This study explores the potential of canine patient-derived organoids (PDOs) in identifying mammary tumor-specific weaknesses by comparing tumor with matching normal mammary PDOs from the same patient. For this purpose, two customized CRISPR/Cas9 screens were performed to identify genes that are essential for tumor cells to survive but dispensable for normal mammary epithelial cells. CDK2 was subsequently identified as an important vulnerability in PDOs derived from a canine mammary tumor patient, which can be targeted by an existing inhibitor, PF3600. Although we only used tumor and normal PDOs from a single patient, our approach exemplifies how to discover novel, more individualized anti-cancer treatments for dogs.
High-throughput omics approaches have long been used to uncover potential vulnerabilities in human personalized oncology, but are often limited by the lack of function-al validation. Therefore, emphasis was put on functional drug testing using patient-derived organoids (PDOs). However, PDOs generated from tumors mostly lack comparison with matching normal tissue and the number of testable drugs is limited. Here, we demonstrate how matching neoplastic and non-neoplastic mammary PDOs derived from the same dog can utilize targeted CRISPR-Cas9 screens to unveil cancer cell specific vulnerabilities.    As a proof-of-principle experiment, we designed two different canine CRISPR/Cas9 sublibraries targeting either the epigenome (n=1,269) or druggable genes (n=834) and completed two independent pooled CRISPR/Cas9 dropout screens in paired PDOs de-rived from both carcinoma and normal mammary tissues from an individual dog. By comparing genes essential for tumor cells to survive but dispensable for non-neoplastic cells, we identified and validated CDK2 as a functional vulnerability in canine mammary tumors (CMT) that can be targeted with the PF3600 inhibitor. Moreover, we un-raveled additional specific targets that may guide the choice of individualized anti-cancer therapy. Hence, functional screening of individual neoplastic versus non-neoplastic PDOs using targeted CRISPR/Cas9 libraries is a powerful approach to identifying targetable vulnerabilities in dogs.