A MAP1B-cortactin-Tks5 axis regulates TNBC invasion and tumorigenesis.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.

Wnt/ß-catenin-C-kit axis may play a role in adenoid cystic carcinoma prognostication.

Adenoid cystic carcinoma (ACC) is one of the most common malignant salivary gland tumors. ACC is composed of myoepithelial and epithelial neoplastic cells which grow slowly and have a tendency for neural invasion. The long term prognosis is still relatively poor. Although several gene abnormalities, such as fusions involving MYB or MYBL1 oncogenes and the transcription factor gene NFIB, and overexpression of KIT have been reported in ACC, their precise functions in the pathogenesis of ACC remain unclear. We recently demonstrated that the elevated expression of Semaphorin 3A (SEMA3A), specifically expressed in myoepithelial neoplastic cells, might function as a novel oncogene-related molecule to enhance cell proliferation through activated AKT signaling in 9/10 (90%) ACC cases. In the current study, the patient with ACC whose tumor was negative for SEMA3A in the previous study, revisited our hospital with late metastasis of ACC to the cervical lymph node eight years after surgical resection of the primary tumor. We characterized this recurrent ACC, and compared it with the primary ACC using immunohistochemical methods. In the recurrent ACC, the duct lining epithelial cells, not myoepithelial neoplastic cells, showed an elevated Ki-67 index and increased cell membrane expression of C-kit, along with the expression of phosphorylated ERK. Late metastasis ACC specimens were not positive for ß-catenin and lymphocyte enhancer binding factor 1 (LEF1), which were detected in the nuclei of perineural infiltrating cells in primary ACC cells. In addition, experiments with the GSK-3 inhibitor revealed that ß-catenin pathway suppressed not only KIT expression but also proliferation of ACC cells. Moreover, stem cell factor (SCF; also known as KIT ligand, KITL) induced ERK activation in ACC cells. These results suggest that inactivation of Wnt/ß-catenin signaling may promote C-kit-ERK signaling and cell proliferation of in metastatic ACC.

Arl4c is involved in tooth germ development through osteoblastic/ameloblastic differentiation.

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

Stepwise activation of p63 and the MEK/ERK pathway induces the expression of ARL4C to promote oral squamous cell carcinoma cell proliferation.

Carcinogenesis is a multistep process wherein cells accumulate multiple genetic alterations and progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through a preneoplastic lesion/benign tumor to cancer. Histologically, oral squamous cell carcinoma (OSCC) progresses in multiple ordered steps that begin with mucosal epithelial cell hyperplasia, which is followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore hypothesized that genetic alteration-mediated multistep carcinogenesis would be involved in the development of OSCC; however, the detailed molecular mechanisms are unknown. We clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data from a pathological specimen of OSCC (including a non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion). The expression of numerous genes and signal activation were altered in the development of OSCC. Among these, the p63 expression was increased and the MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion and in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or the MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions, than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were frequently merged in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and MEK/ERK-MAPK cooperatively induce the expression of ARL4C and cell growth in OSCC cells. These results suggest that the stepwise activation of p63 and MEK/ERK-MAPK contributes to OSCC tumor cell growth through regulation of ARL4C expression.

High Plasma tPAPAI-1C Levels May Be Related to a Poor Prognosis in Patients with Severe or Critical COVID-19: A Single-Center Retrospective Study.

Severe novel coronavirus disease 2019 (COVID-19) patients have a high incidence of thrombotic complications and mortality. The pathophysiology of coagulopathy involves fibrinolytic system impairment and vascular endothelial damage. This study examined coagulation and fibrinolytic markers as outcome predictors. In an observational study of 164 COVID-19 patients admitted to our emergency intensive care unit, hematological parameters on days 1, 3, 5, and 7 were retrospectively compared between survivors and nonsurvivors. Nonsurvivors had a higher APACHE II score, SOFA score, and age than survivors. Nonsurvivors also had a significantly lower platelet count and significantly higher plasmin/α2plasmin inhibitor complex (PIC), tissue plasminogen activator/plasminogen activator inhibitor-1 complex (tPAPAI-1C), D-dimer, and fibrin/fibrinogen degradation product (FDP) levels than survivors throughout the measurement period. The 7-day maximum or minimum values of the tPAPAI-1C, FDP, and D-dimer levels were significantly higher in nonsurvivors. A multivariate logistic regression analysis showed that the maximum tPAPAI-1C (OR = 1.034; 95% CI,1.014-1.061; p = 0.0041) was an independent factor affecting mortality, with an area under the curve (AUC) of 0.713 (optimum cut-off of 51 ng/mL; sensitivity, 69.2%; and specificity, 68.4%). COVID-19 patients with poor outcomes exhibit exacerbated coagulopathy with fibrinolysis inhibition and endothelial damage. Consequently, plasma tPAPAI-1C might be a useful predictor of the prognosis in patients with severe or critical COVID-19.

Identification of glioblastoma-specific antigens expressed in patient-derived tumor cells as candidate targets for chimeric antigen receptor T cell therapy.

Background: New therapies for glioblastoma (GBM) are urgently needed because the disease prognosis is poor. Chimeric antigen receptor (CAR)-T cell therapy that targets GBM-specific cell surface antigens is a promising therapeutic strategy. However, extensive transcriptome analyses have uncovered few GBM-specific target antigens. Methods: We established a library of monoclonal antibodies (mAbs) against a tumor cell line derived from a patient with GBM. We identified mAbs that reacted with tumor cell lines from patients with GBM but not with nonmalignant human brain cells. We then detected the antigens they recognized using expression cloning. CAR-T cells derived from a candidate mAb were generated and tested in vitro and in vivo. Results: We detected 507 mAbs that bound to tumor cell lines from patients with GBM. Among them, E61 and A13 reacted with tumor cell lines from most patients with GBM, but not with nonmalignant human brain cells. We found that B7-H3 was the antigen recognized but E61. CAR-T cells were established using the antigen-recognition domain of E61-secreted cytokines and exerted cytotoxicity in co-culture with tumor cells from patients with GBM. Conclusions: Cancer-specific targets for CAR-T cells were identified using a mAb library raised against primary GBM tumor cells from a patient. We identified a GBM-specific mAb and its antigen. More mAbs against various GBM samples and novel target antigens are expected to be identified using this strategy.

WT1 Trio Peptide-Based Cancer Vaccine for Rare Cancers Expressing Shared Target WT1.

No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers.

Wnt signaling promotes tooth germ development through YAP1-TGF-ß signaling.

Tooth germ development involves continuous and sequential steps with reciprocal interactions between odontogenic epithelium and the adjacent mesenchyme. Several growth factors, including Wnt, are essential for tooth germ development. Molecular mechanisms underlying Wnt/ß-catenin-regulated tooth germ development are poorly understood. In tooth germ rudiments culture, we recently demonstrated that Semaphorin 3A (Sema3A), an axonal guidance factor, stimulation reversed Wnt/ß-catenin signaling-dependent decreased cell proliferation but did not completely rescue the morphological anomalies of tooth germ, suggesting that an uncharacterized signaling pathway may be essential in Wnt/ß-catenin signaling-dependent tooth germ development. Herein, an enrichment analysis using DNA microarray data, which was obtained in our previous research, revealed that Wnt/ß-catenin signaling negatively regulates YAP1 and/or TGF-ß signalings. In odontogenic epithelial cells and tooth germ rudiments, Wnt/ß-catenin signaling activation reduced YAP1 expression, thereby suppressing YAP1 and TGF-ß signalings sequentially. Additionally, YAP1 signaling induced TGF-ß2 expression to promote TGF-ß signaling in the cells. Finally, Wnt/ß-catenin signaling-dependent disorganized tooth germ development, in which YAP1 signaling was suppressed, was reversed by TGF-ß stimulation. These results suggest that Wnt/ß-catenin signaling contributes to the tooth germ development through YAP1-TGF-ß signaling.

The Semaphorin 3A-AKT axis-mediated cell proliferation in salivary gland morphogenesis and adenoid cystic carcinoma pathogenesis.

We recently demonstrated that Semaphorin 3 A (Sema3A), the expression of which is negatively regulated by Wnt/ß-catenin signaling, promotes odontogenic epithelial cell proliferation, suggesting the involvement of Sema3A in tooth germ development. Salivary glands have a similar developmental process to tooth germ development, in which reciprocal interactions between the oral epithelium and adjacent mesenchyme proceeds via stimulation with several growth factors; however, the role of Sema3A in the development of salivary glands is unknown. There may thus be a common mechanism between epithelial morphogenesis and pathogenesis; however, the role of Sema3A in salivary gland tumors is also unclear. The current study investigated the involvement of Sema3A in submandibular gland (SMG) development and its expression in adenoid cystic carcinoma (ACC) specimens. Quantitative RT-PCR and immunohistochemical analyses revealed that Sema3A was expressed both in epithelium and in mesenchyme in the initial developmental stages of SMG and their expressions were decreased during the developmental processes. Loss-of-function experiments using an inhibitor revealed that Sema3A was required for AKT activation-mediated cellular growth and formation of cleft and bud in SMG rudiment culture. In addition, Wnt/ß-catenin signaling decreased the Sema3A expression in the rudiment culture. ACC arising from salivary glands frequently exhibits malignant potential. Immunohistochemical analyses of tissue specimens obtained from 10 ACC patients showed that Sema3A was hardly observed in non-tumor regions but was strongly expressed in tumor lesions, especially in myoepithelial neoplastic cells, at high frequencies where phosphorylated AKT expression was frequently detected. These results suggest that the Sema3A-AKT axis promotes cell growth, thereby contributing to morphogenesis and pathogenesis, at least in ACC, of salivary glands.

Clear cell squamous cell carcinoma of the tongue exhibits characteristics as an undifferentiated squamous cell carcinoma.

Clear cell squamous cell carcinoma (CCSCC), where cells show abundant clear cytoplasm, -is a variant of squamous cell carcinoma (SCC) and a rare entity in the oral cavity. The characteristics of CCSCC, especially in immunohistochemical features, remain unclear. We characterized a case of CCSCC arising from the oral mucosal epithelium of tongue, where the clear cell lesion accounted for a predominant portion of the tumor. This CCSCC, which was partially surrounded by conventional SCC, exhibited cellular atypia immunohistopathologically and histopathologically with a high Ki-67 index, increased number of mitotic figures and enlarged nuclei. Intravascular invasion of the carcinoma cells was also observed. Furthermore, the CCSCC recurred and metastasized to the cervical lymph nodes and both lungs three months after resection. Immunohistochemical analyses demonstrated decreased expression of p40 (an isoform of SCC marker p63), ADP-ribosylation factor (ARF)-like 4c (ARL4C), yes-associated protein (YAP) and 5-methylcytosine (5mC) in the CCSCC lesion compared with the surrounding SCC lesion, where the expression of ARL4C was upregulated compared with non-tumor region and YAP showed nuclear translocation. In addition, siRNA loss-of-function experiments revealed that p63 expression was required for ARL4C expression and DNA methylation was induced by p63 and YAP/transcriptional co-activator with PDZ-binding motif (TAZ) signaling in oral SCC cell lines. These results suggest that CCSCC, in which several markers of SCC-associated intracellular signaling pathways are downregulated, together with evidence of altered epigenetic regulation, is characterized as an undifferentiated SCC variant.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.

Effect of Tocilizumab Treatment on Patients with Coronavirus Disease 2019 and Bacteremia: A Retrospective Cohort Study.

INTRODUCTION: This study aimed to determine if tocilizumab treatment for coronavirus disease 2019 (COVID-19) increases bacteremia and suppresses fever and inflammatory reactants. METHODS: In this single-center, retrospective, observational study, all patients with COVID-19 admitted to our emergency intensive care unit from March 2020 to August 2021 were categorized into tocilizumab-treated and tocilizumab-naïve groups, and the incidence of bacteremia and other factors between the two groups were compared. Patients with bacteremia were further classified into tocilizumab-treated and tocilizumab-naïve groups to determine if fever and inflammatory reactants were suppressed. RESULTS: Overall, 144 patients were included in the study, 51 of whom received tocilizumab, which was administered on the day of admission. Further, of the 24 (16.7%) patients with bacteremia, 13 were in the tocilizumab-treated group. Results revealed a significant difference in the C-reactive protein level (p < 0.001) at the onset of bacteremia between the tocilizumab-treated group [median 0.42 mg/dL (0.27-0.44 mg/dL)] and the tocilizumab-naïve group [7.48 mg/dL (4.56-13.9 mg/dL)]. The median number of days from admission to onset of bacteremia was not significantly different between the tocilizumab-treated group [10 days (9-12 days)] and the tocilizumab-naïve group [9 days (7.5-11 days)] (p = 0.48). There was no significant difference in fever between the groups. Multivariate logistic analysis showed that tocilizumab treatment did not affect the probability of bacteremia. CONCLUSION: Treatment of patients with COVID-19 with tocilizumab does not increase the risk of bacteremia. Tocilizumab suppresses C-reactive protein levels but not fever. Therefore, careful monitoring of fever can reduce the risk of missed bacteremia.

Effect of Tocilizumab Treatment on Patients with Coronavirus Disease 2019 and Bacteremia: A Retrospective Cohort Study.

INTRODUCTION: This study aimed to determine if tocilizumab treatment for coronavirus disease 2019 (COVID-19) increases bacteremia and suppresses fever and inflammatory reactants. METHODS: In this single-center, retrospective, observational study, all patients with COVID-19 admitted to our emergency intensive care unit from March 2020 to August 2021 were categorized into tocilizumab-treated and tocilizumab-naïve groups, and the incidence of bacteremia and other factors between the two groups were compared. Patients with bacteremia were further classified into tocilizumab-treated and tocilizumab-naïve groups to determine if fever and inflammatory reactants were suppressed. RESULTS: Overall, 144 patients were included in the study, 51 of whom received tocilizumab, which was administered on the day of admission. Further, of the 24 (16.7%) patients with bacteremia, 13 were in the tocilizumab-treated group. Results revealed a significant difference in the C-reactive protein level (p < 0.001) at the onset of bacteremia between the tocilizumab-treated group [median 0.42 mg/dL (0.27-0.44 mg/dL)] and the tocilizumab-naïve group [7.48 mg/dL (4.56-13.9 mg/dL)]. The median number of days from admission to onset of bacteremia was not significantly different between the tocilizumab-treated group [10 days (9-12 days)] and the tocilizumab-naïve group [9 days (7.5-11 days)] (p = 0.48). There was no significant difference in fever between the groups. Multivariate logistic analysis showed that tocilizumab treatment did not affect the probability of bacteremia. CONCLUSION: Treatment of patients with COVID-19 with tocilizumab does not increase the risk of bacteremia. Tocilizumab suppresses C-reactive protein levels but not fever. Therefore, careful monitoring of fever can reduce the risk of missed bacteremia.

Identification of mouse helper epitopes for WT1-specific CD4+ T cells.

Helper T lymphocytes (HTLs) play a central role in cancer immunity because they can not only help the induction and proliferation of cytotoxic T lymphocytes (CTLs) but also their differentiation into cytotoxic CD4+ T cells and directly kill the target cells.This study describes the identification of three novel mouse Th epitope peptides, WT135-52, WT186-102 and WT1294-312, derived from WT1 protein, which is the most potent tumor-associated antigen. Compared to immunization with WT1 CTL peptide alone, immunization with the addition of these WT1-specific Th peptides strongly induced WT1-specific CTLs, continued to maintain them, and efficiently rejected the challenge of WT1-expressing tumor cells. Importantly, the majority of WT1-specific CTLs induced by the co-immunization with WT1 CTL and the WT1-specific Th peptides were CD44+CD62L- effector memory CD8+ T cells, which played a central role in tumor rejection. Establishment of mouse models suitable for the analysis of the detailed mechanism of these functions of HTLs is very important. These results clearly showed that WT1-specific HTLs perform an essential function in WT1-specific tumor immunity. Therefore, the WT1-specific Th peptides identified here should make a major contribution to elucidation of the mutual roles of WT1-specific CTLs and HTLs in cancer immunity in in vivo mouse models.

Successful Embolization of Collaterals from the Round Ligament Artery during Uterine Artery Embolization for Traumatic Uterine Leiomyoma Rupture: A Case Report.

We describe the case of a 48-year-old woman who presented with traumatic rupture of a giant leiomyoma and massive hemoperitoneum caused by slipping and falling in the bathroom. She was in shock on arrival, and resuscitation was performed. Contrast-enhanced computed tomography showed massive intra-abdominal hematoma and extravasation from the subserous leiomyoma. Uterine artery embolization was performed, but she went into shock again after 6 h. The second contrast-enhanced computed tomography revealed persistence of extravasation. During 2nd UAE, an angiogram revealed extravasation originating from left round ligament artery. After the embolization of the left round ligament and bilateral uterine arteries, the patient recovered from shock. Total abdominal hysterectomy was performed on day 2 of admission to prevent re-bleeding and infection, then she discharged on day 19 of admission.

YAP signaling induces PIEZO1 to promote oral squamous cell carcinoma cell proliferation.

Most cancer cells are exposed to altered extracellular environments, such as an increase in extracellular matrix (ECM) stiffness and soluble signals consisting of growth factors and cytokines. It is therefore conceivable that changes in tumor extracellular environments affect tumor cell behavior. The Hippo pathway reportedly responds to the extracellular environment and regulates the nuclear localization of the transcription co-activator, yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ). Inactivation of the Hippo pathway with nuclear translocation of YAP/TAZ stimulates cell proliferation. Its pathway also regulates gene expression, but the precise molecule(s) meditating the cell-proliferating effect of YAP signaling on oral squamous cell carcinoma (OSCC) is unclear. First, we examined the effects of YAP signaling on OSCC tumorigenesis. Loss-of-function experiments using siRNA or an inhibitor, and immunohistochemical analyses of tissue specimens obtained from OSCC patients demonstrated that YAP signaling was involved in OSCC cell proliferation. Second, we identified Piezo-type mechanosensitive ion channel component 1 (PIEZO1), a Ca2+ channel, as a transcriptional target of YAP signaling and showed that elevated PIEZO1 was required for PIEZO1 agonist-dependent Ca2+ entry and cell proliferation in OSCC cells. Experiments using three-dimensional and suspension culture revealed that PIEZO1 was involved in OSCC cellular growth. Finally, YAP overexpression in the nucleus and/or cytoplasm was immunohistochemically detected in tumor lesions with frequent expression of both PIEZO1 and Ki-67, but not in non-tumor regions of OSCC specimens. These results suggest that the YAP/PIEZO1 axis promotes OSCC cell growth. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

Extracellular vesicles derived from inflamed murine colorectal tissue induce fibroblast proliferation via epidermal growth factor receptor.

Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract. Although IBDs increase the risk of colitis-associated colon cancer, the underlying mechanisms are not fully understood. Extracellular vesicles (EVs) are lipid-bound sacs that transport proteins, RNA, and lipids between cells and are key mediators of cellular communication in both physiological and pathological settings. EVs have been implicated in many cancer hallmarks, including uncontrolled tumor growth and metastasis. In this study, we investigated the effects of colon-derived EVs on the proliferation of fibroblasts. We used comparative proteomics to characterize protein profiles of colorectal EVs isolated from healthy mice (Con-EVs) and those with dextran sulfate sodium-induced colitis (IBD-EVs). The results showed that 109 proteins were upregulated in IBD-EVs. Notably, expression of epidermal growth factor receptor (EGFR), which plays important roles in cell proliferation and development, was increased in IBD-EVs. We then examined the effect of EVs on murine NIH3T3 fibroblasts and found that IBD-EVs significantly promoted cell proliferation in EGFR- and ERK-dependent manner. Our findings suggest that inflamed colon-derived EVs promote tumor development thorough activation of fibroblasts.

Enhanced immune reaction resulting from co-vaccination of WT1 helper peptide assessed on PET-CT.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells.

Periodontal ligament (PDL) stem cells (PDLSCs) have been reported as a useful cell source for periodontal tissue regeneration. However, one of the issues is the difficulty of obtaining a sufficient number of PDLSCs for clinical application because very few PDLSCs can be isolated from PDL tissue of donors. Therefore, we aimed to identify a specific factor that converts human PDL cells into stem-like cells. In this study, microarray analysis comparing the gene profiles of human PDLSC lines (2-14 and 2-23) with those of a cell line with a low differentiation potential (2-52) identified the imprinted gene mesoderm-specific transcript (MEST). MEST was expressed in the cytoplasm of 2-23 cells. Knockdown of MEST by siRNA in 2-23 cells inhibited the expression of stem cell markers, such as CD105, CD146, p75NTR, N-cadherin, and NANOG; the proliferative potential; and multidifferentiation capacity for osteoblasts, adipocytes, and chondrocytes. On the other hand, overexpression of MEST in 2-52 cells enhanced the expression of stem cell markers and PDL-related markers and the multidifferentiation capacity. In addition, MEST-overexpressing 2-52 cells exhibited a change in morphology from a spindle shape to a stem cell-like round shape that was similar to 2-14 and 2-23 cell morphologies. These results suggest that MEST plays a critical role in the maintenance of stemness in PDLSCs and converts PDL cells into PDLSC-like cells. Therefore, this study indicates that MEST may be a therapeutic factor for periodontal tissue regeneration by inducing PDLSCs.