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By retaining genetic, histological, and functional characteristics of the original tumor, including intra\u2010 and interpatient heterogeneity, they provide powerful tools to investigate therapy resistance. While genomic profiling is well established in organoid\u2010based screening, non\u2010genetic 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\n                    <jats:italic>Apc<\/jats:italic>\n                    ,\n                    <jats:italic>Braf<\/jats:italic>\n                    , and\n                    <jats:italic>Trp53<\/jats:italic>\n                    mutations. Using our automated Pick\u2010Flow\u2010Drop 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\u2010deprived conditions, correlating with distinct Wnt\u20103a profiles in the culture supernatant. Solid organoids were more trametinib\u2010sensitive and exhibited higher Wnt\u20103a levels, suggesting divergent cell compositions and pathway dependencies. Our findings highlight the functional relevance of non\u2010genetic variability in organoid cultures and establish a framework to improve reproducibility and biological insight in PDO\u2010based drug screening.\n                  <\/jats:p>","DOI":"10.1002\/ijc.70453","type":"journal-article","created":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T03:48:02Z","timestamp":1773632882000},"page":"490-500","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Impact of non\u2010genetic heterogeneity of\n                    <scp>BRAF<\/scp>\n                    \u2010mutant colon cancer organoids on growth kinetics, drug sensitivity and Wnt dynamics"],"prefix":"10.1002","volume":"159","author":[{"given":"Viktoria","family":"Zieger","sequence":"first","affiliation":[{"name":"Laboratory for MEMS Applications, IMTEK \u2010 Department of Microsystems Engineering University of Freiburg  Freiburg 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