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Quantifying single-cell responses to irradiation in 3D.

Joshua François1, Alina Simerzin1, Ashwini Jambhekar1

  • 1Department of Systems Biology, Blavantik Institute, Harvard Medical School, Boston, MA, United States.

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|April 10, 2026
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Summary
This summary is machine-generated.

This study developed advanced tools to track cell responses in 3D cultures, revealing that cell signaling kinetics differ significantly in spheroids compared to 2D systems after irradiation.

Keywords:
3Dimagingmatrigel assaymicroscopyp21 (CDKN1A)spheroids

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Area of Science:

  • Cell Biology
  • Biophysics
  • Cancer Research

Background:

  • Three-dimensional (3D) cell cultures better mimic physiological conditions than 2D systems, enabling investigation of cell-cell and cell-extracellular matrix interactions.
  • Quantifying cellular signaling dynamics in 3D environments is challenging due to imaging and computational limitations.

Purpose of the Study:

  • To develop and validate experimental and computational tools for tracking individual cell responses in 3D.
  • To investigate the dynamics of p21 activation in human breast cancer cells following irradiation in 3D cultures.

Main Methods:

  • Developed a dual-Matrigel assay to embed individual cells and spheroids.
  • Utilized light sheet fluorescence microscopy (LSFM) for high-resolution imaging.
  • Created computational pipelines for quantitative analysis of p21 dynamics.

Main Results:

  • Dispersed cells showed a gradual increase in p21-positive cells post-irradiation, while spheroids exhibited transient decrease followed by a delayed rise.
  • p21 intensity remained constant, suggesting regulation of transcription probability rather than magnitude.
  • Spheroids displayed slower p21 induction kinetics compared to 2D and 3D single-cell cultures.

Conclusions:

  • The developed platform enables dissection of signaling dynamics in physiologically relevant 3D contexts.
  • Differences in p21 induction kinetics in spheroids highlight the impact of 3D architecture and cell-extracellular matrix interactions.
  • The framework can be extended to study drug effects on complex multicellular structures.