Impact of non‐genetic heterogeneity of <scp>BRAF</scp> ‐mutant colon cancer organoids on growth kinetics, drug sensitivity and Wnt dynamics

Patient-derived organoid (PDO) models are powerful systems for studying tumor biology and drug response. By retaining genetic, histological, and functional characteristics of the original tumor, including intra- and interpatient heterogeneity, they provide powerful tools to investigate therapy resistance. While genomic profiling is well established in organoid-based screening, non-genetic parameters such as organoid size, seeding density, and morphology remain underexplored, despite their potential to influence functional readouts. Here, we systematically examined how these factors affect proliferation, drug sensitivity, and Wnt responses in a murine colorectal cancer organoid model carrying oncogenic Apc, Braf, and Trp53 mutations. Using our automated Pick-Flow-Drop handling platform, we implemented a reproducible plating workflow enabling precise control over organoid selection and screening conditions. We found that higher seeding density and larger organoid size reduced metabolic activity and decreased sensitivity to the MEK inhibitor trametinib. Moreover, distinct morphological subgroups, such as solid and cystic organoids, displayed differential drug responses under growth factor-deprived conditions, correlating with distinct Wnt-3a profiles in the culture supernatant. Solid organoids were more trametinib-sensitive and exhibited higher Wnt-3a levels, suggesting divergent cell compositions and pathway dependencies. Our findings highlight the functional relevance of non-genetic variability in organoid cultures and establish a framework to improve reproducibility and biological insight in PDO-based drug screening.