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Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.A biobank of ovarian cancer organoids recapitulates the histopathological and molecular hallmarks of patient tumors and provides a resource for preclinical research.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine. Pre-clinical cell culture models capturing the heterogeneity of childhood kidney tumours are limited. Here, the authors establish and characterise an organoid biobank of tumour and matched normal organoid cultures from over 50 children with different subtypes of kidney cancer.

High-grade serous ovarian cancer (HG-SOC)—often referred to as a “silent killer”—is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53 , Brca1 , Nf1 and Pten . Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients. The relative contribution of fallopian tube (FT) or ovarian surface epithelium (OSE) to high-grade serous ovarian cancer (HG-SOC) development is unclear. Here, the authors establish organoid models from murine oviductal and OSE tissues that allow cancer modeling via CRISPR-Cas9 genome editing, and report a dual origin of murine HG-SOC.

Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.

Most currently available colorectal cancer (CRC) mouse models are not suitable for studying progression toward the metastatic stage. Recently, establishment of tumor organoid lines, either from murine CRC models or patients, and the possibility of engineering them with genome-editing technologies, have provided a large collection of tumor material faithfully recapitulating phenotypic and genetic heterogeneity of native tumors. To study tumor progression in the natural in vivo environment, we developed an orthotopic approach based on transplantation of CRC organoids into the cecal epithelium. The 20-min procedure is described in detail here and enables growth of transplanted organoids into a single tumor mass within the intestinal tract. Due to long latency, tumor cells are capable of spreading through the blood circulation and forming metastases at distant sites. This method is designed to generate tumors suitable for studying CRC progression, thereby providing the opportunity to visualize tumor cell dynamics in vivo in real time by intravital microscopy.

In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-β signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize—i.e., to colonize distant sites—is the direct consequence of the loss of dependency on specific niche signals.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt‐agonistic R‐spondins (RSPOs). Intestinal, stomach and liver Lgr5 + stem cells grow in 3D cultures to form ever‐expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro , duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1‐based cultures, and develop into budding cyst‐like structures (organoids) that expand five‐fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi‐potentiality. The establishment of conditions for long‐term culture and expansion of adult, bi‐potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5⁺) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5⁺ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5⁺ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4⁺ DCS cells promote organoid formation of single Lgr5⁺ colon stem cells. DCS cells can be massively produced from Lgr5⁺ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4⁺ DCS cells serve as Paneth cell equivalents in the colon crypt niche.

Intestinal cancer: stem-cell destinies Inappropriate activation of the Wnt signalling pathway in intestinal stem cells causes them to become cancerous. Two papers in this issue help identify the cell type at the root of this cancer, which should in turn aid therapeutic design. Zhu et al . report that prominin 1, a surface protein found on both normal stem cells and cancer stem cells, is a marker for stem cells that are prone to neoplastic transformation. Barker et al . show that in cells expressing Lgr5 , previously identified as a marker for intestinal stem cells, activation of Wnt signalling is sufficient to initiate tumour formation. Intestinal tumours can originate from Lgr5 + intestinal stem cells after genetic activation of the Wnt signalling pathway. Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli ( APC ). As in most cancers, the cell of origin has remained elusive. In a previously established Lgr5 (leucine-rich-repeat containing G-protein-coupled receptor 5) knockin mouse model, a tamoxifen-inducible Cre recombinase is expressed in long-lived intestinal stem cells 1 . Here we show that deletion of Apc in these stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fuelling a growing microadenoma. These microadenomas show unimpeded growth and develop into macroscopic adenomas within 3-5weeks. The distribution of Lgr5 + cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. When Apc is deleted in short-lived transit-amplifying cells using a different cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30weeks, large adenomas are very rare in these mice. We conclude that stem-cell-specific loss of Apc results in progressively growing neoplasia.

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes. CRISPR base editing technologies can be used for disease modelling. Here the authors use various base editing tools to generate tumour models in human adult stem cell-derived hepatocyte, endometrial and intestinal organoids.