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Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-γ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-γ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-γ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death.

Although bronchiolitis obliterans syndrome (BOS) is a major cause of death after lung transplantation, an effective drug therapy for BOS has not yet developed. Here, we assessed the effectiveness of a neutralizing anti-S100 calcium binding protein (S100) A8/A9 antibody against BOS. A murine model of heterotopic tracheal transplantation was used. Mice were intraperitoneally administered control IgG or the S100A8/A9 antibody on day 0 and twice per week until they were sacrificed. Tissue sections were used to evaluate the obstruction ratio, epithelium-preservation ratio, α-smooth muscle actin (SMA)-positive myofibroblast infiltration, and luminal cell death. Quantitative reverse transcriptase-polymerase chain reaction analysis was performed to analyze the mRNA-expression levels of collagen, inflammatory cytokines, and chemokines on days 7, 14, and 21. The anti-S100A8/A9 antibody significantly improved the obstruction ratio and epithelium-preservation ratio, with less α-SMA-positive myofibroblast infiltration compared to the control group. Antibody treatment reduced the type-III collagen: type-I collagen gene-expression ratio. The antibody also significantly suppressed the number of dead cells in the graft lumen. The expression levels of tumor growth factor β1 and C-C motif chemokine 2 on day 21, but not those of interleukin-1β, interleukin-6, and tumor necrosis factor α, were significantly suppressed by S100A8/A9 antibody treatment. These findings suggest that S100A8/A9 may be a potential therapeutic target for BOS after lung transplantation.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

Abstract The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3—namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. Key messages • REIC/Dkk-3 protein effectively suppresses breast cancer progression through an acceleration of PD-L1 degradation. • PD-L1 stability on the cancer cell membrane is kept high by binding with mainly CMTM6. • Competitive binding of REIC/Dkk-3 protein with CMTM6 liberates PD-L1, leading to PD-L1 degradation.

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

In patients with interstitial pneumonia, pulmonary fibrosis is an irreversible condition that can cause respiratory failure. Novel treatments for pulmonary fibrosis are necessary. Inflammation is thought to activate lung fibroblasts, resulting in pulmonary fibrosis. Of the known inflammatory molecules, we have focused on S100A8/A9 from the onset of inflammation to the subsequent progression of inflammation. Our findings confirmed the high expression of S100A8/A9 in specimens from patients with pulmonary fibrosis. An active role of S100A8/A9 was demonstrated not only in the proliferation of fibroblasts but also in the fibroblasts' differentiation to myofibroblasts (the active form of fibroblasts). S100A8/A9 also forced fibroblasts to upregulate the production of collagen. These effects were induced via the receptor of S100A8/A9, i.e., the receptor for advanced glycation end products (RAGE), on fibroblasts. The anti-S100A8/A9 neutralizing antibody inhibited the effects of S100A8/A9 on fibroblasts and suppressed the progression of fibrosis in bleomycin (BLM)-induced pulmonary fibrosis mouse model. Our findings strongly suggest a crucial role of S100A8/A9 in pulmonary fibrosis and the usefulness of S100A8/A9-targeting therapy for fibrosis interstitial pneumonia. HIGHLIGHTS: S100A8/A9 level is highly upregulated in the IPF patients' lungs as well as the blood. S100A8/A9 promotes not only the growth of fibroblasts but also differentiation to myofibroblasts. The cell surface RAGE acts as a crucial receptor to the extracellular S100A8/A9 in fibroblasts. The anti-S100A8/A9 antibody effectively suppresses the progression of IPF in a mouse model. In idiopathic pulmonary fibrosis (IPF), S100A8/A9, a heterodimer composed of S100A8 and S100A9 proteins, plays a crucial role in the onset of inflammation and the subsequent formation of a feed-forward inflammatory loop that promotes fibrosis. (1) The local, pronounced increase in S100A8/A9 in the injured inflammatory lung region-which is provided mainly by the activated neutrophils and macrophages-exerts strong inflammatory signals accompanied by dozens of inflammatory soluble factors including cytokines, chemokines, and growth factors that further act to produce and secrete S100A8/A9, eventually making a sustainable inflammatory circuit that supplies an indefinite presence of S100A8/A9 in the extracellular space with a mal-increased level. (2) The elevated S100A8/A9 compels fibroblasts to activate through receptor for advanced glycation end products (RAGE), one of the major S100A8/A9 receptors, resulting in the activation of NFκB, leading to fibroblast mal-events (e.g., elevated cell proliferation and transdifferentiation to myofibroblasts) that actively produce not only inflammatory cytokines but also collagen matrices. (3) Finally, the S100A8/A9-derived activation of lung fibroblasts under a chronic inflammation state leads to fibrosis events and constantly worsens fibrosis in the lung. Taken together, these findings suggest that the extracellular S100A8/A9 heterodimer protein is a novel mainstay soluble factor for IPF that exerts many functions as described above (1-3). Against this background, we herein applied the developed S100A8/A9 neutralizing antibody to prevent IPF. The IPF imitating lung fibrosis in an IPF mouse model was effectively blocked by treatment with the antibody, leading to enhanced survival. The developed S100A8/A9 antibody, as an innovative novel biologic, may help shed light on the difficulties encountered with IPF therapy in clinical settings.

Background and Aim: Kidney regeneration is required for dogs with end-stage renal failure. Decellularization is one of the bioengineering techniques, which involves the removal of all tissue cells and cellular components and conservation of the extracellular matrix (ECM). Studies in rats have shown that decellularized kidney has regenerative potential; however, there are no reports on renal decellularization in dogs. Here, we showed the decellularization of the canine kidney. Materials and Methods: The renal artery of the cadaveric canine kidney was cannulated and the whole kidney was frozen at –80°C. After completely thawing, it was perfused with physiological saline and sodium dodecyl sulfate (0.5%, 6 h) through the cannulated renal artery to achieve decellularization. To assess the efficiency of the decellularization protocol, histological and immunohistochemical analysis of decellularized kidney was performed. Results: The results of hematoxylin and eosin (H and E) staining revealed that the decellularized canine kidney had no apparent cellular components. In addition, 4’,6-diamidino-2-phenylindole (DAPI) staining showed no visible nuclear components within the whole decellularized kidney. Therefore, both H and E and DAPI staining showed decellularization of the canine kidney. Our decellularization protocol also preserved the basement membrane of glomerulus, shown by periodic acid methenamine silver, periodic acid–Schiff, fibronectin, and collagen type IV stain. Conclusion: Our decellularization protocol could eliminate cellular components and remaining native ECM structures of canine kidney. These results could promote further research into canine kidney regeneration, which may be the first small step to regenerate the canine kidney waiting for renal transplantation.

Previous studies of dolphin electrocardiograms showed that they are mainly composed of increased negative waves similar to ungulates. The electrocardiogram waveform is determined by the distribution of the location of the Purkinje fibers. Based on the waveform of the dolphin electrocardiogram, Hamlin predicted that the distribution of Purkinje fibers would be within the ventricular muscle, as in ungulates. In this study, bottlenose dolphin heart were observed both visually and histologically, and the effects of Purkinje fiber distribution and cardiac morphology on electrocardiogram waveforms were considered. This study showed that the Purkinje fibers of dolphins run just below the endocardium as in humans, dogs, and cats, whose electrocardiograms show mainly positive waves. When the cardiac morphology of dolphins was observed carefully again, the right ventricle was extremely dilated compared to terrestrial mammals. In human recreational divers, right ventricular dilatation is induced by diving. We hypothesized that the dolphin’s heart is in a similar state to that of right heart dilatation in terrestrial animals. The dolphin electrocardiogram waveform is considered to be due to right axis deviation. Based on the above, we concluded that the dolphin electrocardiogram waveform was due to its living in water. We found that the dolphin is genetically related to the ungulates, especially the hippopotamus, but that their hearts have evolved differently.

The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

S100A11, a member of the S100 family of proteins, is actively secreted from pancreatic ductal adenocarcinoma (PDAC) cells. However, the role of the extracellular S100A11 in PDAC progression remains unclear. In the present study, we investigated the extracellular role of S100A11 in crosstalking between PDAC cells and surrounding fibroblasts in PDAC progression. An abundant S100A11 secreted from pancreatic cancer cells stimulated neighboring fibroblasts through receptor for advanced glycation end products (RAGE) upon S100A11 binding and was followed by not only an enhanced cancer cell motility in vitro but also an increased number of the PDAC-derived circulating tumor cells (CTCs) in vivo. Mechanistic investigation of RAGE downstream in fibroblasts revealed a novel contribution of a mitogen-activated protein kinase kinase kinase (MAPKKK), tumor progression locus 2 (TPL2), which is required for positive regulation of PDAC cell motility through induction of cyclooxygenase 2 (COX2) and its catalyzed production of prostaglandin E2 (PGE2), a strong chemoattractive fatty acid. The extracellularly released PGE2 from fibroblasts was required for the rise in cellular migration as well as infiltration of their adjacent PDAC cells in a coculture setting. Taken together, our data reveal a novel role of the secretory S100A11 in PDAC disseminative progression through activation of surrounding fibroblasts triggered by the S100A11‐RAGE‐TPL2‐COX2 pathway. The findings of this study will contribute to the establishment of a novel therapeutic antidote to PDACs that are difficult to treat by regulating cancer-associated fibroblasts (CAFs) through targeting the identified pathway.

The aim of this study was to verify the safety and efficacy of transvaginal mesh by analyzing the 2-year follow-up data of patients performed by a surgeon with a high volume of procedures.A total of 617 patients with pelvic organ prolapse underwent transvaginal mesh by a single surgeon. Complications and anatomical status of each patient were examined up to 24 months after surgery. Risk factors for the recurrence were also analyzed.Regarding complications, we experienced 10 patients (3.8%) of bladder injuries in anterior transvaginal mesh and eight (3.4%) in anterior and posterior transvaginal mesh. Massive blood loss was observed in four patients, but there was no case of blood transfusion. Mesh exposures were seen in seven patients (1.2%). A total of 100 patients (16.2%) had prolapse recurrence, defined as the Pelvic Organ Prolapse Quantification System stage ≥II. As to recurrences on the operated compartments, we observed five patients (2.0%) for anterior transvaginal mesh, three (6.5%) for posterior transvaginal mesh, five (7.4%) for combined transvaginal mesh, and 31 (14.2%) in anterior and posterior transvaginal mesh. Regarding Point C before operation in the anterior and posterior transvaginal mesh, the recurrence rates were more than 23% in patients with a Point C of 4 or more. Binominal regression analyses showed that higher body mass index, younger age, and higher stage of uterine prolapse were significant risk factors.The transvaginal mesh surgery is safe when conducted by experts. However, the recurrence rate may exceed 20% for high-stage uterine prolapse even when conducted by experts.

International audience; Advances in organ-on-chip technology allowed the recapitulation of two or more organs interaction thanks to dedicated microbioreactors interconnected by microfluidic network. Here, we developed pancreas-liver coculture model in a microfluidic biochip, using rat Langerhans islets and hepatocytes. The behavior and functionality of the model were compared to islets and hepatocytes (with/without insulin) monocultures. We confirmed the insulin, glucagon and C-peptide secretions by islets monoculture and coculture. Furthermore, C-peptide and insulin secretions were higher in coculture after 5 days of culture. The islets coculture presented downregulation of Pdx1, Glut2, Gcg, App, Ins1, Neurod, Neurog3 and Gcgr genes, compared to monocultures. In hepatic compartment, the monocultures without insulin were negative to CK18 staining and displayed a weaker production of albumin when compared to monocultures with insulin. They also presented a moderate protein expression of CYP3A2, GLUT2, INSR and modulation of several hepatic genes. In coculture model, hepatocytes displayed albumin productions similar to those in monoculture with insulin. The hepatocytes cocultures were highly positive to INSR, GLUT2, CK18 and CYP3A2 immunostaining and allowed to recover mRNA levels similar to monoculture with insulin (Gcgr, Insr, Hnf4a, Igfbp1 and Alb). The result illustrated that islets can produce insulin to supplement the culture medium and recover hepatic functionality. We believe that our model illustrated the potential of organ-on-chip technology to reproduce cross-talk between liver and pancreas.

Microneedle (MN) is a key technology of the biomedical engineering field due to its capability of accessing the biological information in a minimally invasive manner. One of the huge demands for next-generation healthcare monitoring is continuous monitoring, especially of blood glucose concentration. For this, MN should be kept inserted into the human skin for a certain period of time, enduring stresses induced by daily human motion and at the same time measuring biomarkers in ISF. However, conventional MNs for biosensing are not suitable for a long term insertion due to the rigid structure and biological risks of MN breakage. In this study, a novel MN structure is proposed and investigated by combining flexible "sponge-like" porous PDMS matrix and coating by biodissolving hyaluronic acid (HA). The fabricated porous MNs coated with HA show ideal mechanical characteristics, by which the MNs are rigid enough to penetrate the skin and become flexible after insertion into the skin. It is also shown that the MN array successfully extracts ISF in vitro and in vivo not by capillary action but by repeated compressions. The results show the applicability of the flexible MNs to continuous blood glucose monitoring.

Compiling evidence indicates an unusual role of extracellular S100A8/A9 in cancer metastasis. S100A8/A9 secreted from either cancer cells or normal cells including epithelial and inflammatory cells stimulates cancer cells through S100A8/A9 sensor receptors in an autocrine or paracrine manner, leading to cancer cell metastatic progression. We previously reported a novel S100A8/A9 receptor, neuroplastin-β (NPTNβ), which plays a critical role in atopic dermatitis when it is highly activated in keratinocytes by an excess amount of extracellular S100A8/A9 in the inflammatory skin lesion. Interestingly, our expression profiling of NPTNβ showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNβ as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNβ has strong ability to induce cancer-related cellular events, including anchorage-independent growth, motility and invasiveness, in lung cancer cells in response to extracellular S100A8/A9, eventually leading to the expression of a cancer disseminative phenotype in lung tissue in vivo. Mechanistic investigation revealed that binding of S100A8/A9 to NPTNβ mediates activation of NFIA and NFIB and following SPDEF transcription factors through orchestrated upstream signals from TRAF2 and RAS, which is linked to anchorage-independent growth, motility and invasiveness. Overall, our results indicate the importance of the S100A8/A9-NPTNβ axis in lung cancer disseminative progression and reveal a pivotal role of its newly identified downstream signaling, TRAF2/RAS-NFIA/NFIB-SPDEF, in linking to the aggressive development of lung cancers.

The molecular mechanisms underlying the 'seed and soil' theory are unknown. S100A8/A9 (a heterodimer complex of S100A8 and S100A9 proteins that exhibits a 'soil signal') is a ligand for Toll-like receptor 4, causing distant melanoma cells to approach the lung as a 'seeding' site. Unknown soil sensors for S100A8/A9 may exist, e.g., extracellular matrix metalloproteinase inducer, neuroplastin, activated leukocyte cell adhesion molecule, and melanoma cell adhesion molecule. We call these receptor proteins 'novel S100 soil sensor receptors (novel SSSRs).' Here we review and summarize a crucial role of the S100A8/A9-novel SSSRs' axis in cancer metastasis. The binding of S100A8/A9 to individual SSSRs is important in cancer metastasis via upregulations of the epithelial-mesenchymal transition, cellular motility, and cancer cell invasiveness, plus the formation of an inflammatory immune suppressive environment in metastatic organ(s). These metastatic cellular events are caused by the SSSR-featured signal transductions we identified that provide cancer cells a driving force for metastasis. To deprive cancer cells of these metastatic forces, we developed novel biologics that prevent the interaction of S100A8/A9 with SSSRs, followed by the efficient suppression of S100A8/A9-mediated lung-tropic metastasis in vivo.

Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNβ, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".

Mitochondrial dysfunction is a key pathological feature of many different types of neurodegenerative disease. Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) has been attracting much attention as an important molecule for inducing axonal degeneration and neuronal cell death by causing loss of NAD (NADH). However, it has remained unclear what exactly regulates the SARM1 activity. Here, we report that NAD+ cleavage activity of SARM1 is regulated by its own phosphorylation at serine 548. The phosphorylation of SARM1 was mediated by c-jun N-terminal kinase (JNK) under oxidative stress conditions, resulting in inhibition of mitochondrial respiration concomitant with enhanced activity of NAD+ cleavage. Nonphosphorylatable mutation of Ser-548 or treatment with a JNK inhibitor decreased SARM1 activity. Furthermore, neuronal cells derived from a familial Parkinson's disease (PD) patient showed a congenitally increased level of SARM1 phosphorylation compared with that in neuronal cells from a healthy person and were highly sensitive to oxidative stress. These results indicate that JNK-mediated phosphorylation of SARM1 at Ser-548 is a regulator of SARM1 leading to inhibition of mitochondrial respiration. These findings suggest that an abnormal regulation of SARM1 phosphorylation is involved in the pathogenesis of Parkinson's disease and possibly other neurodegenerative diseases.

We previously identified novel S100A8/A9 receptors, extracellular matrix metalloproteinase inducer (EMMPRIN), melanoma cell adhesion molecule (MCAM), activated leukocyte cell adhesion molecule (ALCAM), and neuroplastin (NPTN) , that are critically involved in S100A8/A9-mediated cancer metastasis and inflammation when expressed at high levels. However, little is known about the presence of any cancer-specific mechanism(s) that modifies these receptors, further inducing upregulation at protein levels without any transcriptional regulation. Expression levels of glycosyltransferase-encoding genes were examined by a PCR-based profiling array followed by confirmation with quantitative real-time PCR. Cell migration and invasion were assessed using a Boyden chamber. Western blotting was used to examine the protein level, and the RNA level was examined by Northern blotting. Immunohistochemistry was used to examine the expression pattern of -1,3-galactosyl-O-glycosyl-glycoprotein -1,6-N-acetylglucosaminyltransferase 3 (GCNT3) and MCAM in melanoma tissue. We found that GCNT3 is overexpressed in highly metastatic melanomas. Silencing and functional inhibition of GCNT3 greatly suppressed migration and invasion of melanoma cells, resulting in the loss of S100A8/A9 responsiveness. Among the novel S100A8/A9 receptors, GCNT3 favorably glycosylates the MCAM receptor, extending its half-life and leading to further elevation of S100A8/A9-mediated cellular motility in melanoma cells. GCNT3 expression is positively correlated to MCAM expression in patients with high-grade melanomas. Collectively, our results showed that GCNT3 is an upstream regulator of MCAM protein and indicate the possibility of a potential molecular target in melanoma therapeutics through abrogation of the S100A8/A9MCAM axis.

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2-) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2-, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2- generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.

Herbal medicines and their bioactive compounds are increasingly being recognized as useful drugs for cancer treatments. The parasitic fungus Cordyceps militaris is an attractive anticancer herbal since it shows very powerful anticancer activity due to its phytocompound cordycepin. We previously discovered and reported that a high amount of xylitol is present in Cordyceps militaris extract, and that xylitol unexpectedly showed anticancer activity in a cancer-selective manner. We thus hypothesized that xylitol could become a useful supplement to help prevent various cancers, if we can clarify the specific machinery by which xylitol induces cancer cell death. It is also unclear whether xylitol acts on cancer suppression in vivo as well as in vitro. Here we show for the first time that induction of the glutathione-degrading enzyme CHAC1 is the main cause of xylitol-induced apoptotic cell death in cancer cells. The induction of CHAC1 is required for the endoplasmic reticulum (ER) stress that is triggered by xylitol in cancer cells, and is linked to a second induction of oxidative stress in the treated cells, and eventually leads to apoptotic cell death. Our in vivo approach also demonstrated that an intravenous injection of xylitol had a tumor-suppressing effect in mice, to which the xylitol-triggered ER stress also greatly contributed. We also observed that xylitol efficiently sensitized cancer cells to chemotherapeutic drugs. Based on our findings, a chemotherapeutic strategy combined with xylitol might improve the outcomes of patients facing cancer.

Metastasis is the major cause of treatment failure in cancer patients and of cancer-associated death so that therapeutic regulation of metastasis is very important subject for the cancer treatment. We have been reported that S100A8/A9, a heterodimer complex of S100A8 and S100A9, and its receptors play a crucial role in the lung tropic cancer metastasis, i.e., S100A8/A9 is actively secreted from the lung when cancer mass exists even at remote area from the lung and then functions to attract the distant cancer cells to the lung since cancer cells own the S100A8/A9 receptor(s) on their cell surface. Interestingly, one of the newly developed decoys, exMCAM-Fc, a Fc fusion protein with the extracellular region of melanoma cell adhesion molecule (MCAM), one of the S100A8/A9 receptors, that could prevent the interaction of S100A8/A9 with MCAM, efficiently suppressed the lung tropic cancer metastasis through exerting the several inhibitory effects on the S100A8/A9-mediated cancer cell events including enhanced mobility, invasion and attachment to the endothelial cells. However, it still remains to clarify if the decoy will reduce the number of circulating tumor cells (CTCs) that are defined as substantial cells in the context of organ tropic cancer metastasis. Here, we first show that exMCAM-Fc effectively reduces the number of CTCs in the blood flow of the melanoma bearing mice. The novel finding reinforces the suppressive role of exMCAM-Fc on the cancer metastasis. We therefore expect that exMCAM-Fc may greatly contribute to reduce treatment failure by the efficient blocking of the life threatening cancer metastasis.

The fertile stroma in pancreatic ductal adenocarcinomas (PDACs) has been suspected to greatly contribute to PDAC progression. Since the main cell constituents of the stroma are fibroblasts, there is crosstalking(s) between PDAC cells and surrounding fibroblasts in the stroma, which induces a fibroblast proliferation burst. We have reported that several malignant cancer cells including PDAC cells secrete a pronounced level of S100A11, which in turn stimulates proliferation of cancer cells via the receptor for advanced glycation end products (RAGE) in an autocrine manner. Owing to the RAGE+ expression in fibroblasts, the extracellular abundant S100A11 will affect adjacent fibroblasts. In this study, we investigated the significance of the paracrine axis of S100A11‐RAGE in fibroblasts for their proliferation activity. In in vitro settings, extracellular S100A11 induced upregulation of fibroblast proliferation. Our mechanistic studies revealed that the induction is through RAGE‐MyD88‐mTOR‐p70 S6 kinase upon S100A11 stimulation. The paracrine effect on fibroblasts is linked mainly to triggering growth but not cellular motility. Thus, the identified pathway might become a potential therapeutic target to suppress PDAC progression through preventing PDAC-associated fibroblast proliferation.

The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-κB activation and ROS formation. Notably, MCAM not only activated NF-κB more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems., Graphical abstract fx1, Highlights • S100A8/A9 heterodimer efficiently formed by coexpression in E. coli. • S100A8/A9 heterodimer preferentially assembled as compared to homodimer. • Simplified purification procedure for recombinant S100A8/A9 without fusion of affinity tag. • Convenient recombination method for construction of coexpression vector.

Since metastasis accounts for the majority of cancer-associated deaths, studies on the mechanisms of metastasis are needed to establish innovative strategies for cancer treatment. We previously reported that melanoma cell adhesion molecule (MCAM) functions as a critical receptor for S100A8/A9, and binding of S100A8/A9 to MCAM results in the migration of melanoma cells to lung tissue. However, the critical role of MCAM in the original melanoma skin lesion is still not clear. In this study, we aimed to determine the importance of the S100A8/A9-MCAM axis in melanoma dissemination in a skin lesion as a critical early step for metastasis. Mechanistic studies revealed the downstream signaling of MCAM that signaled the induction of metastasis. S100A8/A9-MCAM binding activates mitogen-activated protein kinase kinase kinase 8 (MAP3K8), also termed TPL2, leading to strong activation of the transcription factor ETV4 and subsequent induction of matrix metalloproteinase-25 (MMP25), and finally to induction of melanoma lung tropic metastasis. Collectively, our results demonstrate a crucial role of the S100A8/A9-MCAM signaling axis in metastatic onset of melanoma cells and indicate that strategies targeting the identified pathway may be useful for the establishment of innovative anti-cancer therapies.

This paper proposed a novel treatment for stomatitis using microneedles-based patches. We purposed to test whether appropriate physical stimulation by microneedles has a promotion effect on the healing of stomatitis. At the present stage, we have made a modeling of stomatitis last for more than one week on the oral mucosa of Sprague-Dawly (SD) rats and recorded the spontaneous recovery process.

Embigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, is involved in prostate and mammary gland development. As embigin&rsquo, s roles in cancer remain elusive, we studied its biological functions and interaction with extracellular S100A4 in prostate cancer progression. We found by a pull-down assay that embigin is a novel receptor for S100A4, which is one of the vital cancer microenvironment milleu. Binding of extracellular S100A4 to embigin mediates prostate cancer progression by inhibition of AMPK activity, activation of NF-&kappa, B, MMP9 and mTORC1 signaling, and inhibition of autophagy, which increase prostate cancer cell motility. We also found that embigin promotes prostate cancer growth, spheroid- and colony-forming ability, and survival upon chemotherapy independently of S100A4. An in vivo growth mouse model confirmed the importance of embigin and its cytoplasmic tail in mediating prostate tumor growth. Moreover, embigin and p21WAF1 can be used to predict survival of prostate cancer patients. Our results demonstrated for the first time that the S100A4-embigin/AMPK/mTORC1/p21WAF1 and NF-&kappa, B/MMP9 axis is a vital oncogenic molecular cascade for prostate cancer progression. We proposed that embigin and p21WAF1 could be used as prognostic biomarkers and a strategy to inhibit S100A4-embigin binding could be a therapeutic approach for prostate cancer patients.

The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.