Ravian L. van Ineveld, Michiel Kleinnijenhuis, Maria Alieva, Sam de Blank, Mario Barrera Roman, Esmée J. van Vliet, Clara Martínez Mir, Hannah R. Johnson, Frank L. Bos, Raimond Heukers, Susana M. Chuva de Sousa Lopes, Jarno Drost, Johanna F. Dekkers, Ellen J. Wehrens, Anne C. Rios

Jun 3, 2021
Nature Biotechnology
Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning–based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor’s spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.
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