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Abstract Pulmonary fibrosis (PF), a condition characterized by inflammation and collagen deposition in the alveolar interstitium, causes dyspnea and fatal outcomes. Although the bleomycin-induced PF mouse model has improved our understanding of exogenous factor-induced fibrosis, the mechanism governing endogenous factor-induced fibrosis remains unknown. Here, we find that Ifngr1 -/- Rag2 -/- mice, which lack the critical suppression factor for group 2 innate lymphoid cells (ILC2), develop PF spontaneously. The onset phase of fibrosis includes ILC2 subpopulations with a high Il1rl1 (IL-33 receptor) expression, and fibrosis does not develop in ILC-deficient or IL-33-deficient mice. Although ILC2s are normally localized near bronchioles and blood vessels, ILC2s are increased in fibrotic areas along with IL-33 positive fibroblasts during fibrosis. Co-culture analysis shows that activated-ILC2s directly induce collagen production from fibroblasts. Furthermore, increased IL1RL1 and decreased IFNGR1 expressions are confirmed in ILC2s from individuals with idiopathic PF, highlighting the applicability of Ifngr1 -/- Rag2 -/- mice as a mouse model for fibrosis research.

Abstract Background The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs. Methods sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined. Results The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced. Conclusion In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Abstract Cholesteatoma, which potentially results from tympanic membrane retraction, is characterized by intractable local bone erosion and subsequent hearing loss and brain abscess formation. However, the pathophysiological mechanisms underlying bone destruction remain elusive. Here, we performed a single-cell RNA sequencing analysis on human cholesteatoma samples and identify a pathogenic fibroblast subset characterized by abundant expression of inhibin βA. We demonstrate that activin A, a homodimer of inhibin βA, promotes osteoclast differentiation. Furthermore, the deletion of inhibin βA /activin A in these fibroblasts results in decreased osteoclast differentiation in a murine model of cholesteatoma. Moreover, follistatin, an antagonist of activin A, reduces osteoclastogenesis and resultant bone erosion in cholesteatoma. Collectively, these findings indicate that unique activin A-producing fibroblasts present in human cholesteatoma tissues are accountable for bone destruction via the induction of local osteoclastogenesis, suggesting a potential therapeutic target.

Summary: The in situ behavior of living cells can be visualized by two-photon microscopy. Here, we present a protocol for the live imaging of transferred mouse bone marrow cells by two-photon microscopy. We describe steps for staining and injecting target cells into mice, fixing the skull bone to a head holder and stage, and 4D imaging bone marrow using multi-photon microscopy. We then detail procedures for creating images and analyzing cells.For complete details on the use and execution of this protocol, please refer to Sudo et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Rheumatoid arthritis (RA) is characterized by chronic inflammation and resultant cartilage/bone destruction because of aberrantly activated osteoclasts. Recently, novel treatments with several Janus kinase (JAK) inhibitors have been shown to successfully ameliorate arthritis-related inflammation and bone erosion, although their mechanisms of action for limiting bone destruction remain unclear. Here, we examined the effects of a JAK inhibitor on mature osteoclasts and their precursors by intravital multiphoton imaging. Methods Inflammatory bone destruction was induced by local injection of lipopolysaccharides into transgenic mice carrying reporters for mature osteoclasts or their precursors. Mice were treated with the JAK inhibitor, ABT-317, which selectively inhibits the activation of JAK1, and then subjected to intravital imaging with multiphoton microscopy. We also used RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanism underlying the effects of the JAK inhibitor on osteoclasts. Results The JAK inhibitor, ABT-317, suppressed bone resorption by blocking the function of mature osteoclasts and by targeting the migratory behaviors of osteoclast precursors to the bone surface. Further exhaustive RNA-Seq analysis demonstrated that Ccr1 expression on osteoclast precursors was suppressed in the JAK inhibitor-treated mice; the CCR1 antagonist, J-113863, altered the migratory behaviors of osteoclast precursors, which led to the inhibition of bone destruction under inflammatory conditions. Conclusions This is the first study to determine the pharmacological actions by which a JAK inhibitor blocks bone destruction under inflammatory conditions; this inhibition is beneficial because of its dual effects on both mature osteoclasts and immature osteoclast precursors.

Alveolar macrophages represent a cell type that is physiologic to the lung immune landscape, however, it is not known whether they play an active role to maintain the tumour immune microenvironment. Here authors show by single cell RNA sequencing and functional experiments, that intra-tumour alveolar macrophages are phenotypically and transcriptionally different from the healthy ones, and likely play an aetio-pathologic role in tumorigenesis.

Abstract Osteoclasts are myeloid lineage cells with a unique bone-destroying ability that maintains bone homeostasis together with bone formation by osteoblasts. An advanced intravital imaging system using a two-photon microscopy has enabled the observation and evaluation of osteoclast dynamics and behaviors in the bone marrow of living mice. Using this system, it has become clear that pathological osteoclasts under inflamed conditions differ from physiological osteoclasts under a steady-state. Recently, we identified novel osteoclast precursors in arthritis, called arthritis-associated osteoclastogenic macrophages (AtoMs), which differentiate into pathological osteoclasts and induce inflammatory bone destruction. In this review, we introduce the in vivo imaging of physiological and pathological osteoclasts and their differentiation mechanism.

Abstract Oxygen is a key regulator of both development and homeostasis. To study the role of oxygen, a variety of in vitro and ex vivo cell and tissue models have been used in biomedical research. However, because of ambiguity surrounding the level of oxygen that cells experience in vivo, the cellular pathway related to oxygenation state and hypoxia have been inadequately studied in many of these models. Here, we devised a method to determine the oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy with the cell-penetrating phosphorescent probe, BTPDM1. Phosphorescence lifetime imaging revealed the physiological level of oxygen tension in monocytes to be 5.3% in live mice exposed to normal air. When the mice inhaled hypoxic air, the level of oxygen tension in bone marrow monocytes decreased to 2.4%. By performing in vitro cell culture experiment within the physiological range of oxygen tension, hypoxia changed the molecular phenotype of monocytes, leading to enhanced the expression of CD169 and CD206, which are markers of a unique subset of macrophages in bone marrow, osteal macrophages. This current study enables the determination of the physiological range of oxygen tension in bone marrow with spatial resolution at a cellular level and application of this information on oxygen tension in vivo to in vitro assays. Quantifying oxygen tension in tissues can provide invaluable information on metabolism under physiological and pathophyisological conditions. This method will open new avenues for research on oxygen biology.

Abstract STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP concentration-dependent manner. Although STA551 is assumed to show higher target binding in tumor tissues than in normal tissues, quantitative detection of the target binding of the switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered fluorescently labeled antibodies. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy were conducted. Higher CD137 expression in tumor than in spleen tissues was detected by flow cytometry analysis, and T cells and NK cells were the major CD137-expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibodies showed binding in tumors. However, in the spleen, the fluorescence of the switch antibody was much weaker than that of the conventional anti-CD137 antibody and comparable with that of the isotype control. In conclusion, we were able to assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggest that the tumor-selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.

Bone remodeling involves a switch between bone formation and resorption, but the mechanisms is unclear. Here, the authors show that intercellular communication via extracellular vesicles secreted by mature osteoblasts is a key factor for the switching, via a microRNA-mediated mechanism.

Rheumatoid arthritis is a chronic form of arthritis that causes bone destruction in joints such as the knees and fingers. Over the past two decades, the clinical outcomes of rheumatoid arthritis have improved substantially with the development of biological agents and Janus kinase inhibitors. Osteoclasts are myeloid lineage cells with a unique bone-destroying ability that can lead to joint destruction. On the other hand, osteoclasts play an important role in skeletal homeostasis by supporting bone remodeling together with osteoblasts in the bone marrow under steady-state conditions. However, the same osteoclasts are considered to participate in physiological bone remodeling and joint destruction. We found that pathological osteoclasts have different differentiation pathways and regulatory transcription factors compared to physiological osteoclasts. We also identified arthritis-associated osteoclastogenic macrophages (AtoMs), which are common progenitors of pathological osteoclasts in mice and humans that develop specifically in inflamed synovial tissue. This review presents details of the newly identified AtoMs and the original intravital imaging systems that can visualize synovial tissue and pathological osteoclasts at the pannus–bone interface.

The mechanism by which parathyroid hormone mediates the switch from bone resorption to bone formation is unclear. Here, the authors show that SLPI regulates the communication between osteoblasts and osteoclasts to promote the anabolic effect of parathyroid hormone.

Abstract Mesenchymal stromal cells (MSCs) are used for ameliorating liver fibrosis and aiding liver regeneration after cirrhosis; Here, we analyzed the therapeutic potential of small extracellular vesicles (sEVs) derived from interferon-γ (IFN-γ) pre-conditioned MSCs (γ-sEVs). γ-sEVs effectively induced anti-inflammatory macrophages with high motility and phagocytic abilities in vitro, while not preventing hepatic stellate cell (HSC; the major source of collagen fiber) activation in vitro. The proteome analysis of MSC-derived sEVs revealed anti-inflammatory macrophage inducible proteins (e.g., annexin-A1, lactotransferrin, and aminopeptidase N) upon IFN-γ stimulation. Furthermore, by enabling CX3CR1+ macrophage accumulation in the damaged area, γ-sEVs ameliorated inflammation and fibrosis in the cirrhosis mouse model more effectively than sEVs. Single cell RNA-Seq analysis revealed diverse effects, such as induction of anti-inflammatory macrophages and regulatory T cells, in the cirrhotic liver after γ-sEV administration. Overall, IFN-γ pre-conditioning altered sEVs resulted in efficient tissue repair indicating a new therapeutic strategy.

Abstract Bone homeostasis is dynamically regulated by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. Visualizing and evaluating the dynamics of bone cells in vivo remain difficult using conventional technologies, including histomorphometry and imaging analysis. Over the past two decades, multiphoton microscopy, which can penetrate thick specimens, has been utilized in the field of biological imaging. Using this innovative technique, the in vivo dynamic motion of bone metabolism-related cells and their interactions has been revealed. In this review, we summarize previous approaches used for bone imaging and provide an overview of current bone tissue imaging methods using multiphoton excitation microscopy.

Abstract There have been many attempts to visualize the inflamed joints using multiphoton microscopy. However, due to the hypervascular and multilayered structure of the inflamed synovium, intravital imaging of the deep synovial tissue has been difficult. Here, we established original intravital imaging systems to visualize synovial tissue and pathological osteoclasts at the pannus–bone interface using multiphoton microscopy. Combined with fluorescence-labeling of CTLA-4 Ig, a biological agent used for the treatment of rheumatoid arthritis, we identified that CTLA-4 Ig was distributed predominantly within the inflamed synovium and bound to CX3CR1+ macrophages and CD140a+ fibroblasts 6 h after injection, but not to mature osteoclasts. Intravital imaging of blood and lymphatic vessels in the inflamed synovium further showed that extravasated CTLA-4 Ig was immediately drained through lymphatic vessels under acute arthritic conditions, but the drainage activity was retarded under chronic conditions. These results indicate that this intravital synovial imaging system can serve as a platform for exploring the dynamics of immune cells, osteoclasts, and biological agents within the synovial microenvironment in vivo.

Abstract Background Dormant chemotherapy-resistant leukemia cells can survive for an extended period before relapse. Nevertheless, the mechanisms underlying the development of chemoresistance in vivo remain unclear. Methods Using intravital bone imaging, we characterized the behavior of murine acute myeloid leukemia (AML) cells (C1498) in the bone marrow before and after chemotherapy with cytarabine. Results Proliferative C1498 cells exhibited high motility in the bone marrow. Cytarabine treatment impaired the motility of residual C1498 cells. However, C1498 cells regained their migration potential after relapse. RNA sequencing revealed that cytarabine treatment promoted MRTF-SRF pathway activation. MRTF inhibition using CCG-203971 augmented the anti-tumor effects of chemotherapy in our AML mouse model, as well as suppressed the migration of chemoresistant C1498 cells. Conclusions These results provide novel insight into the role of cell migration arrest on the development of chemoresistance in AML, as well as provide a strong rationale for the modulation of cellular motility as a therapeutic target for refractory AML.

Thrombomodulin (TM) is an integral membrane protein expressed on the surface of vascular endothelial cells that suppresses blood coagulation. Recent studies have shown that TM exhibits anti-inflammatory effects by inhibiting leukocyte recruitment. However, the actual modes of action of TM in vivo remain unclear. Here, we describe the pharmacological effects of recombinant human soluble TM (TM alfa) on leukocyte dynamics in living mice using intravital imaging techniques. Under control conditions, neutrophils exhibited three distinct types of adhesion behavior in vessels: 1) “non-adhesion”, in which cells flowed without vessel adhesion; 2) “rolling adhesion”, in which cells transiently interacted with the endothelium; and 3) “tight binding”, in which cells bound strongly to the endothelial cells. Compared to control conditions, local lipopolysaccharide stimulation resulted in an increased frequency of rolling adhesion that was not homogeneously distributed on vessel walls but occurred at specific endothelial sites. Under inflammatory conditions, TM alfa, particularly the D1 domain which is a lectin-like region of TM, significantly decreased the frequency of rolling adhesion, but did not influence the number of tight bindings. This was the first study to demonstrate that TM alfa exerts anti-inflammatory effects by inhibiting rolling adhesion of neutrophils to vascular endothelial cells in living mice. Keywords: Thrombomodulin, Rolling adhesion, Leukocyte, Endothelial cell, Intravital imaging

Background: Mesenchymal stem cells (MSCs) can be easily expanded. They can be acquired from medical waste such as adipose and umbilical cord tissues, are influenced by culturing conditions, and exert anti-inflammatory, antioxidant, anti-fibrotic, and angiogenic effects. We analyzed the multi-directional effects of MSCs cultured under hypoxic conditions and their underlying mechanisms in the treatment of liver cirrhosis in a mouse model. Methods: Human bone marrow-derived MSCs cultured under hypoxic (5% O2; hypoMSCs) and normoxic (21% O2; norMSCs) conditions were compared by cap analysis of gene expression (CAGE) with or without serum from liver cirrhosis patients. The therapeutic effects of MSCs, including serum liver enzyme induction, fibrosis regression, and hepatic oxidative stress, were evaluated by injecting 1 × 106, 2 × 105, or 4 × 104 MSCs/mouse into the tail veins of mice with carbon tetrachloride (CCl4)-induced liver cirrhosis. Intravital imaging was performed with a two-photon excitation microscope to confirm the various MSC migration paths to the liver. Results: CAGE analysis revealed that the RNA expression levels of prostaglandin E synthase (Ptges) and miR210 were significantly higher in hypoMSCs than in norMSCs. In vivo analysis revealed that both hypoMSCs and norMSCs reduced serum alanine aminotransferase, oxidative stress, and fibrosis compared to that in control mice in a dose-dependent manner. However, hypoMSCs had stronger therapeutic effects than norMSCs. We confirmed this observation by an in vitro study in which hypoMSCs changed macrophage polarity to an anti-inflammatory phenotype via prostaglandin E2 (PGE2) stimulation. In addition, miR210 reduced the rate of hepatocyte apoptosis. Intravital imaging after MSC administration showed that both cell types were primarily trapped in the lungs. Relatively a few hypoMSCs and norMSCs migrated to the liver. There were no significant differences in their distributions. Conclusion: The therapeutic effect of hypoMSCs was mediated by PGE2 and miR210 production and was greater than that of norMSCs. Therefore, MSCs can be manipulated to improve their therapeutic efficacy in the treatment of liver cirrhosis and could potentially serve in effective cell therapy. MSCs produce several factors with multidirectional effects and function as “conducting cells” in liver cirrhosis. Keywords: Mesenchymal stem cells, Hypoxic condition, Liver cirrhosis, PGE2, miR210

Communication between osteoblasts and osteoclasts is essential for bone homeostasis, but the mode of interaction is unclear. The authors use intravital two-photon microscopy in mice to show that these cells directly interact, regulating activity of osteoclasts, and that the interaction is modulated by parathyroid hormone administration.

Immune cells, including dendritic cells, macrophages, and T and B cells, express the vitamin D receptor and 1α-hydroxylase. In vitro studies have shown that 1,25-dihydroxyvitamin D, the active form of vitamin D, has an anti-inflammatory effect. Recent epidemiological evidence has indicated a significant association between vitamin D deficiency and an increased incidence, or aggravation, of infectious diseases and inflammatory autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. However, the impact of vitamin D on treatment and prevention, particularly in infectious diseases such as the 2019 coronavirus disease (COVID-19), remains controversial. Here, we review recent evidence associated with the relationship between vitamin D and inflammatory diseases and describe the underlying immunomodulatory effect of vitamin D.

Abstract Multiphoton excitation microscopy (MPM) is regarded as an effective tool that enables the visualization of deep regions within living tissues and organs, with little damage. Here, we report novel non-labeling MPM (NL-MPM) imaging of fresh human colorectal mucosa, which is useful for discriminating cancer lesions from normal tissues quantitatively without any need for resection, fixation, or staining. Using NL-MPM, we visualized three components in human colorectal mucosa, epithelial cells, immune cells, and basement membranes, based on their characteristic patterns of fluorescence. These patterns are characterized by the different auto-fluorescence properties of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and flavin adenine dinucleotide and from second harmonic generation (SHG). NL-MPM images were at least as informative to pathologists as were ‘conventional’ images of fixed tissue sections stained with hematoxylin and eosin. Additionally, two quantitative parameters extracted from NL-MPM images – the nucleus diameter (index N) and the intensity of SHG in the basement membrane (index S) – rendered it possible to diagnose cancer regions effectively. In conclusion, NL-MPM is a novel, promising method for real-time clinical diagnosis of colorectal cancers, and is associated with minimal invasiveness.

Obesity promotes infiltration of inflammatory cells into various tissues, leading to parenchymal and stromal cell interaction and development of cellular and organ dysfunction. Liver sinusoidal endothelial cells (LSECs) are the first cells that contact portal blood cells and substances in the liver, but their functions in the development of obesity-associated glucose metabolism remain unclear. Here, we find that LSECs are involved in obesity-associated accumulation of myeloid cells via VLA-4-dependent cell-cell adhesion. VLA-4 blockade in mice fed a high-fat diet attenuated myeloid cell accumulation in the liver to improve hepatic inflammation and systemic glucose intolerance. Ex vivo studies further show that cell-cell contact between intrahepatic leukocytes and parenchymal hepatocytes induces gluconeogenesis via a Notch-dependent pathway. These findings suggest that cell-cell interaction between parenchymal and stromal cells regulates hepatic glucose metabolism and offers potential strategies for treatment or prevention of obesity-associated glucose intolerance.

Bone is a highly dynamic organ that is continuously being remodeled by the reciprocal interactions between bone and immune cells. We have originally established an advanced imaging system for visualizing the in vivo behavior of osteoclasts and their precursors in the bone marrow cavity using two-photon microscopy. Using this system, we found that the blood-enriched lipid mediator, sphingosine-1-phosphate, controlled the migratory behavior of osteoclast precursors. We also developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo, and identified two distinct functional states of differentiated osteoclasts, “bone-resorptive” and “non-resorptive.” Here, we summarize our studies on the dynamics and functions of bone and immune cells within the bone marrow. We further discuss how our intravital imaging techniques can be applied to evaluate the mechanisms of action of biological agents in inflammatory bone destruction. Our intravital imaging techniques would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would also be useful for evaluating novel therapies in animal models of bone-destroying diseases.

Polarized epithelial morphogenesis is an essential process in animal development. While this process is mostly attributed to directional cell intercalation, it can also be induced by other mechanisms. Using live-imaging analysis and a three-dimensional vertex model, we identified ‘cell sliding,’ a novel mechanism driving epithelial morphogenesis, in which cells directionally change their position relative to their subjacent (posterior) neighbors by sliding in one direction. In Drosophila embryonic hindgut, an initial left-right (LR) asymmetry of the cell shape (cell chirality in three dimensions), which occurs intrinsically before tissue deformation, is converted through LR asymmetric cell sliding into a directional axial twisting of the epithelial tube. In a Drosophila inversion mutant showing inverted cell chirality and hindgut rotation, cell sliding occurs in the opposite direction to that in wild-type. Unlike directional cell intercalation, cell sliding does not require junctional remodeling. Cell sliding may also be involved in other cases of LR-polarized epithelial morphogenesis.

Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

Intravital multiphoton microscopy has opened a new era in the field of biological imaging. Focal excitation of fluorophores by simultaneous attack of multiple (normally “two”) photons generates images with high spatial resolution, and use of near-infrared lasers for multiphoton excitation allows penetration of thicker specimens, enabling biologists to visualize living cellular dynamics deep inside tissues and organs without thin sectioning. Moreover, the minimized photo-bleaching and toxicity associated with multiphoton techniques is beneficial for imaging of live specimens for extended observation periods. Here we focus on recent findings using intravital multiphoton imaging of dynamic biological systems such as the immune system and bone homeostasis. The immune system comprises highly dynamic networks, in which many cell types actively travel throughout the body and interact with each other in specific areas. Therefore, real-time intravital imaging represents a powerful tool for understanding the mechanisms underlying this dynamic system. Keywords:: intravital imaging, multiphoton microscopy, cellular dynamics, bone, inflammation

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.