Single-cell RNA sequencing profiles drug activity within spatially engineered 3D cultures

Spatial transcriptomic techniques provide a wealth of information useful in guiding drug development, while three-dimensional (3D) cell cultures have demonstrated power in accelerating drug approvals. However, techniques for robust spatial analysis of 3D cultures are limited. Here, we present a transfection-based method for constructing cellular spheroids through a layer-by-layer approach, in which DNA barcodes encode the spatial positioning of cells. Our technique facilitates multiplex single-cell RNA sequencing, providing spatial maps of gene expression and drug response, while correlative imaging reveals the locations of barcoded cell populations and quantifies local tissue elasticity. We show that model HeLa 3D spheroids display heterogeneous responses to drugs, which may arise through diffusion gradients of the drug, or from differences in metabolism, nutrient supply, and cellular stressors. The ability to create spatially encoded cellular assemblies may help to reveal spatial variation in gene expression within 3D culture models.

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Publication Details

Journal
Nature Communications
Published
2026-06-19
DOI
https://doi.org/10.1038/s41467-026-74587-8
Primary Topic
Single-cell and spatial transcriptomics
Type
article
Field-Weighted Citation Impact
0.00

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article

Single-cell RNA sequencing profiles drug activity within spatially engineered 3D cultures

Yu Suk Choi, Nishka Bhalla, Marck Nörret, Cameron W. Evans et al.
Nature Communications
Single-cell and spatial transcriptomics
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Single-cell RNA sequencing profiles drug activity within spatially engineered 3D cultures

Yu Suk Choi, Nishka Bhalla, Marck Nörret, Cameron W. Evans, Jessica J. King, Sebastian E. Amos, Alireza Mowla, Ulrich D. Kadolsky, K. Swaminathan Iyer, Alka Saxena, Jessica A. Kretzmann, Brendan J. Kennedy, Munir Iqbal, Giselle Sugianto, Nicole M. Smith
article en

Abstract

Spatial transcriptomic techniques provide a wealth of information useful in guiding drug development, while three-dimensional (3D) cell cultures have demonstrated power in accelerating drug approvals. However, techniques for robust spatial analysis of 3D cultures are limited. Here, we present a transfection-based method for constructing cellular spheroids through a layer-by-layer approach, in which DNA barcodes encode the spatial positioning of cells. Our technique facilitates multiplex single-cell RNA sequencing, providing spatial maps of gene expression and drug response, while correlative imaging reveals the locations of barcoded cell populations and quantifies local tissue elasticity. We show that model HeLa 3D spheroids display heterogeneous responses to drugs, which may arise through diffusion gradients of the drug, or from differences in metabolism, nutrient supply, and cellular stressors. The ability to create spatially encoded cellular assemblies may help to reveal spatial variation in gene expression within 3D culture models.

Nature Communications
University of Tasmania (AU), The University of Western Australia (AU), Harry Perkins Institute of Medical Research (AU), Nicolaus Copernicus University (PL), Institute of Physics (PL), ARC Centre of Excellence in Plant Energy Biology (AU), Tasmanian Land Conservancy (AU)
Australian Cancer Research Foundation, Government of Western Australia
Good health and well-being
Openalex Percentile: Top 11%
Single-cell and spatial transcriptomics
DOIOpen Access PDFOpenAlex
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