As tumours grow, complexity increases, resulting in heterogeneous cellular organizations encompassing many populations of intertwined cancer and stromal cells. These intimate connections can confer survival or growth advantages upon tumour cells, as well as enable resistance to treatment. Therefore, the spatial analysis of tumours is crucial to profile their cellular and structural composition and to fully understand the implication of cell localization, interconnection and morphology for cancer progression and treatment design.
Multispectral large-scale single-cell resolution three-dimensional (mLSR-3D) imaging is a new addition to the volumetric microscopy toolbox that enables simultaneous spatial recording of 8–10 molecular markers in intact biological samples. To further exploit its spatial profiling potential, mLSR-3D was combined with a newly developed artificial intelligence (AI)-based analytical pipeline, segmentation analysis by parallelization of 3D datasets (STAPL-3D), enabling extraction of hundreds of molecular, spatial and volumetric features from millions of cells imaged in 3D. With this pipeline, we were able to perform in situ single cell profiling without forfeiting spatial information, or cell loss, which occurs with tissue dissociation. Using mLSR-3D and STAPL-3D, we could unravel the structural organization of human tumours and how this relates to organ development. For example, we profiled the developmental trajectory of a pediatric kidney Wilms tumour (WT), in relation to healthy kidney development. This revealed a highly disorganized spatial pattern in the WT compared to a healthy fetal kidney. Yet, cell identity could be matched to fetal cells, with the identification of an enlarged committed progenitor cluster fuelled by intrinsic cycling properties, as well as a transitioning population derived from undifferentiated progenitors. Furthermore, we identified previously unreported tumour-specific cell subpopulations, uniquely retrieved by their spatial embedding and morphological attributes. This included a stromal cell subset with mesenchymal cell shape and SIX2 expression, and thereby, likely to display enhanced stemness and invasive properties. Hence, mLSR-3D and STAPL-3D has the potential to uncover new tumour cell populations that might impact on tumour aggressiveness and prognosis.
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