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Cancer cell–intrinsic properties caused by oncogenic mutations have been well characterized; however, how specific oncogenes and tumor suppressors impact the tumor microenvironment (TME) is not well understood. Here, we present a novel non–cell-autonomous function of the retinoblastoma (RB) tumor suppressor in controlling the TME. RB inactivation stimulated tumor growth and neoangiogenesis in a syngeneic and orthotropic murine soft-tissue sarcoma model, which was associated with recruitment of tumor-associated macrophages (TAM) and immunosuppressive cells such as Gr1+CD11b+ myeloid-derived suppressor cells (MDSC) or Foxp3+ regulatory T cells (Treg). Gene expression profiling and analysis of genetically engineered mouse models revealed that RB inactivation increased secretion of the chemoattractant CCL2. Furthermore, activation of the CCL2–CCR2 axis in the TME promoted tumor angiogenesis and recruitment of TAMs and MDSCs into the TME in several tumor types including sarcoma and breast cancer. Loss of RB increased fatty acid oxidation (FAO) by activating AMP-activated protein kinase that led to inactivation of acetyl-CoA carboxylase, which suppresses FAO. This promoted mitochondrial superoxide production and JNK activation, which enhanced CCL2 expression. These findings indicate that the CCL2–CCR2 axis could be an effective therapeutic target in RB-deficient tumors.Significance:These findings demonstrate the cell-nonautonomous role of the tumor suppressor retinoblastoma in the tumor microenvironment, linking retinoblastoma loss to immunosuppression.

RPKM value of CCL2, CCL5 and RB1 gene in each breast cancer cell line based on Cancer Cell Line Encyclopedia (CCLE) database.

This file contains additional data to further support the conclusions in the main manuscript. Supplementary figure S1 shows the data related to figure 1,2,3. Supplementary figure S2 shows the data related to figure 4,5,6.

Cancer stem cells (CSCs) are a class of cancer cells characterized by self-renewal, differentiation and tumorigenic potential. We previously established a model of CSCs by culturing mouse induced pluripotent stem cells (miPSCs) for four weeks in the presence of a conditioned medium (CM) of cancer cell lines, which functioned as the tumor microenvironment. Based on this methodology of developing CSCs from miPSCs, we assessed the risk of 110 non-mutagenic chemical compounds, most of which are known as inhibitors of cytoplasmic signaling pathways, as potential carcinogens. We treated miPSCs with each compound for one week in the presence of a CM of Lewis lung carcinoma (LLC) cells. However, one-week period was too short for the CM to convert miPSCs into CSCs. Consequently, PDO325901 (MEK inhibitor), CHIR99021 (GSK-3β inhibitor) and Dasatinib (Abl, Src and c-Kit inhibitor) were found to confer miPSCs with the CSC phenotype in one week. The tumor cells that survived exhibited stemness markers, spheroid formation and tumorigenesis in Balb/c nude mice. Hence, we concluded that the three signal inhibitors accelerated the conversion of miPSCs into CSCs. Similarly to our previous study, we found that the PI3K-Akt signaling pathway was upregulated in the CSCs. Herein, we focused on the expression of relative genes after the treatment with these three inhibitors. Our results demonstrated an increased expression of pik3ca, pik3cb, pik3r5 and pik3r1 genes indicating class IA PI3K as the responsible signaling pathway. Hence, AKT phosphorylation was found to be up-regulated in the obtained CSCs. Inhibition of Erk1/2, tyrosine kinase, and/or GSK-3β was implied to be involved in the enhancement of the PI3K-AKT signaling pathway in the undifferentiated cells, resulting in the sustained stemness, and subsequent conversion of miPSCs into CSCs in the tumor microenvironment.

Paclitaxel (PTX) is one of the front-line drugs approved for the treatment of ovarian cancer. However, the application of PTX is limited due to the significant hydrophobicity and poor pharmacokinetics. We previously reported target-directed liposomes carrying tumor-selective conjugated antibody and encapsulated glycosylated PTX (gPTX-L) which successfully overcome the PTX limitation. The tubulin stabilizing activity of gPTX was equivalent to that of PTX while the cytotoxic activity of gPTX was reduced. In human ovarian cancer cell lines, SK-OV-3 and OVK18, the concentration at which cell growth was inhibited by 50% (IC50) for gPTX range from 15&ndash, 20 nM, which was sensitive enough to address gPTX-L with tumor-selective antibody coupling for ovarian cancer therapy. The cell membrane receptor CD44 is associated with cancer progression and has been recognized as a cancer stem cell marker including ovarian cancer, becoming a suitable candidate to be targeted by gPTX-L therapy. In this study, gPTX-loading liposomes conjugated with anti-CD44 antibody (gPTX-IL) were assessed for the efficacy of targeting CD44-positive ovarian cancer cells. We successfully encapsulated gPTX into liposomes with the loading efficiency (LE) more than 80% in both of gPTX-L and gPTX-IL with a diameter of approximately 100 nm with efficacy of enhanced cytotoxicity in vitro and of convenient treatment in vivo. As the result, gPTX-IL efficiently suppressed tumor growth in vivo. Therefore gPTX-IL could be a promising formulation for effective ovarian cancer therapies.

Cancer cell–intrinsic properties caused by oncogenic mutations have been well characterized; however, how specific oncogenes and tumor suppressors impact the tumor microenvironment (TME) is not well understood. Here, we present a novel non–cell-autonomous function of the retinoblastoma (RB) tumor suppressor in controlling the TME. RB inactivation stimulated tumor growth and neoangiogenesis in a syngeneic and orthotropic murine soft-tissue sarcoma model, which was associated with recruitment of tumor-associated macrophages (TAM) and immunosuppressive cells such as Gr1+CD11b+ myeloid-derived suppressor cells (MDSC) or Foxp3+ regulatory T cells (Treg). Gene expression profiling and analysis of genetically engineered mouse models revealed that RB inactivation increased secretion of the chemoattractant CCL2. Furthermore, activation of the CCL2–CCR2 axis in the TME promoted tumor angiogenesis and recruitment of TAMs and MDSCs into the TME in several tumor types including sarcoma and breast cancer. Loss of RB increased fatty acid oxidation (FAO) by activating AMP-activated protein kinase that led to inactivation of acetyl-CoA carboxylase, which suppresses FAO. This promoted mitochondrial superoxide production and JNK activation, which enhanced CCL2 expression. These findings indicate that the CCL2–CCR2 axis could be an effective therapeutic target in RB-deficient tumors. Significance: These findings demonstrate the cell-nonautonomous role of the tumor suppressor retinoblastoma in the tumor microenvironment, linking retinoblastoma loss to immunosuppression.

Cancer stem cells (CSCs) represent the subpopulation of cancer cells with the ability to differentiate into other cell phenotypes and initiated tumorigenesis. Previously, we reported generating CSCs from mouse induced pluripotent stem cells (miPSCs). Here, we investigated the ability of the CSCs to differentiate into hematopoietic cells. First, the primary cells were isolated from malignant tumors that were formed by the CSCs. Non-adherent cells (NACs) that arose from adherent cells were collected and their viability, as well as the morphology and expression of hematopoietic cell markers, were analyzed. Moreover, NACs were injected into the tail vein of busulfan conditioned Balb/c nude mice. Finally, CSCs were induced to differentiate to macrophages while using IL3 and SCF. The round nucleated NACs were found to be viable, positive for hematopoietic lineage markers and CD34, and expressed hematopoietic markers, just like homing to the bone marrow. When NACs were injected into mice, Wright&ndash, Giemsa staining showed that the number of white blood cells got higher than those in the control mice after four weeks. CSCs also showed the ability to differentiate toward macrophages. CSCs were demonstrated to have the potential to provide progenies with hematopoietic markers, morphology, and homing ability to the bone marrow, which could give new insight into the tumor microenvironment according to the plasticity of CSCs.

The cancer stem cell (CSC) hypothesis suggests that tumors are sustained exclusively by a small population of the cells with stem cell properties. CSCs have been identified in most tumors and are responsible for the initiation, recurrence, and resistance of different cancers. In vitro CSC models will be of great help in revisiting the mechanism of cancer development, as well as the tumor microenvironment and the heterogeneity of cancer and metastasis. Our group recently described the generation of CSCs from induced pluripotent stem cells (iPSCs), which were reprogrammed from normal cells, and/or embryonic stem cells (ESCs). This procedure will improve the understanding of the essential niche involved in cancer initiation. The composition of this cancer-inducing niche, if identified, will let us know how normal cells convert to malignant in the body and how, in turn, cancer prevention could be achieved. Further, once developed, CSCs demonstrate the ability to differentiate into endothelial cells, cancer-associated fibroblasts, and other phenotypes establishing the CSC niche. These will be good materials for developing novel cancer treatments. In this protocol, we describe how to handle mouse iPSCs/ESCs and how to choose the critical time for starting the conversion into CSCs. This CSC generation protocol is essential for understanding the role of CSC in cancer initiation and progress.

Somatic reprogramming induced by defined transcription factors is a low efficiency process that is enhanced by p53 deficiency 1-5. To date, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1, 3, suggesting additional p53 targets may regulate this process. Here, we demonstrated that mir-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. mir-34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of mir-34a promoted iPSC generation without compromising self-renewal and differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three mir-34 miRNAs acted cooperatively in this process. Taken together, our findings identified mir-34 miRNAs as novel p53 targets that play an essential role in restraining somatic reprogramming.

電解質溶液の液膜厚みが10～100 μmの場合の腐食速度は酸素拡散律速となる．更に液膜厚みが10 μm以下となった場合には液膜への酸素溶解速度が律速となる．このような薄液膜下におけるガルバニック腐食の数値解析モデルの開発を行った．このモデルでは，酸素のSalting-out効果，及び導電率，酸素溶解度と酸素溶解速度の温度依存性を考慮した．Fe/Znガルバニック電極を用いた塩水噴霧試験（SST）とサイクル腐食試験（CCT）を行い，ガルバニック電流の測定を行った．ガルバニック電流は，乾燥湿潤過程において最大値を示した．実測されたガルバニック電流は，数値解析結果と良く一致した．CCTにおける液膜厚みを数値解析により推定した結果，ガルバニック電流が最大となる液膜厚みは3～10 μmであった．

The purpose of this study is to clarify the effect of dry and wet processes on the mechanisms of edge corrosion of hot dip 55 mass% Al–Zn alloy coated steel sheet. The corrosion tests employed were continuous salt-spray tests and cyclic corrosion tests using NaCl solution or artificial seawater. It was confirmed that the edge corrosion is suppressed during the salt-spray test using artificial seawater. Furthermore, the suppression was not prevented by dry and wet processes. In order to reveal the corrosion mechanisms, the galvanic current and the cathodic polarization curves at exposed steel were measured and the corrosion products were characterized. It is suggested that the artificial seawater forms anti-corrosive corrosion products and that suppress the cathodic reaction on the steel which is exposed on the shear cut edge. The corrosion products are not changed by dry and wet processes. The numerical analysis also revealed the influence of the dry and wet process on edge corrosion.

Numerical analysis models for galvanic corrosion has been developed in recent years, but most of the ordinary models only can calculate current density and potential distribution in an electrolyte solution. In order to make clear corrosion mechanism, more information such as pH, ion and corrosion product distributions are necessary. However, it is difficult to compute these phenomena by mathematical model because various kinds of ions and complex reactions exist in corrosion progress. Calculating ion movement with satisfying charge conservation is especially difficult.We have developed a new numerical analysis model for galvanic corrosion that can calculate ions movement and reactions. In this model, ion density distribution is corrected by solving Poisson's equation to satisfy charge conservation. Equilibrium reactions are computed after calculating ion transportation.Galvanic corrosion of a model Zn/steel couple in a NaCl solution was calculated with this model. The difference of ion and corrosion product distribution in 50 ppm and 50000 ppm NaCl solutions was discussed. Corrosion product distribution obtained by this numerical analysis model was well agreed with OH− distribution on a galvanic corrosion specimen measured by FT-IR method qualitatively.

This article proposes multiple self-organizing maps (SOMs) for control of a visuo-motor system that consists of a redundant manipulator and multiple cameras in an unstructured environment. The maps control the manipulator so that it reaches its end-effector at targets given in the camera images. The maps also make the manipulator take obstacle-free poses. Multiple cameras are introduced to avoid occlusions, and multiple SOMs are introduced to deal with multiple camera images. Some simulation results are shown.

Hot-dip 55%Al–Zn alloy coated steel sheet has superior corrosion resistance both on flat panel and near shear cut edge in various atmospheric environments. However, the corrosion often occurs near the shear cut edge in continuous wet conditions, for example, NaCl solution spray test (SST, JIS Z2371), and this has been explained by galvanic model. In this paper, it is clarified that the corrosion near shear cut edge is suppressed by artificial sea water and its second ingredient, MgCl2.To make clear the corrosion mechanisms on MgCl2 suppression of the corrosion near shear cut edge, the measurement of cathodic polarization curve on the Fe electrode after the corrosion test using a AZ/Fe/AZ galvanic electrode, the estimation of corrosion products by numerical analysis in consideration of the substances migration and the precipitation reactions, and the analysis of corrosion products by FT-IR spectroscopy were conducted. As a result, in the galvanic condition, it is clarified that Mg(OH)2 precipitates on the Fe exposed cut edge. Moreover, the corrosion near shear cut edge is suppressed by the effect of the cathodic reaction suppression of Mg(OH)2.

A problem of probabilistic vision-based multi-person tracking with a monocular camera in a cluttered office environment is addressed. In such a case, it is often difficult to obtain reliable measurements of the absolute position of a person on the floor plane due to the inherent uncertainty in depth. To deal with this problem, a novel measurement technique based on occlusion reasoning is proposed and combined with a standard particle filtering technique. This technique is based on the relative position of a person with respect to background objects. The effectiveness of the proposed technique is investigated in an experiment where two persons overlap on the image plane.

This paper presents a method to coordinate an uncalibrated visuo-motor system in a 3D space. In order to handle spaces occluded by obstacles, a 3-camera system and two related self-organizing maps (SOMs) are employed. The self-organizing maps are directly connected to the camera system and are trained to perform position control. Based on the visibility of targets given in the workspace, an appropriate map is adopted. The maps determine the joint angles of the manipulator which make the end effector reach the targets precisely, and make the manipulator take obstacle-free poses. The proposed learning method ensures that the manipulator moves smoothly and consistently in whole workspace even though we use two maps to control it. In our visuo-motor system, neither any priori knowledge about the manipulator nor the camera parameters is required. In addition, the system is robust to change in its geometry. Simulation results are presented to demonstrate the effectiveness of the proposed method and the robustness of the system.

A convenient - and quick to realize - approach for the synthesis of tetrabutylammonium hexachlorometalates (Bu4N)2[MCl6] is suggested. Since the preparation of (Bu4N)2[MCl6] (M=Re, Os, Ir) by simple one-pot method is hitherto unreported, we successfully synthesized it by simply mixing the tetrabutylammonium hydroxide with acidic solution of potassium salt of hexachlorometalates.

It is fundamental problem for mobile robots to detect 2D free regions where they can move by local sensing. In many indoor environments, there may be lots of objects, such as chairs, desks, etc., and much portion of the free region can not be directly detected. To resolve this problem, we have developed a method for detecting free regions in which a robot visually tracks walking human, and regards 2D regions swept by the human as free regions. Based on this idea, we propose a method for building a map of obstacles and free regions, and navigation with an distance transform based path planning algorithm. The free regions were obtained reliably by combining stereo vision and visual tracking techniques.

One of the fundamental problems of the mobile robots is self-localization, i.e. to estimate the self-position by comparing sensor data and a map. In non-stationary enviroments, a robot should avoid to use changed objects as landmarks in the localization. In this paper, we propose a self-localization method that is robust against changes in environments. The method identifies changes from noisy and ambiguous sensor data. Since an object with a random shape may be added at a random position, it genetates and utilizes multiple hypotheses about the changes. A number of simulation experiments have been performed in various environments, to demonstrate the effectiveness of the method.

A range finder realizable with an easy circuit composition is proposed. The range finder is based on the slit-ray projection method. In the system, positions of slit-ray images on an image plane are detected by using pattern masks electronically provided to the image plane. Due to using the electronic pattern masks, the range finder realizes high cost performance, highspeed measurement and small size. In order to estimate measurement speed, a prototype circuit has been developed. The experimental results obtained by the circuit have indicated that the range finder will be able to take a range image of which resolution is 64 × 64 and more in 1/30[s] or less in the future. A prototype range finder which has a 32 × 32 photo diode array and a laser slit marker has been also developed, and range images have been actually taken using it. In this paper, with emphasis on indicating the availability of the proposed method, the range finder system and experimental results by the prototype circuit and the prototype range finder will be shown.

Here, by combining lipidomics with transcriptome analysis, we demonstrate that Rb depletion in mouse embryonic fibroblastss induces significant alterations in their lipid composition. We discovered that Rb depletion induced increase in lysophosphatidylserine, diacylglycerol (DAG), fatty acid (FA), acylcarnitine, phosphatidylcholine (PC), arachidonoyl ethanolamine, and decrease in phosphatidylglycerol, monoacylglycerol, without change in total lipid per protein levels. Analysis of the acyl chain composition of DAG, PC and phosphatidylserine revealed increase of saturated and mono-unsaturated acyl chains with specific carbon chain length. Consistently, we observed that Rb depletion increased the levels of fatty acids with the corresponding carbon chain length and number of carbon–carbon double bondssuch as myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0) and all forms of FA 18:1. Microarray analysis revealed that Rb depletion induced significant upregulation of enzymes involved in elongation and desaturation of fatty acids. Among these, we found that elongation of long chain fatty acid family member 6 (Elovl6) and stearoyl-CoA desaturase 1 (Scd1) are the most robustly controlled by Rb possibly through E2F and sterol regulatory element-binding protein transcription factors. Depletion of Elovl6 or Scd1 significantly suppressed colony formation, sphere formation and xenograft tumor growth of Rb-deficient tumor cells. Suppression of self-renewal by the SCD1 inhibitor was rescued upon supplementation of the mono-unsaturated fatty acids generated by this enzyme. This study suggests a novel role for Rb in suppressing the malignant progression of tumors by controlling the lipid composition.

As bona fide p53 transcriptional targets, miR-34 microRNAs (miRNAs) exhibit frequent alterations in many human tumor types and elicit multiple p53 downstream effects upon overexpression. Unexpectedly, miR-34 deletion alone fails to impair multiple p53-mediated tumor suppressor effects in mice, possibly due to the considerable redundancy in the p53 pathway. Here, we demonstrate that miR-34a represses HDM4, a potent negative regulator of p53, creating a positive feedback loop acting on p53. In a Kras-induced mouse lung cancer model, miR-34a deficiency alone does not exhibit a strong oncogenic effect. However, miR-34a deficiency strongly promotes tumorigenesis when p53 is haploinsufficient, suggesting that the defective p53–miR-34 feedback loop can enhance oncogenesis in a specific context. The importance of the p53/miR-34/HDM4 feedback loop is further confirmed by an inverse correlation between miR-34 and full-length HDM4 in human lung adenocarcinomas. In addition, human lung adenocarcinomas generate an elevated level of a short HDM4 isoform through alternative polyadenylation. This short HDM4 isoform lacks miR-34-binding sites in the 3′ untranslated region (UTR), thereby evading miR-34 regulation to disable the p53-miR-34 positive feedback. Taken together, our results elucidated the intricate cross-talk between p53 and miR-34 miRNAs and revealed an important tumor suppressor effect generated by this positive feedback loop.

Summary LATS2 (Large tumor suppressor 2), a member of the conserved AGC Ser/Thr (S/T) kinase family, is a human tumor suppressor gene. Here, we show that in response to ultraviolet radiation, Lats2 is phosphorylated by Chk1 at Ser835 (S835), which is located in the kinase domain of Lats2. This phosphorylation enhances Lats2 kinase activity. Subsequently, Lats2 phosphorylates p21 at S146. p21 (CDKN1A) is a cyclin-dependent kinase (CDK) inhibitor, which not only regulates the cell cycle by inhibition of CDK, but also inhibits apoptosis by binding to procaspase-3 in the cytoplasm. Phosphorylation by Lats2 induces degradation of p21 and promotes apoptosis. Accordingly, Lats2 overexpression induces p21 degradation, activation of caspase-3 and caspase-9, and apoptosis. These findings describe a novel Lats2-dependent mechanism for induction of cell death in response to severe DNA damage.

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.

Proper response to DNA damage is essential for maintaining the integrity of the genome. Here we show that in response to ultraviolet (UV) radiation, the Lats2 tumor suppressor protein is phosphorylated predominantly by Chk1 and weakly by Chk2 at S408 in vivo, and that this process occurs at all stages of the cell cycle and leads to phosphorylation of 14-3-3γ on S59 by Lats2. Interaction of Lats2 and 14-3-3γ in vivo was confirmed by immunoprecipitation and western blot analysis. Phosphorylated 14-3-3γ translocates to the P-body, where mRNA degradation, translational repression and mRNA surveillance take place. Depletion of Lats2 or 14-3-3γ by siRNA inhibits P-body formation in response to UV, newly implicating Lats2 and 14-3-3 as regulators of P-body formation. By contrast, siRNA-mediated depletion of Lats1, a mammalian paralog of Lats2, showed no such effect. On the basis of these findings, we propose that the Chk1/2–Lats2–14-3-3 axis identified here plays an important role in connecting DNA damage signals to P-body assembly.

We developed a visuo-motor system with multiple self-organizing maps. The system consists of a redundant manipulator, multiple cameras and multiple SOMs corresponding to the cameras. By using the cameras, the system can control the manipulator in an environment with obstacles. To realize the system, we also developed a learning method of the SOMs. The learning method can keep consistency of outputs among the SOMs. We then combined the SOMs and a path planning system to achieve collision avoidance. Since the SOMs outputs collision free poses, the path planning system became very simple. Simulation results showed that the SOMs control the manipulator with obstacle free poses and that the collision avoidance was realized.