High-throughput omics approaches have long been used to uncover potential vulnerabilities in human personalized oncology but are often limited by the lack of functional validation. Therefore, we placed our emphasis 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 the neoplastic and non-neoplastic mammary PDOs derived from the same dog can utilize targeted CRISPR/Cas9 screens to unveil cancer cell specific vulnerabilities. We performed two independent CRISPR/Cas9 dropout screens using sub-libraries targeting the epigenome (n = 1269) or druggable genes (n = 834) in paired PDOs derived from both carcinoma and normal mammary tissues from the same dog. A comparison of essential genes for tumor cells survival identified CDK2 as a functional vulnerability in canine mammary tumors (CMTs) that can be targeted with the PF3600 inhibitor. Additional potential targets were also uncovered, providing insights for personalized cancer treatments in dogs.

Mammary tumors in dogs hold great potential as naturally occurring breast cancer models in translational oncology, as they share the same environmental risk factors, key histological features, hormone receptor expression patterns, prognostic factors, and genetic characteristics as their human counterparts. We aimed to develop in vitro tools that allow functional analysis of canine mammary tumors (CMT), as we have a poor understanding of the underlying biology that drives the growth of these heterogeneous tumors. We established the long-term culture of 24 organoid lines from 16 patients, including organoids derived from normal mammary epithelium or benign lesions. CMT organoids recapitulated key morphological and immunohistological features of the primary tissue from which they were derived, including hormone receptor status. Furthermore, genetic characteristics (driver gene mutations, DNA copy number variations, and single-nucleotide variants) were conserved within tumororganoid pairs. We show how CMT organoids are a suitable model for in vitro drug assays and can be used to investigate whether specific mutations predict therapy outcomes. In addition, we could genetically modify the CMT organoids and use them to perform pooled CRISPR/Cas9 screening, where library representation was accurately maintained. In summary, we present a robust 3D in vitro preclinical model that can be used in translational research, where organoids from normal, benign as well as malignant mammary tissues can be propagated from the same patient to study tumorigenesis.