Multiplexed 3D Analysis of Immune States and Niches in Human Tissue

Tissue homeostasis and the emergence of disease are controlled by changes in the proportions of resident and recruited cells, their organization into cellular neighbourhoods, and their interactions with acellular tissue components. Highly multiplexed tissue profiling (spatial omics)[1][1] makes it possible to study this microenvironment in situ , usually in 4-5 micron thick sections (the standard histopathology format)[2][2]. Microscopy-based tissue profiling is commonly performed at a resolution sufficient to determine cell types but not to detect subtle morphological features associated with cytoskeletal reorganisation, juxtracrine signalling, or membrane trafficking[3][3]. Here we describe a high-resolution 3D imaging approach able to characterize a wide variety of organelles and structures at sub-micron scale while simultaneously quantifying millimetre-scale spatial features. This approach combines cyclic immunofluorescence (CyCIF) imaging[4][4] of over 50 markers with confocal microscopy of archival human tissue thick enough (30-40 microns) to fully encompass two or more layers of intact cells. 3D imaging of entire cell volumes substantially improves the accuracy of cell phenotyping and allows cell proximity to be scored using plasma membrane apposition, not just nuclear position. In pre-invasive melanoma in situ [5][5], precise phenotyping shows that adjacent melanocytic cells are plastic in state and participate in tightly localised niches of interferon signalling near sites of initial invasion into the underlying dermis. In this and metastatic melanoma, mature and precursor T cells engage in an unexpectedly diverse array of juxtracrine and membrane-membrane interactions as well as looser “neighbourhood” associations[6][6] whose morphologies reveal functional states. These data provide new insight into the transitions occurring during early tumour formation and immunoediting and demonstrate the potential for phenotyping of tissues at a level of detail previously restricted to cultured cells and organoids. ### Competing Interest Statement PKS is a co-founder and member of the BOD of Glencoe Software, member of the BOD for Applied Biomath, and member of the SAB for RareCyte, NanoString, Reverb Therapeutics and Montai Health; he holds equity in Glencoe, Applied Biomath, and RareCyte. PKS consults for Merck and the Sorger lab has received research funding from Novartis and Merck in the past five years. The other authors declare no outside interests. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6