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Updated: Mar 7, 2026
Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture
Published on: June 23, 2020
3D in vitro co-culture disc for assessing cancer invasiveness in the tumor stroma microenvironment
Weiyou Sun1, Yutaka Kikuchi1, Haruko Takahashi1
1Graduate School of Integrated Sciences for Life, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526 Japan.
Purpose:
Cancer cell invasion into the stroma is a key step in tumor progression, yet many in vitro models fail to recapitulate stromal architecture or permit quantitative analysis along the thickness (Z) dimension. This study aimed to develop a simple 3D co-culture disc system that maintains fibroblast-rich gels without shrinkage and enables quantitative evaluation of epithelial cancer cell invasion.
Methods:
A thin plastic disc with a central opening was used to support collagen gels containing normal human lung fibroblasts (NHLFs). Noncancerous MCF-10 A and cancer cell lines with different invasive capacities (MCF-7, MDA-MB-231) were seeded on the gel surface. Gel contraction, fibroblast morphology, and invasion depth were assessed using fluorescence labeling and confocal laser scanning microscopy (CLSM). Transforming growth factor-β1 (TGF-β1) was applied to test responsiveness to environmental stimuli.
Results:
Fibroblast-rich gels contracted uniaxially along the Z-axis but remained laterally stable within the disc, enabling long-term co-culture. Cancer cells showed distinct invasion profiles, with MDA-MB-231 invading significantly deeper than MCF-10 A or MCF-7. Invading cells frequently localized near αSMA-high myofibroblast-like fibroblasts. TGF-β treatment increased fibroblast contractility and further enhanced cancer cell invasion, demonstrating that the system quantitatively detects stromal activation and invasion-promoting cues.
Conclusion:
The disc-based 3D co-culture system offers a simple and physiologically relevant platform for quantifying cancer cell invasion and fibroblast activation. By supporting fibroblast-rich stromal environments, resolving Z-axis invasion, and responding to external stimuli such as TGF-β, this system provides a practical tool for studying tumor-stroma interactions and may be applicable to patient-derived cells in future studies.
Supplementary Information:
The online version contains supplementary material available at 10.1007/s44164-026-00106-0.
