In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.

Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.

Intravital imaging of cancer stem cell plasticity in mammary tumors.

It is widely debated whether all tumor cells in mammary tumors have the same potential to propagate and maintain tumor growth or whether there is a hierarchical organization. Evidence for the latter theory is mainly based on the ability or failure of transplanted tumor cells to produce detectable tumors in mice with compromised immune systems; however, this assay has lately been disputed to accurately reflect cell behavior in unperturbed tumors. Lineage tracing experiments have recently shown the existence of a small population of cells, referred to as cancer stem cells (CSCs), that maintains and provides growth of squamous skin tumors and intestinal adenomas. However, the lineage tracing techniques used in these studies provide static images and lack the ability to study whether stem cell properties can be obtained or lost, a process referred to as stem cell plasticity. Here, by intravital lineage tracing, we report for the first time the existence of CSCs in unperturbed mammary tumors and demonstrate CSC plasticity. Our data indicate that existing CSCs disappear and new CSCs form during mammary tumor growth, illustrating the dynamic nature of these 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. 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 Apc mutations 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.

Fsp1-Mediated Lineage Tracing Fails to Detect the Majority of Disseminating Cells Undergoing EMT.

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.