Laila Ritsma

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.

Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen

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.

Intravital microscopy: new insights into metastasis of tumors

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.

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.

Intravital Microscopy Through an Abdominal Imaging Window Reveals a Pre-Micrometastasis Stage During Liver Metastasis

An abdominal imaging window allows in vivo visualization of dynamic cellular processes, including liver metastasis and islet cell transplantation. Peering Into Cancer Understanding what goes on inside the body, as it is happening, is an ongoing challenge in medical imaging. Conventional imaging methods are only “snapshots,” unable to truly capture biology in action or the progression of disease. In this study by Ritsma and colleagues, an abdominal imaging window (AIW) proves to be the answer, allowing the authors to visualize and quantify metastatic processes in real time, in vivo in mice. The AIW consisted of a titanium ring with a glass coverslip, which could be tightly secured to the abdominal wall of a mouse. This window stayed in place for an average of 5 weeks, which is long enough to visualize many biological phenomena, including single-cell activity in the small intestine, spleen, pancreas, and kidney, as demonstrated by Ritsma et al. Although able to image many organs and cells, the authors chose to focus on tumor cell metastasis—specifically, the metastasis of mouse colorectal cancer C26 cells to the liver. By tracking fluorescently labeled C26 cells over the course of 2 weeks, the authors were able to confirm that the majority of metastatic growth was clonal (that is, from a single founder cell) rather than synergistic. The authors also noticed that the cancer cells had different phenotypic properties at different time points: At day 3, the cells were motile and diffuse in the liver tissue, whereas, at day 5, the cells stopped moving and were densely packed. The authors called this phenotypic shift a “pre-micrometastatic” state, followed by the “micrometastatic state.” Blocking cell migration in the pre-micrometastatic stage with a small-molecule inhibitor reduced cell growth and formation of subsequent micrometastases. Ristma and coauthors have developed a powerful in vivo imaging tool to track biological events in real time. This will hopefully lend insight into many diseases that affect abdominal organs. Although their preliminary findings suggest a new target for pharmacological inhibition of cancer growth and migration, additional preclinical and clinical studies will be needed to follow up this pre-micrometastatic hypothesis and to further confirm its presence in humans. 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.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Next-generation biosensors enable in vivo monitoring of ERK activity and detection of RhoA activity in small cellular extensions. Seeing Signaling in Action Biosensors consist of two fluorophores that produce a light signal when in close proximity and a “sensing module,” which is a protein (or protein fragment) that detects a signaling event, such as the activated state of a guanosine triphosphatase (GTPase) or the activity of a kinase. Producing optimal biosensors to monitor specific signaling events is challenging and time-consuming. Fritz et al. constructed a library of FRET vectors that enabled the rapid generation of highly effective biosensors and created an improved biosensor for RhoA GTPase activity that was used to detect spatial regulation of RhoA activity in filopodia and neuronal growth cones and another that monitors activity of the mitogen-activated protein kinase ERK and was used to detect the activity of this enzyme in living zebrafish. 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.

Direct Spatial Control of Epac1 by Cyclic AMP

ABSTRACT Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.

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

Vessel co-option mediates resistance to anti-angiogenic therapy in liver metastases

The efficacy of angiogenesis inhibitors in cancer is limited by resistance mechanisms that are poorly understood. Notably, instead of through the induction of angiogenesis, tumor vascularization can occur through the nonangiogenic mechanism of vessel co-option. Here we show that vessel co-option is associated with a poor response to the anti-angiogenic agent bevacizumab in patients with colorectal cancer liver metastases. Moreover, we find that vessel co-option is also prevalent in human breast cancer liver metastases, a setting in which results with anti-angiogenic therapy have been disappointing. In preclinical mechanistic studies, we found that cancer cell motility mediated by the actin-related protein 2/3 complex (Arp2/3) is required for vessel co-option in liver metastases in vivo and that, in this setting, combined inhibition of angiogenesis and vessel co-option is more effective than the inhibition of angiogenesis alone. Vessel co-option is therefore a clinically relevant mechanism of resistance to anti-angiogenic therapy and combined inhibition of angiogenesis and vessel co-option might be a warranted therapeutic strategy.

Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

ABSTRACT Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.