Clinical implementation of tumor organoids for personalized medicine requires that pure tumor organoids can be reliably established. Here, we present our experience with organoid cultures from >70 non-small cell lung cancer (NSCLC) samples. We systematically evaluate several methods to identify tumor purity of organoids established from intrapulmonary tumors. Eighty percent of organoids from intrapulmonary lesions have a normal copy number profile, suggesting overgrowth by normal airway organoids (AOs). This is further supported by the failure to detect mutations found in the original tumor in organoids. Histomorphology alone is insufficient to determine tumor purity, but when combined with p63 immunostaining, tumor and normal AOs can be distinguished. Taking into account overgrowth by normal AOs, the establishment rate of pure NSCLC organoids is 17%. Therefore, current methods are insufficient to establish pure NSCLC organoids from intrapulmonary lesions. We discourage their use unless steps are taken to prevent overgrowth by normal AOs.
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