The Comprehensive Characterization of B7-H3 Expression in the Tumor Microenvironment of Lung Squamous Cell Carcinoma: A Retrospective Study.

Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer; therefore, new therapeutic approaches are required to improve the prognosis of LSCC. Although immunotherapies targeting B7 family molecules were explored as treatments for several cancer types, the expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated in detail. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression using 110 surgically resected pathological specimens retrospectively. We examined the correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells. High B7-H3 expression in tumor cells was associated with a better prognosis and a significant increase in the number of CD163+PD-L1+ macrophages. Quantitative analysis revealed that there is a positive correlation between B7-H3 and PD-L1 expression in tumor and stromal cells, as well as in intratumoral tumor-infiltrating lymphocytes and tumor-associated macrophages in the same cells. CD68+, CD163+, and CK+ cells with PD-L1+ phenotypes had higher B7-H3 expression compared to PD-L1- cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies.

Mutual interaction between endothelial cells and mural cells enhances BMP9 signaling in endothelial cells.

Hereditary hemorrhagic telangiectasia is characterized by the formation of abnormal vascular networks and caused by the mutation of genes involved in BMP9 signaling. It is also known that the interaction between endothelial cells (ECs) and mural cells (MCs) is critical to maintain vessel integrity. However, it has not yet fully been uncovered whether the EC-MC interaction affects BMP9 signaling or not. To elucidate this point, we analyzed BMP9 signaling in a co-culture of several types of human primary culture ECs and MCs. The co-culture activated the Notch pathway in both types of cells in a co-culture- and BMP9-dependent manner. In HUVECs, the genes induced by BMP9 were significantly and synergistically induced in the presence of pericytes, fibroblasts or mesenchymal stem cells. The synergistic induction was greatly reduced in a non-contact condition. In fibroblasts, PDGFRB expression was potently induced in the presence of HUVECs, and BMP9 additively increased this response. Taken together, these results suggest that the EC-MC interaction potentiates BMP9 signaling both in ECs and MCs and plays a critical role in the maintenance of proper vessel functions.

Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish.

Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs.

Fbxw7 controls angiogenesis by regulating endothelial Notch activity.

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7(iECKO) mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.

Axin localizes to the centrosome and is involved in microtubule nucleation.

Axin is known to have an important role in the degradation of beta-catenin in the Wnt pathway. Here, we reveal a new function of Axin at the centrosome. Axin was localized to the centrosome in various cell lines and formed a complex with gamma-tubulin. Knockdown of Axin reduced the localization of gamma-tubulin and gamma-tubulin complex protein 2-components of the gamma-tubulin ring complex-to the centrosome and the centrosomal microtubule nucleation activity after treatment with nocodazole. These phenotypes could not be rescued by the reduction in the levels of beta-catenin. Although the expression of Axin rescued these phenotypes in Axin-knockdown cells, overexpression of Axin2, which is highly homologous to Axin, could not. Axin2 was also localized to the centrosome, but it did not form a complex with gamma-tubulin. These results suggest that Axin, but not Axin2, is involved in microtubule nucleation by forming a complex with gamma-tubulin at the centrosome.

GSK-3beta regulates proper mitotic spindle formation in cooperation with a component of the gamma-tubulin ring complex, GCP5.

Glycogen synthase kinase-3beta (GSK-3beta) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3beta in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the gamma-tubulin ring complex (gammaTuRC). GCP5 bound directly to GSK-3beta in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and gamma-tubulin in the gammaTuRC fraction of sucrose density gradients and disrupted gamma-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of gammaTuRC and the recruitment of gamma-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of gamma-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of gammaTuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3beta regulates the localization of gammaTuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.

Effects of bisphenol A on the development, growth, and sex ratio of the housefly Musca domestica.

The effects of bisphenol A (BPA) on the life cycle of the housefly Musca domestica were examined. The sex ratio of the imago shifted in favor of males when eggs and larvae were exposed to BPA in culture media at concentrations of 1,000 microg kg(-1) for five generations and 100 microg kg(-1) for seven generations. Notably, at an initial concentration of 100 microg kg(-1), BPA levels in the growth medium decreased 61% at 4 h after egg inoculation, and no BPA was detected after 24 h. Pupal weight increased upon exposure to 100 microg kg(-1) BPA but decreased after exposure to 1,000 microg kg(-1), suggesting highly variable concentration-dependent toxicity. Both the survival ratio of eggs to the third instar larval stage and the ratio of pupae to larvae decreased, indicating that BPA affected both eggs and larvae. A delay in the timing of emergence typically was observed in insects exposed to >100 microg kg(-1) BPA. Compared to the control group, juvenile hormones II and III levels were elevated significantly in larvae between days 4 and 7 in medium spiked with 100 microg kg(-1) BPA. These results suggest that exposure to environmentally relevant concentrations of BPA during the early stages of the housefly life cycle can result in various disorders (pupal weight and sex ratio) that may be a consequence of endocrine disruption.

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration.

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.

Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact.

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and beta-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and beta-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and beta-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of "E-cadherin-mediated cell-cell adhesion --> Rac1 activation --> actin-meshwork formation by IQGAP1 --> increasing E-cadherin-mediated cell-cell adhesion."

Production and immunogenic efficacy of botulinum tetravalent (A, B, E, F) toxoid.

A tetravalent (type A, B, E and F) toxoid was produced and its efficacy and safety were assessed. The toxoid preparation was inoculated from two to five times to 15 healthy adult volunteers participating in botulinum toxin research. The serum samples taken from the toxoid recipients were titrated for the antitoxin potencies by enzyme-linked immunosorbent assay (ELISA) and the neutralization test. The neutralizing and ELISA titers were too low to correlate each other. The mean neutralization titer of four recipients in 9 months after three doses of toxoid was about 0.1IU/ml for each of the four types, whereas, the one receiving five doses possessed a higher titer. Since the amount of the toxin handled in laboratory work is usually not so large, three or more doses of the present toxoid will bestow sufficient immunity on the workers participating in botulinum research. Nevertheless booster injections might be desirable to those at higher risk, handling the toxin of a high concentration.