Translational 3D in vitro models for immunotherapy testing: from reconstituted organoid co-culture assays to autologous ex vivo patient tissues

The lack of patient-relevant, clinically predictive models for early (immuno)-oncology (IO) drug discovery is a significant challenge for drug developers. Advances in three-dimensional (3D) cell culture technologies are narrowing the gap for preclinical drug testing, capturing cell–cell and cell–matrix interactions, extracellular matrix components, and spatial organization absent in 2D systems and crucial in modeling immunotherapy efficacy. Moreover, these advanced systems more accurately reflect tumor genetics, antigen expression levels, tumor heterogeneity, and critically, have proven to be highly predictive of clinical responses. Here, we present two novel high-content imaging-based 3D in vitro platforms for immune-modulating drug screening: a reconstituted organoid-based model that recreates key aspects of the tumor microenvironment under controlled conditions, and a native ex vivo patient tissue platform that retains the original components of the microenvironment, including immune cell composition and tumor heterogeneity, enabling comparative testing of immunotherapies in an autologous setting. Both methods were evaluated using multiple therapeutic modalities, comparing their ability to capture relevant immune responses. By outlining their strengths, limitations, and translational potential, this work highlights how advanced 3D in vitro models can accelerate development of effective immune-modulating therapies, aid in biomarker identifications, and inform personalized treatment strategies.