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Background: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. Results: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopie

Background: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. Results: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness. Conclusions: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.

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 pl

The cancer stem cell (CSC) theory proposes that a tumor is maintained and propagated by a cancer cell with stem cell-like properties. CSCs have been implicated to be the main driver for tumor initiation, propagation, metastasis and recurrence. Although a lot of work has been performed on identifying and characterizing CSCs, no unifiable markers for, and characteristics of CSCs, have been established to date. More recently, a rising number of studies provide evidence that stemness characteristics can be acquired by more differentiated cancer cells, giving rise to de novo CSCs. Therefore, the CSC theory shifts towards a model in which the CSC characteristics can be acquired by stochastic mechanisms, genetic and epigenetic changes and extrinsic factors from the microenvironment, which could also be defined as the CSC niche. Most studies on CSC plasticity have been performed using in vitro studies, serial transplants or xenograft assays. These methods represent another environment than the unperturbed tumor and are static models, making it challenging to study in vivo CSC dynamics. Intravital imaging techniques in combination with genetic lineage tracing will enable researchers to study CSCs over time and in the unperturbed and will most likely prove to be essential in studying CSC dynamics in vivo. To summarize, this thesis reviews current challenges in CSC identification and reviews mechanisms which have been found to induce de novo CSC generation. Additionally, future experimental setups and the therapeutic consequences of de novo generation of CSCs will be discussed.

Taxanes, such as docetaxel, are microtubule-targeting chemotherapeutics that have been successfully used in the treatment of cancer. Based on data obtained from cell cultures, it is believed that taxanes induce tumor cell death by specifically perturbing mitotic progression. Here, we report on data that suggest that this generally accepted view may be too simplified. We describe a high-resolution intravital imaging method to simultaneously visualize mitotic progression and the onset of apoptosis. To directly compare in vitro and in vivo data, we have visualized the effect of docetaxel on mitotic progression in mouse and human colorectal tumor cell lines both in vitro and in isogenic tumors in mice. We show that docetaxel-induced apoptosis in vitro occurs via mitotic cell death, whereas the vast majority of tumor cells in their natural environment die independent of mitotic defects. This demonstrates that docetaxel exerts its anti-tumor effects in vivo through means other than mitotic perturbation. The differences between in vitro and in vivo mechanisms of action of chemotherapeutics may explain the limited response to many of the anti-mitotic agents that are currently validated in clinical trials. Our data illustrate the requirement and power of our intravital imaging technique to study and validate the mode of action of chemotherapeutic agents in vivo, which will be essential to understand and improve their clinical efficacy., Taxanes, such as docetaxel, are microtubule-targeting chemotherapeutics that have been successfully used in the treatment of cancer. Based on data obtained from cell cultures, it is believed that taxanes induce tumor cell death by specifically perturbing mitotic progression. Here, we report on data that suggest that this generally accepted view may be too simplified. We describe a high-resolution intravital imaging method to simultaneously visualize mitotic progression and the onset of apoptosis. To directly compare in vitro and in vivo data, we have visualized the effect of docetaxel on mitotic progression in mouse and human colorectal tumor cell lines both in vitro and in isogenic tumors in mice. We show that docetaxel-induced apoptosis in vitro occurs via mitotic cell death, whereas the vast majority of tumor cells in their natural environment die independent of mitotic defects. This demonstrates that docetaxel exerts its anti-tumor effects in vivo through means other than mitotic perturbation. The differences between in vitro and in vivo mechanisms of action of chemotherapeutics may explain the limited response to many of the anti-mitotic agents that are currently validated in clinical trials. Our data illustrate the requirement and power of our intravital imaging technique to study and validate the mode of action of chemotherapeutic agents in vivo, which will be essential to understand and improve their clinical efficacy.

Intravital microscopy has been used extensively to study dynamic processes in the context of a living animal; however, only a limited number of fluorescent probes and mouse models are available. By contrast, many dyes and antibodies exist for the immuno-labelling of fixed tissue. Here we report a method that combines the advantages of histochemistry and in vivo imaging by correlating cryosection labelling with corresponding intravital microscopy images (CLIM). Using CLIM, we find that the presence of CD3(+) T cells correlates with mammary tumour cell migration. When CD4(+) and CD8(+) T-cell subsets are depleted, reduced tumour cell migration is observed. From these data we conclude that CLIM is a powerful tool to correlate intravital microscopy data with cryosection labelling data., Intravital microscopy has been used extensively to study dynamic processes in the context of a living animal; however, only a limited number of fluorescent probes and mouse models are available. By contrast, many dyes and antibodies exist for the immuno-labelling of fixed tissue. Here we report a method that combines the advantages of histochemistry and in vivo imaging by correlating cryosection labelling with corresponding intravital microscopy images (CLIM). Using CLIM, we find that the presence of CD3(+) T cells correlates with mammary tumour cell migration. When CD4(+) and CD8(+) T-cell subsets are depleted, reduced tumour cell migration is observed. From these data we conclude that CLIM is a powerful tool to correlate intravital microscopy data with cryosection labelling data.

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors., Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

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 approximately 1 h, and subsequent multiphoton imaging, which takes up to 1 month., 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 approximately 1 h, and subsequent multiphoton imaging, which takes up to 1 month.

A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interactions., A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interactions.

Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling., Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling.

Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation., Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.

Recent work has demonstrated that following the clearance of infection a stable population of memory T cells remains present in peripheral organs and contributes to the control of secondary infections. However, little is known about how tissue-resident memory T cells behave in situ and how they encounter newly infected target cells. Here we demonstrate that antigen-specific CD8(+) T cells that remain in skin following herpes simplex virus infection show a steady-state crawling behavior in between keratinocytes. Spatially explicit simulations of the migration of these tissue-resident memory T cells indicate that the migratory dendritic behavior of these cells allows the detection of antigen-expressing target cells in physiologically relevant time frames of minutes to hours. Furthermore, we provide direct evidence for the identification of rare antigen-expressing epithelial cells by skin-patrolling memory T cells in vivo. These data demonstrate the existence of skin patrol by memory T cells and reveal the value of this patrol in the rapid detection of renewed infections at a previously infected site., Recent work has demonstrated that following the clearance of infection a stable population of memory T cells remains present in peripheral organs and contributes to the control of secondary infections. However, little is known about how tissue-resident memory T cells behave in situ and how they encounter newly infected target cells. Here we demonstrate that antigen-specific CD8(+) T cells that remain in skin following herpes simplex virus infection show a steady-state crawling behavior in between keratinocytes. Spatially explicit simulations of the migration of these tissue-resident memory T cells indicate that the migratory dendritic behavior of these cells allows the detection of antigen-expressing target cells in physiologically relevant time frames of minutes to hours. Furthermore, we provide direct evidence for the identification of rare antigen-expressing epithelial cells by skin-patrolling memory T cells in vivo. These data demonstrate the existence of skin patrol by memory T cells and reveal the value of this patrol in the rapid detection of renewed infections at a previously infected site.

Metastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fully understood. Although histological techniques have provided important information, they give only a static image and thus compromise interpretation of this dynamic process. New advances in intravital microscopy (IVM), such as two-photon microscopy, imaging chambers, and multicolor and fluorescent resonance energy transfer imaging, have recently been used to visualize the behavior of single metastasizing cells at subcellular resolution over several days, yielding new and unexpected insights into this process. For example, IVM studies showed that tumor cells can switch between multiple invasion strategies in response to various densities of extracellular matrix. Moreover, other IVM studies showed that tumor cell migration and blood entry take place not only at the invasive front, but also within the tumor mass at tumor-associated vessels that lack an intact basement membrane. In this Commentary, we will give an overview of the recent advances in high-resolution IVM techniques and discuss some of the latest insights in the metastasis field obtained with IVM., Metastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fully understood. Although histological techniques have provided important information, they give only a static image and thus compromise interpretation of this dynamic process. New advances in intravital microscopy (IVM), such as two-photon microscopy, imaging chambers, and multicolor and fluorescent resonance energy transfer imaging, have recently been used to visualize the behavior of single metastasizing cells at subcellular resolution over several days, yielding new and unexpected insights into this process. For example, IVM studies showed that tumor cells can switch between multiple invasion strategies in response to various densities of extracellular matrix. Moreover, other IVM studies showed that tumor cell migration and blood entry take place not only at the invasive front, but also within the tumor mass at tumor-associated vessels that lack an intact basement membrane. In this Commentary, we will give an overview of the recent advances in high-resolution IVM techniques and discuss some of the latest insights in the metastasis field obtained with IVM.

The death receptor CD95 promotes apoptosis through well-defined signalling pathways. In colorectal cancer cells, CD95 primarily stimulates migration and invasion through pathways that are incompletely understood. Here, we identify a new CD95-activated tyrosine kinase pathway that is essential for CD95-stimulated tumour cell invasion. We show that CD95 promotes Tyr 783 phosphorylation of phospholipase C-gamma1 through the platelet-derived growth factor receptor-beta, resulting in ligand-stimulated phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis. PIP(2) hydrolysis liberates the actin-severing protein cofilin from the plasma membrane to initiate cortical actin remodelling. Cofilin activation is required for CD95-stimulated formation of membrane protrusions and increased tumour cell invasion., The death receptor CD95 promotes apoptosis through well-defined signalling pathways. In colorectal cancer cells, CD95 primarily stimulates migration and invasion through pathways that are incompletely understood. Here, we identify a new CD95-activated tyrosine kinase pathway that is essential for CD95-stimulated tumour cell invasion. We show that CD95 promotes Tyr 783 phosphorylation of phospholipase C-gamma1 through the platelet-derived growth factor receptor-beta, resulting in ligand-stimulated phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis. PIP(2) hydrolysis liberates the actin-severing protein cofilin from the plasma membrane to initiate cortical actin remodelling. Cofilin activation is required for CD95-stimulated formation of membrane protrusions and increased tumour cell invasion.

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., 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.

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics., Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.

Metastasis is the most frequent cause of death in cancer patients and is therefore important to target therapeutically. To prevent metastasis it is useful to target invasion. It has been suggested that inhibition of MMPs (matrix metalloproteases) is an effective way to target invasion, since MMPs have many functions in invasion. However, so far MMPIs have failed in clinical trials. The main reason for this failure is unspecific inhibition of MMPs. To be able to create more specific MMP inhibitors, MMP biology in cell migration and invasion should be investigated in vivo.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells, angiogenesis, and immune cells response. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra2 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells., In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells, angiogenesis, and immune cells response. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra2 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

In many cell types, the formation of membrane protrusions and directional migration depend on the spatial and temporal regulation of the actin-binding protein cofilin. Cofilin, which is important for the regulation of actin-polymerization initiation, increases the number of actin free barbed ends through three mechanisms: its intrinsic actin-nucleation activity; binding and severing of existing actin filaments; and recycling actin monomers from old filaments to new ones through its actin-depolymerization activity. The increase in free barbed ends that is caused by cofilin initiates new actin polymerization, which can be amplified by the actin-nucleating ARP2/3 complex. Interestingly, different cell systems seem to have different mechanisms of activating cofilin. The initial activation of cofilin in mammary breast tumors is dependent on PLCgamma, whereas cofilin activation in neutrophils is additionally dependent on dephosphorylation, which is promoted through Rac2 signaling. Although the literature seems to be confusing and inconsistent, we propose that all of the data can be explained by a single activity-cycle model. In this Opinion, we give an overview of cofilin activation in both tumor cells and inflammatory cells, and demonstrate how the differences in cofilin activation that are observed in various cell types can be explained by different starting points in this single common activity cycle., In many cell types, the formation of membrane protrusions and directional migration depend on the spatial and temporal regulation of the actin-binding protein cofilin. Cofilin, which is important for the regulation of actin-polymerization initiation, increases the number of actin free barbed ends through three mechanisms: its intrinsic actin-nucleation activity; binding and severing of existing actin filaments; and recycling actin monomers from old filaments to new ones through its actin-depolymerization activity. The increase in free barbed ends that is caused by cofilin initiates new actin polymerization, which can be amplified by the actin-nucleating ARP2/3 complex. Interestingly, different cell systems seem to have different mechanisms of activating cofilin. The initial activation of cofilin in mammary breast tumors is dependent on PLCgamma, whereas cofilin activation in neutrophils is additionally dependent on dephosphorylation, which is promoted through Rac2 signaling. Although the literature seems to be confusing and inconsistent, we propose that all of the data can be explained by a single activity-cycle model. In this Opinion, we give an overview of cofilin activation in both tumor cells and inflammatory cells, and demonstrate how the differences in cofilin activation that are observed in various cell types can be explained by different starting points in this single common activity cycle.

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation., Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments., Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation., Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.