01812nas a2200217   4500000000100000000000100001008004100002260001500043653002800058653001800086653003500104653002500139100001500164700002000179700002600199245009900225856005500324300000900379520119200388022001401580       2025                            d  c2025-07-2510aCancer microenvironment10aCancer models10aExperimental models of disease10aTumour heterogeneity1 aGat Rauner1 aPiyush B. Gupta1 aCharlotte Kuperwasser00aFrom 2D to 3D and beyond: the evolution and impact of in vitro tumor models in cancer research  uhttps://www.nature.com/articles/s41592-025-02769-1  a1-123 aIn vitro tumor models are essential tools for cancer research, offering key insights into not only tumor biology but also therapeutic responses. The transition from traditional two-dimensional to three-dimensional organoid systems marks a paradigm shift in cancer modeling. Although two-dimensional models have been instrumental in elucidating fundamental molecular and genetic mechanisms, they fail to accurately replicate the intricate three-dimensional architecture and dynamic microenvironment characteristic of human tumors. Here we outline how advanced organoid technologies now enable more faithful recapitulation of tumor heterogeneity that better mimic native tissue mechanics and biochemistry. We discuss emerging methods, including air–liquid interface cultures, microfluidic tumor-on-a-chip devices and high-content imaging integrated with machine learning, which collectively address longstanding challenges such as matrix variability and the limited incorporation of immune and vascular elements. These innovations promise to enhance reproducibility and scalability while providing unprecedented insights into tumor biology, cancer progression and therapeutic strategies.  a1548-7105