Abstract
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.