Fluorescently engineered KRAS-mutant organoids as versatile tools for in vitro and in vivo cancer modeling

Organoids have emerged as advanced models in cancer research, offering superior physiological relevance compared to traditional 2D cell cultures. They mimic the three-dimensional architecture and tumor clonal heterogeneity, providing more accurate insights into tumor biology and drug responses. Despite these advantages, organoid-based studies pose significant technical challenges. In in-vitro studies, imaging and drug screening—particularly in co-culture systems—can be difficult. In in-vivo models, assessing tumor characteristics and organoid populations becomes challenging when using heterogeneous organoid mixtures with different mutation profiles. This challenge is further compounded when organoids are not engineered to express imageable markers. To address these limitations, we developed organoids that express green fluorescent protein (GFP) or red fluorescent protein (RFP). We found that factors such as cell seeding density, medium composition, and dissociation methods significantly affect organoid growth and IC50 values in response to chemotherapy drugs in in-vitro systems. Furthermore, we demonstrated that engineering organoids to express GFP or RFP did not significantly alter their growth, drug response, or gene expression profiles. Lastly, in in-vivo studies, we observed no significant changes in tumor growth, and morphology between engineered organoids and their parental lines. In conclusion, our findings suggest that GFP- and RFP-expressing organoids retain the key characteristics of their parental lines and can serve as robust tools for both in-vitro and in-vivo drug screening studies. • Culture conditions impact organoid growth and response to chemotherapy. • Reduced growth factors medium improves reliability of drug sensitivity assays. • Fluorescent labeling does not alter growth or gene expression of organoids in vitro. • Labeled organoids retain tumor growth and morphology in animal models.