a single section may instead belong to a larger continuous architecture within the tissue.

This finding is particularly relevant for the study of tertiary lymphoid structures and germinal centers, where measurements based on individual sections may not capture their full size, connectivity or spatial organization.

The study also demonstrated that OTLS imaging can be incorporated into a non-destructive workflow. Following 3D imaging, samples were reverse-cleared, re-embedded and processed for clinical-grade H&E and IHC, allowing the 3D tissue architecture to be connected back to conventional histopathology and immune markers from the same specimen.

More broadly, the work links molecular discovery, spatial analysis, and computational pathology to questions of treatment response, biomarker development, and TLS biology in neoadjuvant immunotherapy.

The 3D component adds structural context to those findings by showing how immune architectures extend through the intact tissue volume.