Protocol for generating liver metastasis microtissues to decipher cellular interactions between metastatic intestinal cancer and liver tissue.

Cell competition is a quality control mechanism that promotes elimination of suboptimal cells relative to fitter neighbors. Cancer cells exploit these mechanisms for expansion, but the underlying molecular pathways remain elusive. Here, we present a protocol for generating matrix-free microtissues recapitulating cellular interactions between intestinal cancer and hepatocyte-like cells using microscopy or transcriptomics/proteomics.

NAC regulates metabolism and cell fate in intestinal stem cells.

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity; however, it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process.

Cell competition in primary and metastatic colorectal cancer.

Adult tissues set the scene for a continuous battle between cells, where a comparison of cellular fitness results in the elimination of weaker "loser" cells. This phenomenon, named cell competition, is beneficial for tissue integrity and homeostasis. In fact, cell competition plays a crucial role in tumor suppression, through elimination of early malignant cells, as part of Epithelial Defense Against Cancer.

Cell competition promotes metastatic intestinal cancer through a multistage process.

Cell competition plays an instrumental role in quality control during tissue development and homeostasis. Nevertheless, cancer cells can exploit this process for their own proliferative advantage. In our study, we generated mixed murine organoids and microtissues to explore the impact of cell competition on liver metastasis.

Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

Background: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al.

Ribosome impairment regulates intestinal stem cell identity via ZAKɑ activation.

The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5-expressing intestinal stem cells (ISCs). However, several Lgr5-negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown.

Generation of mixed murine organoids to model cellular interactions.

Cell competition is a mechanism of interaction that dictates cell selection based on differences in cellular fitness. We designed a protocol to generate mixed murine organoids and enteroid monolayers used to study such complex cellular interactions in a mammalian system. This protocol is dedicated to follow the behavior of different cell populations over time, using (time-lapse) microscopy or transcriptome/proteome analysis.

Active elimination of intestinal cells drives oncogenic growth in organoids.

Competitive cell interactions play a crucial role in quality control during development and homeostasis. Here, we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells.

Calorie Restriction Increases the Number of Competing Stem Cells and Decreases Mutation Retention in the Intestine.

Calorie restriction (CR) extends lifespan through several intracellular mechanisms, including increased DNA repair, leading to fewer DNA mutations that cause age-related pathologies. However, it remains unknown how CR acts on mutation retention at the tissue level. Here, we use Cre-mediated DNA recombination of the confetti reporter as proxy for neutral mutations and follow these mutations by intravital microscopy to identify how CR affects retention of mutations in the intestine.

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer.

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5 and formed distant metastases in which Lgr5 CSCs appeared.

A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression.

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.

From good to bad: Intravital imaging of the hijack of physiological processes by cancer cells.

Homeostasis of tissues is tightly regulated at the cellular, tissue and organismal level. Interestingly, tumor cells have found ways to hijack many of these physiological processes at all the different levels. Here we review how intravital microscopy techniques have provided new insights into our understanding of tissue homeostasis and cancer progression.

Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.

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.

Integration of kinase and phosphatase activities by BUBR1 ensures formation of stable kinetochore-microtubule attachments.

Maintenance of chromosomal stability depends on error-free chromosome segregation. The pseudokinase BUBR1 is essential for this, because it is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. We have identified a conserved and highly phosphorylated domain (KARD) in BUBR1 that is crucial for formation of kinetochore-microtubule attachments.

The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase.

Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive.

Molecular causes for BUBR1 dysfunction in the human cancer predisposition syndrome mosaic variegated aneuploidy.

Genetic mutations in the mitotic regulatory kinase BUBR1 are associated with the cancer-susceptible disorder mosaic variegated aneuploidy (MVA). In patients with biallelic mutations, a missense mutation pairs with a truncating mutation. Here, we show that cell lines derived from MVA patients with biallelic mutations have an impaired mitotic checkpoint, chromosome alignment defects, and low overall BUBR1 abundance.

A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.

Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function.

Preventing aneuploidy: the contribution of mitotic checkpoint proteins.

Aneuploidy, an abnormal number of chromosomes, is a trait shared by most solid tumors. Chromosomal instability (CIN) manifested as aneuploidy might promote tumorigenesis and cause increased resistance to anti-cancer therapies. The mitotic checkpoint or spindle assembly checkpoint is a major signaling pathway involved in the prevention of CIN.

The Intestinal Wnt/TCF Signature.

Background & Aims: In colorectal cancer, activating mutations in the Wnt pathway transform epithelial cells through the inappropriate expression of a TCF4 target gene program, which is physiologically expressed in intestinal crypts.

Methods: We have now performed an exhaustive array-based analysis of this target gene program in colorectal cancer cell lines carrying an inducible block of the Wnt cascade. Independently, differential gene-expression profiles of human adenomas and adenocarcinomas vs normal colonic epithelium were obtained.