Protocol for first-hit oncogenic organoid modeling and lentiviral functional screening

We present a protocol for modeling early tumorigenesis using air-liquid interface (ALI) organoids derived from oral, esophageal, and lung epithelia bearing first-hit oncogenic mutations. We describe steps for identifying candidate amplified oncogenes through outlier analysis, establishing ALI organoids, and inducing oncogenic activation. We then perform pooled lentiviral open reading frame (ORF) library delivery, selection, and barcode sequencing for functional screening. This approach enables validation of candidate drivers of neoplastic transformations, addressing a gap between in vitro and in vivo tumorigenesis studies. For complete details on the use and execution of this protocol, please refer to Salahudeen et al.¹.