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Organoid technology provides the possibility of culturing patient-derived colon tissue and colorectal cancers (CRCs) while maintaining all functional and phenotypic characteristics. Labeling stem cells, especially in normal and benign tumor organoids of human colon, is challenging and therefore limitsmaximal exploitation of organoid libraries for human stem cell research. Here, we developed STAR (stem cell Ascl2 reporter), a minimal enhancer/promoter element that reports transcriptional activity of ASCL2, a master regulator of LGR5+intestinal stem cells. Using lentiviral infection, STAR drives specific expression in stem cells of normal organoids and in multiple engineered and patient-derived CRC organoids of different genetic makeup. STAR reveals that differentiation hierarchies and the potential for cell fate plasticity are present at all stages of human CRC development. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research into human adult stem cell biology.

Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT) has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells may adapt transient and reversible states. Here, we have tested the existence and role of epithelial-mesenchymal plasticity in metastasis of mammary tumors without artificially modifying EMT regulators. In these tumors, we found by intravital microscopy that the motile tumor cells have undergone EMT, while their epithelial counterparts were not migratory. Moreover, we found that epithelial-mesenchymal plasticity renders any EMT-induced stemness differences, as reported previously, irrelevant for metastatic outgrowth, because mesenchymal cells that arrive at secondary sites convert to the epithelial state within one or two divisions, thereby obtaining the same stem cell potential as their arrived epithelial counterparts. We conclude that epithelial-mesenchymal plasticity supports migration but additionally eliminates stemness-enhanced metastatic outgrowth differences.

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. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell-state transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. Jun N-terminal kinase (JNK) signaling is activated in competing cells and is required for cell-state change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell-state change in a JNK-dependent manner.

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. We find that CR leads to increased numbers of functional Lgr5+stem cells that compete for niche occupancy, resulting in slower but stronger stem cell competition. Consequently, stem cells carrying neutral or Apcmutations encounter more wild-type competitors, thus increasing the chance that they get displaced from the niche to get lost over time. Thus, our data show that CR not only affects the acquisition of mutations but also leads to lower retention of mutations in the intestine.

Epithelial-to-mesenchymal transition (EMT) has long been thought to be crucial for metastasis. Recently a study challenged this idea by demonstrating that metastases were seeded by tumor cells that were not marked by an EMT lineage-tracing reporter on the basis of the expression of the mesenchymal marker fsp1. However, the results of this study and their interpretation are under debate. Here, we combine the lineage-tracing reporter with our real-time EMT-state reporter and show that the fsp1-based EMT lineage-tracing reporter does not mark all disseminating mesenchymal cells with metastatic potential. Our findings demonstrate that fsp1-mediated lineage tracing does not allow any conclusions about the requirement of EMT for metastasis. Instead our data are fully consistent with previous reports that EMT is not a binary phenomenon but rather a spectrum of cellular states.