SUMMARY Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation and exhibit ductal- and disease stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon1, stomach2 and hair follicles3. A 3D culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids that retain many characteristics of the original epithelial architecture2, 4, 5. A crucial component of the culture medium is the Wnt agonist Rspo16, the recently discovered ligand of Lgr57, 8. Here we show that Lgr5-LacZ is not expressed in healthy adult liver, yet that small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by lineage tracing using a novel Lgr5-ires-CreERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged liver can be clonally expanded as organoids in Rspo1-based culture medium over multiple months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into FAH−/− mice. These findings imply that previous observations on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.

Organoids are self‐organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long‐term‐expanding human airway organoids from broncho‐alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi‐ciliated cells, mucus‐producing secretory cells, and CC10‐secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non‐structural viral NS2 protein, and preferentially recruits neutrophils upon co‐culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.