Comprehensive high-throughput image analysis for therapeutic efficacy of architecturally complex heterotypic organoids.

Bioengineered three-dimensional (3D) tumor models that incorporate heterotypic cellular communication are gaining interest as they can recapitulate key features regarding the intrinsic heterogeneity of cancer tissues. However, the architectural complexity and heterogeneous contents associated with these models pose a challenge for toxicological assays to accurately report treatment outcomes. To address this issue, we describe a comprehensive image analysis procedure for structurally complex organotypic cultures (CALYPSO) applied to fluorescence-based assays to extract multiparametric readouts of treatment effects for heterotypic tumor cultures that enables advanced analyses. The capacity of this approach is exemplified on various 3D models including adherent/suspension, mono-/heterocellular cultures and several disease types. The subsequent analysis revealed specific morphological effects of oxaliplatin chemotherapy, radiotherapy, and photodynamic therapy. The procedure can be readily implemented in most laboratories to facilitate high-throughput toxicological screening of pharmaceutical agents and treatment regimens on organotypic cultures of human disease to expedite drug and therapy development.