Anoek Zomer

Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging

The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior

Summary 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.

Brief Report: 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. STEM CELLS2013;31:602–606

Identity and dynamics of mammary stem cells during branching morphogenesis

During puberty, the mouse mammary gland develops into a highly branched epithelial network. Owing to the absence of exclusive stem cell markers, the location, multiplicity, dynamics and fate of mammary stem cells (MaSCs), which drive branching morphogenesis, are unknown. Here we show that morphogenesis is driven by proliferative terminal end buds that terminate or bifurcate with near equal probability, in a stochastic and time-invariant manner, leading to a heterogeneous epithelial network. We show that the majority of terminal end bud cells function as highly proliferative, lineage-committed MaSCs that are heterogeneous in their expression profile and short-term contribution to ductal extension. Yet, through cell rearrangements during terminal end bud bifurcation, each MaSC is able to contribute actively to long-term growth. Our study shows that the behaviour of MaSCs is not directly linked to a single expression profile. Instead, morphogenesis relies upon lineage-restricted heterogeneous MaSC populations that function as single equipotent pools in the long term.

Studying extracellular vesicle transfer by a Cre-loxP method

Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro– or ex vivo–isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase–expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre+ cells and reporter+ cells, which takes ∼6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼2 months.

Implications of Extracellular Vesicle Transfer on Cellular Heterogeneity in Cancer: What Are the Potential Clinical Ramifications?

The functional and phenotypic heterogeneity of tumor cells represents one of the greatest challenges in the successful treatment of cancer patients, because it increases the risk that certain individual tumor cells possess the ability to, for example, metastasize or to tolerate cytotoxic drugs. This heterogeneity in cellular behavior is driven by genetic and epigenetic changes and environmental differences. Recent studies suggest that an additional layer of complexity of tumor heterogeneity exists, based on the ability of cells to share functional biomolecules through local and systemic transfer of extracellular vesicles (EV), with profound effects on cellular behavior. The transfer of functional biomolecules between various populations of tumor cells and between tumor cells and nontumor cells has large consequences for both the tumor cells and the microenvironment that support the cellular behavior of tumor cells, and therefore for the clinical outcome of cancer. Here, we discuss the latest findings on EV transfer and the potential implications of EV-mediated local and systemic transmission of phenotypic behavior, particularly in the context of tumor heterogeneity, metastatic disease, and treatment response. Cancer Res; 76(8); 2071-5. ©2016 AACR.

Real-time intravital imaging of cancer models

High-resolution intravital imaging (IVM) has proven to be a powerful technique to visualise dynamic processes that are important for tumour progression, such as the interplay between tumour cells and cellular components of the tumour microenvironment. The development of IVM tools, including imaging windows and photo-marking of individual cells, has led to the visualisation of dynamic processes and tracking of individual cells over a time span of days. In order to visualise these dynamic processes, several strategies have been described to develop fluorescent IVM tumour models. Genetic tools to engineer fluorescent tumour cell lines have advanced the applications of cell line-based tumour models to study, for example, changes in behaviour or transcriptional and differentiation state of individual cells in a tumour. In order to study tumour progression, fluorescent genetic mouse models have been engineered that better recapitulate human tumours. These technically challenging tumour models are key in visualising dynamic processes during cancer progression and in the translational aspects of IVM experiments.

Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

Exosomes are small endosome-derived extracellular vesicles implicated in cell–cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB–PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB–PM fusion using live total internal reflection fluorescence and dynamic correlative light–electron microscopy. Quantitative analysis demonstrates that MVB–PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB–PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each others metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre‐LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi‐faceted biological response including cell migration.

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

ABSTRACT Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.