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.

Retrograde movements determine effective stem cell numbers in the intestine.

The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.

Stem cell lineage survival as a noisy competition for niche access.

Understanding to what extent stem cell potential is a cell-intrinsic property or an emergent behavior coming from global tissue dynamics and geometry is a key outstanding question of systems and stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially localized niche, giving rise to a stochastic conveyor-belt model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favorably repositioned. Importantly, even when assuming that all cells in a tissue are molecularly equivalent, we predict a common ("universal") functional dependence of the long-term clonal survival probability on distance from the niche, as well as the emergence of a well-defined number of functional stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets of pubertal mammary gland tips and embryonic kidney tips, as well as homeostatic intestinal crypts. Importantly, we find good agreement for the predicted functional dependency of the competition as a function of position, and thus functional stem cell number in each organ. This argues for a key role of positional fluctuations in dictating stem cell number and dynamics, and we discuss the applicability of this theory to other settings.

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.

Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.

In Vivo Imaging Reveals Existence of Crypt Fission and Fusion in Adult Mouse Intestine.

The intestinal epithelium is a repetitive sheet of crypt and villus units with stem cells at the bottom of the crypts. During postnatal development, crypts multiply via fission, generating 2 daughter crypts from 1 parental crypt. In the adult intestine, crypt fission is observed at a low frequency. Using intravital microscopy in Lgr5EGFP-Ires-CreERT2 mice, we monitored individual crypt dynamics over multiple days with single-cell resolution. We discovered the existence of crypt fusion, an almost exact reverse phenomenon of crypt fission, in which 2 crypts fuse into 1 daughter crypt. Examining 819 crypts in 4 mice, we found that 3.5% ± 0.6% of all crypts were in the process of fission, whereas 4.1 ± 0.9% of all crypts were undergoing crypt fusion. As counteracting processes, crypt fission and fusion could regulate crypt numbers during the lifetime of a mouse. Identifying the mechanisms that regulate rates of crypt fission and fusion could provide insights into intestinal adaptation to altered environmental conditions and disease pathogenesis.

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.

Cell polarity proteins and cancer.

Cell polarity is essential in many biological processes and required for development as well as maintenance of tissue integrity. Loss of polarity is considered both a hallmark and precondition for human cancer. Three conserved polarity protein complexes regulate different modes of polarity that are conserved throughout numerous cell types and species. These complexes are the Crumbs, Par and Scribble complex. Given the importance of cell polarity for normal tissue homeostasis, aberrant polarity signaling is suggested to contribute to the multistep processes of human cancer. Most human cancers are formed from epithelial cells. Evidence confirming the roles for polarity proteins in different phases of the oncogenic trajectory comes from functional studies using mammalian cells as well as Drosophila and zebrafish models. Furthermore, several reports have revealed aberrant expression and localization of polarity proteins in different human tumors. In this review we will give an overview on the current data available that couple polarity signaling to tumorigenesis, particularly in epithelial cells.

Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations.

During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias toward proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations that may be broadly relevant for other tissues.

The vast majority of cells lining our intestine die within three to five days. They are replaced by a small group of stem cells which divide to produce either more stem cells, or cells that stop dividing and transform, or 'differentiate', in to mature cells in the intestine. Stem cells must generate the same number of dividing and differentiated cells. If there is even a slight bias and too many stem cells are produced, this can lead to uncontrolled growth, which is the root cause of cancer. In principal, the best way to achieve this balance is for stem cells to always asymmetrically divide in to two distinct cells: one that will continue to divide, and another that will mature in to an adult cell. However, recent research suggests that this process is much more random, with stem cells also dividing symmetrically, either in to two stem cells or two differentiated cells. So, how does the random nature of stem cell divisions not cause the number of dividing cells to fluctuate unpredictably in the intestine? To investigate, Huelsz-Prince et al. studied stem cells in a miniature model of the mouse intestine, known as an organoid, which can be grown outside of the body in a laboratory. All stem cells and their progeny were tracked for over 65 hours using a microscope to see how many dividing and differentiated cells they formed. This revealed that almost all stem cells in the organoid split symmetrically rather than asymmetrically. Huelsz-Prince et al. then developed a computer model of stem cells in the model intestine and tested the impact of changing the proportion of symmetric and asymmetric divisions. The results showed that having more symmetric divisions reduced fluctuations in the number of dividing cells better than high levels of asymmetric divisions. Other organs rely on a similar system to the intestine to replenish their mature cells. Consequently, the finding that symmetric divisions control fluctuations in the number of stem cells may be applicable to other parts of the body. Further testing with human disease samples, such as cells from cancer patients, using the organoid model system may also shed light on how division is disrupted in these conditions.

The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration.

Migration toward chemoattractants is a hallmark of T-cell trafficking and is essential to produce an efficient immune response. Here, we have analyzed the function of the Rac activator Tiam1 in the control of T-cell trafficking and transendothelial migration. We found that Tiam1 is required for chemokine- and S1P-induced Rac activation and subsequent cell migration. As a result, Tiam1-deficient T cells show reduced chemotaxis in vitro, and impaired homing, egress, and contact hypersensitivity in vivo. Analysis of the T-cell transendothelial migration cascade revealed that PKCzeta/Tiam1/Rac signaling is dispensable for T-cell arrest but is essential for the stabilization of polarization and efficient crawling of T cells on endothelial cells. T cells that lack Tiam1 predominantly transmigrate through individual endothelial cells (transcellular migration) rather than at endothelial junctions (paracellular migration), suggesting that T cells are able to change their route of transendothelial migration according to their polarization status and crawling capacity.

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity.

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.

An unanticipated tumor-suppressive role of the SUMO pathway in the intestine unveiled by Ubc9 haploinsufficiency.

Sumoylation is an essential posttranslational modification in eukaryotes that has emerged as an important pathway in oncogenic processes. Most human cancers display hyperactivated sumoylation and many cancer cells are remarkably sensitive to its inhibition, thus supporting application of chemical sumoylation inhibitors in cancer treatment. Here we show, first, that transformed embryonic fibroblasts derived from mice haploinsufficient for Ubc9, the essential and unique gene encoding the SUMO E2 conjugating enzyme, exhibit enhanced proliferation and transformed phenotypes in vitro and as xenografts ex vivo. To then evaluate the possible impact of loss of one Ubc9 allele in vivo, we used a mouse model of intestinal tumorigenesis. We crossed Ubc9+/- mice with mice harboring a conditional ablation of Apc either all along the crypt-villus axis or only in Lgr5+ crypt-based columnar (CBC) cells, the cell compartment that includes the intestinal stem cells proposed as cells-of-origin of intestinal cancer. While Ubc9+/- mice display no overt phenotypes and no globally visible hyposumoylation in cells of the small intestine, we found, strikingly, that, upon loss of Apc in both models, Ubc9+/- mice develop more (>2-fold) intestinal adenomas and show significantly shortened survival. This is accompanied by reduced global sumoylation levels in the polyps, indicating that Ubc9 levels become critical upon oncogenic stress. Moreover, we found that, in normal conditions, Ubc9+/- mice show a moderate but robust (15%) increase in the number of Lgr5+ CBC cells when compared to their wild-type littermates, and further, that these cells display higher degree of stemness and cancer-related and inflammatory gene expression signatures that, altogether, may contribute to enhanced intestinal tumorigenesis. The phenotypes of Ubc9 haploinsufficiency discovered here indicate an unanticipated tumor-suppressive role of sumoylation, one that may have important implications for optimal use of sumoylation inhibitors in the clinic.

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.

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. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5- cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors.

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.

Rho GTPases: functions and association with cancer.

Rho GTPases are small proteins that act as binary molecular switches in a wide range of signalling pathways upon stimulation of cell surface receptors. Three different classes of regulatory proteins control their activity. In the activated state small GTPases are able to bind a variety of effector proteins and initiate downstream signalling. Rho GTPases regulate important cellular processes ranging from cytoskeletal remodelling and gene expression to cell proliferation and membrane trafficking. Therefore it is not surprising that deregulated Rho signalling can contribute to disturbed cellular phenotypes in a wide range of diseases. The main focus of this review will be the diversity of functions of Rho GTPases and the effects of aberrant Rho GTPase signalling in various aspects of cancer.

Imaging hallmarks of cancer in living mice.

To comprehend the complexity of cancer, the biological characteristics acquired during the initiation and progression of tumours were classified as the 'hallmarks of cancer'. Intravital microscopy techniques have been developed to study individual cells that acquire these crucial traits, by visualizing tissues with cellular or subcellular resolution in living animals. In this Review, we highlight the latest intravital microscopy techniques that have been used in living animals (predominantly mice) to unravel fundamental and dynamic aspects of various hallmarks of cancer. In addition, we discuss the application of intravital microscopy techniques to cancer therapy, as well as limitations and future perspectives for these techniques.

Surgical implantation of an abdominal imaging window for intravital microscopy.

High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; however, long-term imaging studies on cellular processes in abdominal organs are more challenging. These processes include colonization of the liver by metastatic tumor cells and the development of an immune response in the spleen. We have recently developed an abdominal imaging window (AIW) that allows long-term imaging of the liver, the pancreas, the intestine, the kidney and the spleen. Here we describe the detailed protocol for the optimal surgical implantation of the AIW, which takes ∼1 h, and subsequent multiphoton imaging, which takes up to 1 month.

The Rac activator Tiam1 is required for polarized protrusional outgrowth of primary astrocytes by affecting the organization of the microtubule network.

Polarized cell migration is a crucial process in the development and repair of tissues, as well as in pathological conditions, including cancer. Recent studies have elucidated important roles for Rho GTPases in the establishment and maintenance of polarity prior to and during cell migration. Here, we show that Tiam1, a specific activator of the small GTPase Rac, is required for the polarized outgrowth of protrusions in primary astrocytes during the initial phase of cell polarization after scratch-wounding monolayers of cells. Tiam1 deficiency delays closure of wounds in confluent monolayers. Lack of Tiam1 impairs adoption of an asymmetrical cell shape as well as microtubule organization within protrusions. Positioning of the centrosome and Golgi apparatus, however, are independent of Tiam1-Rac signaling. We speculate that the function of Tiam1 in polarized outgrowth of astrocyte protrusions involves regulation of microtubule organization, possibly by stabilizing the microtubule cytoskeleton. Our results add Tiam1 as a player to the growing list of proteins involved in polarized outgrowth of protrusions and further elucidate the signaling pathways leading to cell polarization.

Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells.

Rac1 and Rac3 are highly homologous members of the Rho small GTPase family. Rac1 is ubiquitously expressed and regulates cell adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in brain and may therefore have a specific function in neuronal cells. We found that depletion of Rac1 by short interference RNA leads to decreased cell-matrix adhesions and cell rounding in neuronal N1E-115 cells. By contrast, depletion of Rac3 induces stronger cell adhesions and dramatically increases the outgrowth of neurite-like protrusions, suggesting opposite functions for Rac1 and Rac3 in neuronal cells. Consistent with this, overexpression of Rac1 induces cell spreading, whereas overexpression of Rac3 results in a contractile round morphology. Rac1 is mainly found at the plasma membrane, whereas Rac3 is predominantly localized in the perinuclear region. Residues 185-187, present in the variable polybasic rich region at the carboxyl terminus are responsible for the difference in phenotype induced by Rac1 and Rac3 as well as for their different intracellular localization. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. It rather seems that Rac3 exerts its function through negatively affecting integrin-mediated cell-matrix adhesions. Together, our data reveal that Rac3 opposes Rac1 in the regulation of cell adhesion and differentiation of neuronal cells.