Jeroen Matthijs Bugter

Organoids provide a versatile platform for studying fundamental biology, complementing traditional cell culture and in vivo models. Their unique features, such as self-organization and the possibility for their precise manipulation, have enabled key discoveries in tissue homeostasis, development and disease. This tutorial presents an overview of organoid technologies and protocols across a broad spectrum of applications, from investigating cell fate decisions and cell–cell or cell–microbe interactions to the study of genetic diseases, species evolution and translational/clinical questions. By focusing on adult stem cell–derived intestinal and human pluripotent stem cell–derived brain organoids, we highlight models with distinct characteristics that together reflect the broader spectrum of organoid systems, tissue types and research contexts. For applications not supported by these models, we discuss organoids derived from other tissues. We also outline current challenges in the field and highlight emerging innovations that are promising to shape future directions.

Oncogenes such as KRAS display marked tissue specificity in their oncogenic potential, genetic interactions and phenotypic effects, but the underlying determinants remain largely unresolved ^1–5 . Here, to address these questions, we developed the Mouse Cancer Cell line Atlas, a broad-utility resource of 590 comprehensively characterized models across a wide range of entities ( www.mcca.tum.de ). Comparative and functional studies using this platform, human cohorts and mice identified core principles underlying tissue-specific evolution of KRAS -initiated cancers. First, we show that mutant KRAS dosage gain through allelic imbalance exerts cell-type-specific effects, defining its timing across entities, as exemplified by dosage-sensitive developmental reprogramming during pancreatic cancer initiation. Second, we highlight how tissue- and stage-specific evolutionary requirements, such as block of differentiation in the intestine, select for KRAS -collaborating alterations. Third, we identified context-dependent epistatic KRAS –tumour suppressor interactions and show that reciprocal dosage sensitivities dictate the entity-specific patterns of cancer gene alterations, explaining their frequency, zygosity and acquisition chronology. These findings highlight how intrinsic and acquired determinants instruct cancer evolution in different tissues, with predictable molecular patterns, temporal dynamics and phenotypic outcomes. Our study provides major advances towards a mechanistic understanding of cancer genomes.

Hyperactivation of WNT signaling is a well-established hallmark of cancer. Various epithelial cancers express high levels of WNT7B and WNT10A that are not commonly expressed during tissue homeostasis, but rather associate with tissue development and regeneration. Although increased WNT7B/10A expression correlates with aggressive disease and lower patient survival rates, the mechanism by which these WNTs influence cancer progression remains unknown. Here, we use patient-derived organoids to show that tumor-intrinsic expression of WNT7B/10A drives survival and growth of advanced pancreatic ductal adenocarcinoma (PDAC). Bulk and single-cell profiling reveal that WNT7B drives proliferation and promotes expression of a poor prognosis basal-like state by preventing expression of a more differentiated, classical PDAC signature. By generating WNT7B reporter organoids, we show that heterogeneously distributed WNT-high PDAC cells are shifted towards a more basal-like phenotype and stably co-exist with WNT-low/negative lineages. Furthermore, hybrid co-cultures of WNT7B-proficient and -knockout PDAC organoids demonstrate that WNT-sending cells drive survival and proliferation of neighboring WNT-negative cells within the cancer epithelium via short range, cell contact-dependent signaling. In summary, our work uncovers a prominent role of WNT7B/10A in driving PDAC subtype heterogeneity and argues that WNT inhibition may be applied to force a class switch to a more differentiated, less aggressive cancer subtype that correlates with improved therapeutical response.

The contribution of deubiquitylating enzymes (DUBs) to β-Catenin stabilization in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, and by using an unbiassed screen, we discovered that the DUB USP10 stabilizes β-Catenin specifically in APC-truncated CRC in vitro and in vivo. Mechanistic studies, including in vitro binding together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and is outcompeted by intact APC, favouring β-catenin degradation. However, in APC-truncated cancer cells USP10 binds to β-catenin, increasing its stability which is critical for maintaining an undifferentiated tumour identity. Elimination of USP10 reduces the expression of WNT and stem cell signatures and induces the expression of differentiation genes. Remarkably, silencing of USP10 in murine and patient-derived CRC organoids established that it is essential for NOTUM signalling and the APC super competitor-phenotype, reducing tumorigenic properties of APC-truncated CRC. These findings are clinically relevant as patient-derived organoids are highly dependent on USP10, and abundance of USP10 correlates with poorer prognosis of CRC patients. Our findings reveal, therefore, a role for USP10 in CRC cell identity, stemness, and tumorigenic growth by stabilising β-Catenin, leading to aberrant WNT signalling and degradation resistant tumours. Thus, USP10 emerges as a unique therapeutic target in APC truncated CRC.

The transmembrane E3 ligases RNF43 and ZNRF3 perform key tumour suppressor roles by inducing endocytosis of members of the Frizzled (FZD) family, the primary receptors for WNT. Loss-of-function mutations in RNF43 and ZNRF3 mediate FZD stabilisation and a WNT-hypersensitive growth state in various cancer types. Strikingly, RNF43 and ZNRF3 mutations are differentially distributed across cancer types, raising questions about their functional redundance. Here, we compare the efficacy of RNF43 and ZNRF3 of targeting different FZDs for endocytosis. We find that RNF43 preferentially downregulates FZD1/FZD5/FZD7, while ZNRF3 displays preference towards FZD6/FZD10. We show that the RNF43 transmembrane domain (TMD) is a key molecular determinant for inducing FZD5 endocytosis. Furthermore, a TMD swap between RNF43 and ZNRF3 re-directs their preference for FZD member downregulation. We conclude that RNF43 and ZNRF3 preferentially downregulate specific FZDs by a TMD-dependent mechanism. In accordance, tissue-specific expression patterns of FZD homologues correlate with the incidence of RNF43 or ZNRF3 cancer mutations in those tissues. Consequently, our data point to druggable vulnerabilities of specific FZD receptors in RNF43- or ZNRF3 -mutant human cancers.

The contribution of deubiquitylating enzymes to β-Catenin stabilisation in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, we report the deubiquitylase USP10 as an APC-truncation- specific enhancer of β-Catenin stability, potentiating WNT signalling and cancer stem cells and CRC. Mechanistically, interaction and in vitro binding studies, together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and requires the absence of full-length APC. Reduction of USP10 induces the expression of differentiation genes and opposes the APC-truncated phenotype in an intestinal hyperplasia model. Notably, loss of USP10 in CRISPR engineered intestinal organoids opposed the super competitor-signalling and reduced tumorigenic properties of APC-mutated CRC. Taken together, our findings reveal USP10s role in CRC cell identity, stemness and tumour growth by stabilising β-Catenin, leading to aberrant WNT signalling, and implicate USP10 as a cancer specific therapeutic vulnerability in Apc truncated CRC.

Summary: The contribution of deubiquitylating enzymes to β-Catenin stabilisation in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, we report the deubiquitylase USP10 as an APC-truncation- specific enhancer of β-Catenin stability, potentiating WNT signalling in CRC and cancer stem cells. Mechanistically, interaction studies in various CRC cell lines and in vitro binding studies, together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and requires the absence of full-length APC. Notably, loss of USP10 in CRISPR engineered intestinal organoids reduces tumorigenic properties of CRC and blocks the super competitor-signalling of APC-mutated CRC. Furthermore, reduction of USP10 induces the expression of differentiation genes, and opposes the APC-truncated phenotype in an intestinal hyperplasia model of D.melanogaster . Taken together, our findings reveal USP10s role in intestinal tumourigenesis by stabilising β-Catenin, leading to aberrant WNT signalling, enhancing cancer cell stemness and implicate the DUB USP10 as a cancer specific therapeutic vulnerability in Apc truncated CRC.

Summary: In colorectal cancer (CRC), RNF43 mutations are linked to BRAF V600E-initiated serrated adenomas that advance into mucinous adenocarcinomas with poor prognosis upon metastasis. How RNF43 mutations facilitate a metastasis-prone growth state remains unknown. Here, we addressed this issue by repairing mutant RNF43 in patient-derived BRAF-mutant CRC organoids using gene editing. Upon RNF43 correction, CRC organoids exhibit strongly decreased mucus production and, moreover, display loss of niche factor independence and metastatic capacity upon orthotopic transplantation in mice. Mechanistically, we show that mutant RNF43 promotes cancer cell lineage specification towards a non-dividing niche population that secretes essential growth factors, providing a state of self-sufficiency to the cancer epithelium. We show that phenotypic diversification into tumour-intrinsic niche cells (TINCs) and proliferative cancer stem cells depends on tuneable WNT levels enabled by mutations in RNF43 , but not APC . In patient samples, enhanced TINC profiles correlate with RNF43 -mutant CRC, mucinous histology and metastatic disease, thus representing a general cellular mechanism by which tumours acquire a self-sufficient, pro-metastatic growth state.