SImBA-SiQuAl: a new tool enabling high-content high-throughput phenotypic profiling of 3D microtumours

SUMMARY Three-dimensional microtumour models such as spheroids are increasingly used in cancer research as they better capture tumour architecture, growth and invasion than conventional two-dimensional cultures. However, robust and accessible tools for quantitative analysis remain limited. Here we present SImBA-SiQuAl, an integrated open-source workflow for high-throughput quantitative phenotyping of 3D spheroids and organoids. The pipeline combines SImBA, an automated image-analysis framework for performant quality-controlled image segmentation and multi-feature extraction from spheroid assays, with SiQuAl, a downstream analysis platform that automatically performs comprehensive statistical and multivariate analyses to reveal phenotypic differences between experimental conditions. In a first case study, SImBA-SiQuAl resolves intrinsic invasion phenotypes between cancer cell lines. In a second case study, the workflow quantifies both uniform and heterogeneous responses in a spheroid drug screening assay. Together, SImBA-SiQuAl provides a new, timely tool for high-throughput, high-content microtumour phenomics in cancer research. MOTIVATION 3D-microtumour assays such as spheroids and organoids are increasingly used in preclinical research. These assays generate rich phenotypic imaging data, but quantitative automated analysis remains a major bottleneck. This limits reproducibility, scalability, and broad adoption for large-scale, high-content phenomics studies, but also implies biologically relevant phenotypic (heterogeneous) responses in e.g. perturbation studies may not be comprehensively addressed. SImBA-SiQuAl is developed to address this gap by providing an open-source, integrated workflow offering solutions in both the image processing and downstream analysis. Together, this enables in-depth quantitative analysis of 3D microtumour phenotypes across experimental settings. HIGHLIGHTS SImBA-SiQuAl provides a complete end-to-end workflow for high-throughput, high-content, quantitative 3D microtumour analysis, from quality-controlled image segmentation to statistical, multivariate and cluster-based biological interpretation. SImBA-SiQuAl is broadly applicable across multiple 3D systems and assay types. We demonstrate the workflow can capture biologically meaningful heterogeneity and treatment response at scale, supporting robust and unbiased analysis. By combining accessibility, flexibility and analytical depth, SImBA-SiQuAl addresses a key unmet need for accessible advanced open-source tools in 3D preclinical research.