Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

Prognostic value of CAF marker expression in the intratumoral and marginal areas of soft tissue sarcoma.

INTRODUCTION: The tumor microenvironment of sarcomas has not been studied in detail; in particular, little is known about cancer-associated fibroblasts (CAFs). Sarcoma cells are difficult to distinguish from CAFs, either histomorphologically or immunohistochemically. METHODS: We scored expression of individual CAF markers (fibroblast activating protein (FAP), CD10, and podoplanin) in the intratumoral and marginal areas of 133 sarcomas. We also examined the association between these markers, as well as the number of CD163-positive macrophages (i.e., tumor-associated macrophages), and clinical outcome. RESULTS: In all cases, the log-rank test revealed that those with high marker scores and macrophage counts (except for marginal CD10+ CAFs) showed significantly worse disease-free survival (DFS). Grade 2/3 cases with high CAF scores (excluding the marginal FAP and CD10 scores) showed significantly worse DFS, whereas those with high intratumoral FAP/CD10 and marginal podoplanin scores showed significantly worse metastasis-free survival (MFS), and those with a high intratumoral CD10 score showed significantly worse local recurrence-free survival (LFS). Multivariate analysis identified intratumoral CD10/podoplanin scores and marginal FAP/podoplanin scores as independent prognostic factors for DFS, intratumoral FAP/CD10 and marginal FAP/podoplanin/CD163-positive macrophage scores as independent prognostic factors for MFS, and the intratumoral podoplanin score as an independent prognostic factor for LFS. There was a weak-to-moderate correlation between each score and CD163-positive macrophage counts. CONCLUSION: Patients with high CAF marker expression in the intratumoral and marginal areas have a poorer outcome.

Characteristic tetraspanin expression patterns mark various tissues during early Xenopus development.

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.

Crosstalk of tumor stromal cells orchestrates invasion and spreading of gastric cancer.

Tumors contain various stromal cells that support cancer progression. Some types of cancer, such as scirrhous gastric cancer, are characterized by large areas of fibrosis accompanied by cancer-associated fibroblasts (CAFs). Asporin (ASPN) is a small leucine-rich proteoglycan highly expressed in CAFs of various tumors. ASPN accelerates CAF migration and invasion, resulting in CAF-led cancer cell invasion. In addition, ASPN further upregulated the expression of genes specific to a characteristic subgroup of fibroblasts in tumors. These cells were preferentially located at the tumor periphery and could be generated by a unique mechanism involving the CAF-mediated education of normal fibroblasts (CEFs). In this review, we at first describe recent findings regarding the function of ASPN in the tumor microenvironment, as well as the mechanism involved in the generation of CEFs. CAFs are derived from heterogeneous origins besides resident normal fibroblasts. Among them, CAFs derived from mesothelial cells (mesothelial cell-derived CAF [MC-CAFs]) play pivotal roles in peritoneal carcinomatosis. We observed that MC-CAFs on the surfaces of organs also participate in tumor formation by infiltrating into the parenchyma, promoting local invasion by gastric cancers. This review also highlights the potential functions of macrophages in the formation of MC-CAFs in gastric cancers, by transfer the contents of cancer cell-derived extracellular vesicles.

Expression of asporin reprograms cancer cells to acquire resistance to oxidative stress.

Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1α and upregulated lactate dehydrogenase A (LDHA) and PDH-E1α, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1α in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN-/- mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1α-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.

Curcumin analog, GO-Y078, overcomes resistance to tumor angiogenesis inhibitors.

Tumor angiogenesis inhibition is one of the most potent strategies in cancer chemotherapy. From past clinical studies, inhibition of the vascular endothelial growth factor pathway successfully treats malignant tumors. However, vascular endothelial growth factor inhibitors alone cannot cure tumors. Moreover, resistance to small molecule inhibitors has also been reported. Herein, we show the antiangiogenic potential of a newly synthesized curcumin analog, GO-Y078, that possibly functions through inhibition of actin stress fiber formation, resulting in mobility inhibition; this mechanism is different from that of vascular endothelial growth factor inhibition. In addition, we examined the detailed mechanism of action of the antiangiogenesis potential of GO-Y078 using human umbilical venous epithelial cells resistant to angiogenesis inhibitors (HUVEC-R). GO-Y078 inhibited the growth and mobility of HUVEC-R at 0.75 µmol/L concentration. Expression analyses by microarray and RT-PCR showed that expressions of genes including that of fibronectin 1 were significantly suppressed. Among these genes, fibronectin 1 is abundantly expressed and, therefore, seems to be a good target for GO-Y078. In a knockdown experiment using Si-oligo of fibronectin 1 (FN1), FN1 expression was decreased to half of that in mock experiments as well as GO-Y078. Knockdown of FN1 resulted in the suppression of HUVEC-R growth at 24 hours after treatment. Fibronectin is a key molecule contributing to angiogenesis that could be inhibited by GO-Y078. Thus, resistance to vascular endothelial growth factor inhibition can be overcome using GO-Y078.

Spatiotemporal expression of UPK3B and its promoter activity during embryogenesis and spermatogenesis.

Uroplakin (Upk) 3 is one of the main structural components of the urothelium tissue. Although expression of UPK3B is seen in a wider variety of the tissues and organs than UPK3A, tissue-specific expression has not yet been analyzed. Here, we analyzed the Cre recombinase activity driven by the Upk3b promoter in transgenic mice and the endogenous localization of UPK3B. We generated Tg(Upk3b-Cre)/R26tdTomato mice by crossing ROSA26tm14(CAG-tdTomato) (R26tdTomato) mice with Tg(Upk3b-Cre) mice and investigated the spatiotemporal distribution of tdTomato in embryonic and adult mice. In embryos, we detected Cre recombinase activity in neural crest cells and the heart, liver, kidneys, and lungs. In adult mice, Cre recombinase activity was detected in male and female genital organs; however, the activity was absent in the bladder. Histological analyses revealed that both tdTomato and UPK3B were present in testicular and epididymal sperm; however, tdTomato was not present in the ductus epididymis, where the endogenous expression of UPK3B was detected. In female siblings, both tdTomato and UPK3B expressions were detected in the follicles of the ovary, whereas no tdTomato expression was found in the mucosal epithelium of the fallopian tubes, where the endogenous UPK3B was expressed. These data suggest that UPK3B may play a pivotal role in the maturation of gametes and gamete-delivery organs.

Identification of anti-cancer chemical compounds using Xenopus embryos.

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro-invasive ability through epithelial-mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti-cancer compounds that prevent cancer invasion. From the initial test of known anti-cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind-screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C-157 (an analog of podophyllotoxin) and D-572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C-157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C-157 and D-572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical-screening approach based on Xenopus embryos is an effective method for isolating anti-cancer drugs and, in particular, targeting cancer cell invasion and proliferation.

The Src substrate SKAP2 regulates actin assembly by interacting with WAVE2 and cortactin proteins.

In our attempt to screen for substrates of Src family kinases in glioblastoma, Src kinase-associated phosphoprotein 2 (SKAP2) was identified. Although SKAP2 has been suggested to be associated with integrin-mediated adhesion of hematopoietic cells, little is known about its molecular function and the effects in other types of cells and tumors. Here, we demonstrate that SKAP2 physically associates with actin assembly factors WAVE2 and cortactin and inhibits their interaction. Cortactin is required for the membrane localization of WAVE2, and SKAP2 suppresses actin polymerization mediated by WAVE2 and cortactin in vitro. Knockdown of SKAP2 in NIH3T3 accelerated cell migration and enhanced translocation of WAVE2 to the cell membrane, and those effects of SKAP2 depend on the binding activity of SKAP2 to WAVE2. Furthermore, reduction of SKAP2 in the glioblastoma promoted tumor invasion both in ex vivo organotypic rat brain slices and immune-deficient mouse brains. These results suggest that SKAP2 negatively regulates cell migration and tumor invasion in fibroblasts and glioblastoma cells by suppressing actin assembly induced by the WAVE2-cortactin complex, indicating that SKAP2 may be a novel candidate for the suppressor of tumor progression.

Nm23-H1 regulates contact inhibition of locomotion, which is affected by ephrin-B1.

Contact inhibition of locomotion (CIL) is the process by which cells stop the continual migration in the same direction after collision with another cell. Highly invasive malignant cells exhibit diminished CIL when they contact stromal cells, which allows invasion of the tissue by tumors. We show that Nm23-H1 is essential for the suppression of Rac1 through inactivation of Tiam1 at the sites of cell-cell contact, which plays a pivotal role in CIL. U87MG cells show CIL when they contact normal glia. In spheroid confrontation assays U87MG cells showed only limited invasion of the glial population, but reduction of Nm23-H1 expression in U87MG cells abrogated CIL resulting in invasion. In U87MG cells, Nm23-H1 is translocated to the sites of contact with glia through association with α-catenin and N-cadherin. Mutants of Nm23-H1, which lacked the binding ability with Tiam1, or α-catenin did not restore CIL. Moreover, the expression of ephrin-B1 in tumor cells disrupted CIL and promoted invasion. As one mechanism, ephrin-B1 inhibits the association of Nm23-H1 with Tiam1, which contributes for activation of Rac1. These results indicate a novel function of Nm23-H1 to control CIL, and its negative regulation by ephrin-B1.

Exosomes secreted by ST3GAL5high cancer cells promote peritoneal dissemination by establishing a premetastatic microenvironment.

Peritoneal dissemination of cancer affects patient survival. The behavior of peritoneal mesothelial cells (PMCs) and immune cells influences the establishment of a microenvironment that promotes cancer cell metastasis in the peritoneum. Here, we investigated the roles of lactosylceramide alpha-2,3-sialyltransferase (ST3G5; also known as ST3GAL5 and GM3 synthase) in the exosome-mediated premetastatic niche in peritoneal milky spots (MSs). Exosomes secreted from ST3G5high cancer cells (ST3G5high -cExos) were found to contain high levels of hypoxia-inducible factor 1-alpha (HIF1α) and accumulated in MSs via uptake in macrophages (MΦs) owing to increased expression of sialic acid-binding Ig-like lectin 1 (CD169; also known as SIGLEC1). ST3G5high -cExos induced pro-inflammatory cytokines and glucose metabolic changes in MΦs, and the interaction of these MΦs with PMCs promoted mesothelial-mesenchymal transition (MMT) in PMCs, thereby generating αSMA+ myofibroblasts. ST3G5high -cExos also increased the expression of immune checkpoint molecules and T-cell exhaustion in MSs, which accelerated metastasis to the omentum. These events were prevented following ST3G5 depletion in cancer cells. Mechanistically, ST3G5high -cExos upregulated chemokines, including CC-chemokine ligand 5 (CCL5), in recipient MΦs and dendritic cells (DCs), which induced MMT and immunosuppression via activation of signal transducer and activator of transcription 3 (STAT3). Maraviroc, a C-C chemokine receptor type 5 (CCR5) antagonist, prevented ST3G5high -cExo-mediated MMT, T-cell suppression, and metastasis in MSs. Our results suggest ST3G5 as a suitable therapeutic target for preventing cExo-mediated peritoneal dissemination.

Effects of contrast medium viscosity into flushing port on artefacts during optical coherence tomography imaging.

BACKGROUND: Optical coherence tomography (OCT) is becoming the standard imaging modality for percutaneous coronary intervention (PCI) because of its high resolution. To perform appropriate OCT-guided PCI, it is necessary to avoid artefacts and obtain high-quality images. We investigated the relationship between artefacts and the viscosity of contrast media, which were used to remove air before OCT imaging catheter was inserted into guiding catheter. METHODS: We retrospectively analyzed every pullback of OCT examinations from January 2020 to September 2021. Cases were divided into two groups according to the type of contrast media used for catheter flushing: low-viscosity (Iopamidol-300, Bayer, Nordrhein-Westfalen, Germany) vs. high-viscosity (Iopamidol-370, Bayer). We evaluated the artefacts and quality of each OCT image and performed ex vivo experiments to compare differences in artefact frequencies using the two contrast media. RESULTS: A total of 140 pullbacks in the low-viscosity group and 73 pullbacks in the high-viscosity group were analyzed. The percentage of grade 2 and 3 images (with good quality) in the low-viscosity group was significantly lower (68.1 % vs. 94.5 %, p < 0.001). Rotational artefacts were significantly more common in the low-viscosity group (49.3 % vs. 8.2 %, p < 0.001). In multivariate analysis, using low-viscosity contrast media was a significant factor influencing the appearance of rotational artefacts and affecting image quality (odds ratio, 9.42; 95 % confidence interval, 3.58 to 24.8; p < 0.001). In ex vivo experiments, using low-viscosity contrast media was also a significant predictor of artefact occurrence during OCT (p < 0.01). CONCLUSIONS: The viscosity of the contrast agent used while flushing the OCT imaging catheter contributes to the appearance of OCT artefacts.

CCDC85A is regulated by miR-224-3p and augments cancer cell resistance to endoplasmic reticulum stress.

MicroRNAs (miRNAs) play pivotal roles in the tumor microenvironment. Here, we analyzed miRNAs in tumor stromal fibroblasts. Expression of miR-224-3p in cancer-associated fibroblasts (CAF) from scirrhous gastric cancer patients was lower than in normal fibroblasts (NF). Introduction of a miR-224-3p mimic attenuated migration and invasion of CAF. Coiled-coil domain containing 85A (CCDC85A), whose function in tumors is not understood, was the target gene of miR-224-3p. Immunohistological analysis revealed that CCDC85A is expressed to varying degrees by cancer cells and CAFs in gastric and pancreatic carcinomas. Downregulation of CCDC85A in cancer cells revealed that these cells are vulnerable to endoplasmic reticulum (ER) stress induced by thapsigargin or tunicamycin, which were ameliorated after addback of CCDC85A. Injection of NF-derived exosomes containing miR-224-3p into the xenograft tumor increased tumor shrinkage by cisplatin treatment. Mechanistically, CCDC85A associated with the molecular chaperone GRP78 and GRP94, thereby inhibiting association of these negative regulators of the unfolded protein response (UPR), leading to sustained activation of PERK and downstream eIF2〈 and ATF4 upon ER stress. These data suggest a novel miR-224-3p-mediated function for CCDC85A: protection from ER stress and cisplatin resistance.

Macrophage numbers in the marginal area of sarcomas predict clinical prognosis.

Even when treated comprehensively by surgery, chemotherapy, and radiotherapy, soft-tissue sarcoma has an unfavorable outcome. Because soft-tissue sarcoma is rare, it is the subject of fewer clinicopathological studies, which are important for clarifying pathophysiology. Here, we examined tumor-associated macrophages in the intratumoral and marginal areas of sarcomas to increase our knowledge about the pathophysiology. Seventy-five sarcoma specimens (not limited to a single histological type), resected at our institution, were collected, and the number of CD68-, CD163-, and CD204-positive macrophages in the intratumoral and marginal areas was counted. We then performed statistical analysis to examine links between macrophage numbers, clinical factors, and outcomes. A high number of macrophages positive for all markers in both areas was associated with worse disease-free survival (DFS). Next, we divided cases according to the FNCLCC classification (Grade 1 and Grades 2/3). In the Grade 1 group, there was no significant association between macrophage number and DFS. However, in the Grade 2/3 group, high numbers of CD163- and CD204-positive macrophages in the marginal area were associated with poor DFS. By contrast, there was no significant difference between the groups with respect to high or low numbers of CD68-, CD163-, or CD204-positive macrophages in the intratumoral area. Multivariate analysis identified the number of CD163- and CD204-positive macrophages in the marginal area as an independent prognostic factor. Macrophage numbers in the marginal area of soft-tissue sarcoma may better reflect clinical behavior.

Type I and Type II mechanisms of antimicrobial photodynamic therapy: an in vitro study on gram-negative and gram-positive bacteria.

BACKGROUND AND OBJECTIVES: Antimicrobial photodynamic therapy (APDT) employs a non-toxic photosensitizer (PS) and visible light, which in the presence of oxygen produce reactive oxygen species (ROS), such as singlet oxygen ((1) O(2), produced via Type II mechanism) and hydroxyl radical (HO(.), produced via Type I mechanism). This study examined the relative contributions of (1) O(2) and HO(.) to APDT killing of Gram-positive and Gram-negative bacteria. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence probes, 3'-(p-hydroxyphenyl)-fluorescein (HPF) and singlet oxygen sensor green reagent (SOSG) were used to determine HO(.) and (1) O(2) produced by illumination of two PS: tris-cationic-buckminsterfullerene (BB6) and a conjugate between polyethylenimine and chlorin(e6) (PEI-ce6). Dimethylthiourea is a HO(.) scavenger, while sodium azide (NaN(3)) is a quencher of (1) O(2). Both APDT and killing by Fenton reaction (chemical generation of HO(.)) were carried out on Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). RESULTS: Conjugate PEI-ce6 mainly produced (1) O(2) (quenched by NaN(3)), while BB6 produced HO(.) in addition to (1) O(2) when NaN(3) potentiated probe activation. NaN(3) also potentiated HPF activation by Fenton reagent. All bacteria were killed by Fenton reagent but Gram-positive bacteria needed a higher concentration than Gram-negatives. NaN(3) potentiated Fenton-mediated killing of all bacteria. The ratio of APDT killing between Gram-positive and Gram-negative bacteria was 2 or 4:1 for BB6 and 25:1 for conjugate PEI-ce6. There was a NaN(3) dose-dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN(3) almost failed to inhibit killing of Gram-positive bacteria. CONCLUSION: Azidyl radicals may be formed from NaN(3) and HO(.). It may be that Gram-negative bacteria are more susceptible to HO(.) while Gram-positive bacteria are more susceptible to (1) O(2). The differences in NaN(3) inhibition may reflect differences in the extent of PS binding to bacteria (microenvironment) or differences in penetration of NaN(3) into cell walls of bacteria.

Pigment Epithelium Derived Factor Is Involved in the Late Phase of Osteosarcoma Metastasis by Increasing Extravasation and Cell-Cell Adhesion.

Organ tropism of metastatic cells is not well understood. To determine the key factors involved in the selection of a specific organ upon metastasis, we established metastatic cell lines and analyzed their homing to specific tissues. Toward this, 143B osteosarcoma cells were injected intracardially until the kidney-metastasizing sub-cell line Bkid was established, which significantly differed from the parental 143B cells. The candidate genes responsible for kidney metastasis were validated, and SerpinF1/Pigment epithelium derived factor (PEDF) was identified as the primary target. Bkid cells with PEDF knockdown injected intracardially did not metastasize to the kidneys. In contrast, PEDF overexpressing 143B cells injected into femur metastasized to the lungs and kidneys. PEDF triggered mesenchymal-to-epithelial transition (MET) in vitro as well as in vivo. Based on these results, we hypothesized that the MET might be a potential barrier to extravasation. PEDF overexpression in various osteosarcoma cell lines increased their extravasation to the kidneys and lungs. Moreover, when cultured close to the renal endothelial cell line TKD2, Bkid cells disturbed the TKD2 layer and hindered wound healing via the PEDF-laminin receptor (lamR) axis. Furthermore, novel interactions were observed among PEDF, lamR, lysyl oxidase-like 1 (Loxl1), and SNAI3 (Snail-like transcription factor) during endothelial-to-mesenchymal transition (EndoMT). Collectively, our results show that PEDF induces cancer cell extravasation by increasing the permeability of kidney and lung vasculature acting via lamR and its downstream genes. We also speculate that PEDF promotes extravasation via inhibiting EndoMT, and this warrants investigation in future studies.

Weak spin-flip scattering in Pd89Ni11 interlayer of NbN-based ferromagnetic Josephson junctions.

We studied niobium nitride (NbN)-based π-junctions with a diluted ferromagnetic Pd89Ni11 interlayer (NbN/PdNi/NbN junctions). In the NbN/PdNi/NbN junctions with various PdNi thicknesses, we observed a non-monotonic dependence of the critical currents on PdNi thickness, indicating the effects of the exchange interaction on the superconducting order parameter. From theoretical fitting of the experimental data, we found that the NbN/PdNi/NbN junctions showed a significantly smaller degree of spin-flip scattering in the PdNi interlayer than in the CuNi interlayer of NbN/CuNi/NbN junctions reported previously. The weak spin-flip scattering leads to a longer decay length of the Josephson critical current, so the critical currents were observed over a wide range of PdNi thicknesses (10-40 nm). We also fabricated superconducting quantum interference devices (SQUIDs) including the NbN/PdNi/NbN junction, using a PdNi thickness in which the π-state was expected. A half-flux-quantum shift, as evidence of the π-state, was observed in the magnetic field-dependent critical currents of the SQUIDs. This result represents an important step towards the practical application of NbN-based π-Josephson junctions.

SKAP2 suppresses inflammation-mediated tumorigenesis by regulating SHP-1 and SHP-2.

Inflammatory bowel diseases, like ulcerative colitis and Crohn's disease are frequently accompanied by colorectal cancers. However, the mechanisms underlying colitis-associated cancers are not fully understood. Src Kinase Associated Phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages. Here, we examined the effects of SKAP2 on inflammatory responses in a mouse model of tumorigenesis with colitis induced by azoxymethane/dextran sulfate sodium. SKAP2 knockout increased the severity of colitis and tumorigenesis, as well as lipopolysaccharide (LPS) induced acute inflammation. SKAP2 attenuated inflammatory signaling in macrophages induced by uptake of cancer cell-derived exosomes. SKAP2-/- mice were characterized by the activation of NF-κB signaling and the upregulation and release of cytokines including TNFα, IL-1ß, IL-6, CXCL-9/-10/-13, and sICAM1; SKAP2 overexpression attenuated NF-κB activation. Mechanistically, SKAP2 formed a complex with the SHP-1 tyrosine phosphatase via association with the Sirpα transmembrane receptor. SKAP2 also physically associated with the TIR domain of MyD88, TIRAP, and TRAM, adaptors of toll-like receptor 4 (TLR4). SKAP2-mediated recruitment of the Sirpα/SHP-1 complex to TLR4 attenuated inflammatory responses, whereas direct interaction of SKAP2 with SHP-2 decreased SHP-2 activation. SHP-2 is required for efficient NF-κB activation and suppresses the TRAM/TRIF-INFß pathway; therefore, SKAP2-mediated SHP-2 inhibition affected two signaling axes from TLR4. The present findings indicate that SKAP2 prevents excess inflammation by inhibiting the TLR4-NF-κB pathway, and it activates the TLR4-IFNß pathway through SHP-1 and SHP-2, thereby suppressing inflammation-mediated tumorigenesis.

Cancer-associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T-cell suppression.

In some tumors, a small number of cancer cells are scattered in a large fibrotic stroma. Here, we demonstrate a novel mechanism for expansion of pro-tumor fibroblasts via cancer-associated fibroblast (CAF)-mediated education of normal fibroblasts (NFs). When NFs were incubated with conditioned medium from CAFs, the resulting CAF-educated fibroblasts (CEFs) generated reactive oxygen species, which induced NF-κB-mediated expression of inflammatory cytokines and the extracellular matrix protein asporin (ASPN), while expression of a common CAF marker gene, α-SMA, was not increased. ASPN further increased CEF expression of downstream molecules, including indoleamine 2,3-dioxygenase 1 (IDO-1), kynureninase (KYNU), and pregnancy-associated plasma protein-A (PAPP-A). These CEFs induce cytocidal effects against CD8+ T cells and IGF-I activation in cancer cells. CEFs were generated without cancer cells by the direct mixture of NFs and CAFs in mouse xenografts, and once CEFs were generated, they sequentially educated NFs, leading to continuous generation of CEFs. In diffuse-type gastric cancers, ASPNhigh /IDO-1high /KYNUhigh /α-SMA- CEFs were located at the distal invading front. These CEFs expanded in the fibrotic stroma and caused dissemination of cancer cells. ASPN may therefore be a key molecule in facilitating tumor spreading and T-cell suppression.

Photodynamic therapy using intra-articular Photofrin for murine MRSA arthritis: biphasic light dose response for neutrophil-mediated antibacterial effect.

BACKGROUND AND OBJECTIVE: Bacterial arthritis does not respond well to antibiotics and moreover multidrug resistance is spreading. We previously tested photodynamic therapy (PDT) mediated by systemic Photofrin® in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) arthritis, but found that neutrophils were killed by PDT and therefore the infection was potentiated. STUDY DESIGN/MATERIALS AND METHODS: The present study used an intra-articular injection of Photofrin® and optimized the light dosimetry in order to maximize bacterial killing and minimize killing of host neutrophils. MRSA (5 × 10(7) CFU) was injected into the mouse knee followed 3 days later by 1 µg of Photofrin® and 635-nm diode laser illumination with a range of fluences within 5 minutes. Synovial fluid was sampled 6 hours or 1-3, 5, and 7 days after PDT to determine MRSA colony-forming units (CFU), neutrophil numbers, and levels of cytokines. RESULTS: A biphasic light dose response was observed with the greatest reduction of MRSA CFU seen with a fluence of 20 J cm(-2), whereas lower antibacterial efficacy was observed with fluences that were either lower or higher. Consistent with these results, a significantly higher concentration of macrophage inflammatory protein-2, a CXC chemokine, and greater accumulation of neutrophils were seen in the infected knee joint after PDT with a fluence of 20 J cm(-2) compared to fluences of 5 or 70 J cm(-2). CONCLUSION: PDT for murine MRSA arthritis requires appropriate light dosimetry to simultaneously maximize bacterial killing and neutrophil accumulation into the infected site, while too little light does not kill sufficient bacteria and too much light kills neutrophils and damages host tissue as well as bacteria and allows bacteria to grow unimpeded by host defense.