MiR-21 Regulates Growth and Migration of Cervical Cancer Cells by RECK Signaling Pathway.

Expression of miR-21 has been found to be altered in almost all types of cancers, and it has been classified as an oncogenic microRNA. In addition, the expression of tumor suppressor gene RECK is associated with miR-21 overexpression in high-grade cervical lesions. In the present study, we analyze the role of miR-21 in RECK gene regulation in cervical cancer cells. To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression using siRNAs. We analyzed the expression of miR-21 and RECK, as well as functional effects on cell proliferation and migration. We found that in cervical cancer cells, there was an inverse correlation between miR-21 expression and RECK mRNA and protein expression. SiRNAs to miR-21 increased luciferase reporter activity in construct plasmids containing the RECK-3'-UTR microRNA response elements MRE21-1, MRE21-2, and MRE21-3. The role of miR-21 in cell proliferation was also analyzed, and cancer cells transfected with siRNAs exhibited a markedly reduced cell proliferation and migration. Our findings indicate that miR-21 post-transcriptionally down-regulates the expression of RECK to promote cell proliferation and cell migration inhibition in cervical cancer cell survival. Therefore, miR-21 and RECK may be potential therapeutic targets in gene therapy for cervical cancer.

Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET.

BACKGROUND: Brain metastasis (BM) is common among cases of advanced non-small cell lung cancer (NSCLC) and is the leading cause of death for these patients. Mesothelin (MSLN), a tumor-associated antigen expressed in many solid tumors, has been reported to be involved in the progression of multiple tumors. However, its potential involvement in BM of NSCLC and the underlying mechanism remain unknown. METHODS: The expression of MSLN was validated in clinical tissue and serum samples using immunohistochemistry and enzyme-linked immunosorbent assay. The ability of NSCLC cells to penetrate the blood-brain barrier (BBB) was examined using an in vitro Transwell model and an ex vivo multi-organ microfluidic bionic chip. Immunofluorescence staining and western blotting were used to detect the disruption of tight junctions. In vivo BBB leakiness assay was performed to assess the barrier integrity. MET expression and activation was detected by western blotting. The therapeutic efficacy of drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) on BM was evaluated in animal studies. RESULTS: MSLN expression was significantly elevated in both serum and tumor tissue samples from NSCLC patients with BM and correlated with a poor clinical prognosis. MSLN significantly enhanced the brain metastatic abilities of NSCLC cells, especially BBB extravasation. Mechanistically, MSLN facilitated the expression and activation of MET through the c-Jun N-terminal kinase (JNK) signaling pathway, which allowed tumor cells to disrupt tight junctions and the integrity of the BBB and thereby penetrate the barrier. Drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) effectively blocked the development of BM and prolonged the survival of mice. CONCLUSIONS: Our results demonstrate that MSLN plays a critical role in BM of NSCLC by modulating the JNK/MET signaling network and thus, provides a potential novel therapeutic target for preventing BM in NSCLC patients.

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown. METHODS: The expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols. RESULTS: MSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells. CONCLUSIONS: Collectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

Designing and validating of a questionnaire measuring perceived self-care ability (PSCA) in chronic stroke patients at home.

BACKGROUND: Patients with a stroke often cannot care for themselves after hospital discharge. Assessment of their self-care ability is the first step in planning post-discharge home care. This study aimed to design and validate a measure of perceived self-care ability (PSCA) in stroke patients. METHODS: A sequential-exploratory mixed method was conducted in Tehran, Iran, in 2020-2021. The qualitative phase involved in-depth semi-structured interviews with 12 participants. Transcripts were content analyzed. The results guided the development of 81 items. psychometric properties such as face validity (Impact Score > 1.5), content validity ratio (CVR > 0.63), content validity index (Item Content Validity Index: ICVI > 0.78, Scale Content Validity Index/Average: SCVI/Ave > 0.8) and Kappa value (Kappa > 0.7), internal consistency (Cronbach's alpha > 0.7), relative reliability (ICC: inter class correlation coefficient), absolute reliability (Standard Error of Measurement: SEM and Minimal Detectable Changes: MDC), convergent validity (Correlation Coefficient between 0.4-0.7), interpretability, responsiveness, feasibility, and ceiling and floor effects were assessed. RESULTS: Content analysis of the qualitative interviews yielded 5 major categories and 9 subcategories that reflected "Perceptual stability", "Cognitive fluctuations", "Sensory, Motor and Physical health"," The subjective nature" and "The dynamic nature" of PSCA. Results of face and content validity reduced the number of items to 32, capturing three dimensions of PSCA in chronic stroke patients; these dimensions included perceptual ability, threatened health status, and sensory, motor, and cognitive ability. The findings supported the reliability and validity of the measure. CONCLUSIONS: The PSCA questionnaire was developed and validated within the Iranian culture. It is useful in assessing the self-care of patients with stroke and in informing practice.

Lymphatic endothelial cell-mediated accumulation of CD177+Treg cells suppresses antitumor immunity in human esophageal squamous cell carcinoma.

Regulatory T (Treg) cells are critical in shaping an immunosuppressive microenvironment to favor tumor progression and resistance to therapies. However, the heterogeneity and function of Treg cells in esophageal squamous cell carcinoma (ESCC) remain underexplored. We identified CD177 as a tumor-infiltrating Treg cell marker in ESCC. Interestingly, expression levels of CD177 and PD-1 were mutually exclusive in tumor Treg cells. CD177+ Treg cells expressed high levels of IL35, in association with CD8+ T cell exhaustion, whereas PD-1+ Treg cells expressed high levels of IL10. Pan-cancer analysis revealed that CD177+ Treg cells display increased clonal expansion compared to PD-1+ and double-negative (DN) Treg cells, and CD177+ and PD-1+ Treg cells develop from the same DN Treg cell origin. Importantly, we found CD177+ Treg cell infiltration to be associated with poor overall survival and poor response to anti-PD-1 immunotherapy plus chemotherapy in ESCC patients. Finally, we found that lymphatic endothelial cells are associated with CD177+ Treg cell accumulation in ESCC tumors, which are also decreased after anti-PD-1 immunotherapy plus chemotherapy. Our work identifies CD177+ Treg cell as a tumor-specific Treg cell subset and highlights their potential value as a prognostic marker of survival and response to immunotherapy and a therapeutic target in ESCC.

The Risk Genes for Neuropsychiatric Disorders negr1 and opcml Are Expressed throughout Zebrafish Brain Development.

The immunoglobulin LAMP/OBCAM/NTM (IgLON) family of cell adhesion molecules comprises five members known for their involvement in establishing neural circuit connectivity, fine-tuning, and maintenance. Mutations in IgLON genes result in alterations in these processes and can lead to neuropsychiatric disorders. The two IgLON family members NEGR1 and OPCML share common links with several of them, such as schizophrenia, autism, and major depressive disorder. However, the onset and the underlying molecular mechanisms have remained largely unresolved, hampering progress in developing therapies. NEGR1 and OPCML are evolutionarily conserved in teleosts like the zebrafish (Danio rerio), which is excellently suited for disease modelling and large-scale screening for disease-ameliorating compounds. To explore the potential applicability of zebrafish for extending our knowledge on NEGR1- and OPCML-linked disorders and to develop new therapeutic strategies, we investigated the spatio-temporal expression of the two genes during early stages of development. negr1 and opcml are expressed maternally and subsequently in partially distinct domains of conserved brain regions. Other areas of expression in zebrafish have not been reported in mammals to date. Our results indicate that NEGR1 and OPCML may play roles in neural circuit development and function at stages earlier than previously anticipated. A detailed functional analysis of the two genes based on our findings could contribute to understanding the mechanistic basis of related psychiatric disorders.

Differential Gene Regulatory Network Analysis between Azacitidine-Sensitive and -Resistant Cell Lines.

Azacitidine, a DNA methylation inhibitor, is employed for the treatment of acute myeloid leukemia (AML). However, drug resistance remains a major challenge for effective azacitidine chemotherapy, though several studies have attempted to uncover the mechanisms of azacitidine resistance. With the aim to identify the mechanisms underlying acquired azacitidine resistance in cancer cell lines, we developed a computational strategy that can identify differentially regulated gene networks between drug-sensitive and -resistant cell lines by extending the existing method, differentially coexpressed gene sets (DiffCoEx). The technique specifically focuses on cell line-specific gene network analysis. We applied our method to gene networks specific to azacitidine sensitivity and identified differentially regulated gene networks between azacitidine-sensitive and -resistant cell lines. The molecular interplay between the metallothionein gene family, C19orf33, ELF3, GRB7, IL18, NRN1, and RBM47 were identified as differentially regulated gene network in drug resistant cell lines. The biological mechanisms associated with azacitidine and AML for the markers in the identified networks were verified through the literature. Our results suggest that controlling the identified genes (e.g., the metallothionein gene family) and "cellular response"-related pathways ("cellular response to zinc ion", "cellular response to copper ion", and "cellular response to cadmium ion", where the enriched functional-related genes are MT2A, MT1F, MT1G, and MT1E) may provide crucial clues to address azacitidine resistance in patients with AML. We expect that our strategy will be a useful tool to uncover patient-specific molecular interplay that provides crucial clues for precision medicine in not only gastric cancer but also complex diseases.

FUT8-mediated aberrant N-glycosylation of SEMA7A promotes head and neck squamous cell carcinoma progression.

SEMA7A belongs to the Semaphorin family and is involved in the oncogenesis and tumor progression. Aberrant glycosylation has been intricately linked with immune escape and tumor growth. SEMA7A is a highly glycosylated protein with five glycosylated sites. The underlying mechanisms of SEMA7A glycosylation and its contribution to immunosuppression and tumorigenesis are unclear. Here, we identify overexpression and aberrant N-glycosylation of SEMA7A in head and neck squamous cell carcinoma, and elucidate fucosyltransferase FUT8 catalyzes aberrant core fucosylation in SEMA7A at N-linked oligosaccharides (Asn 105, 157, 258, 330, and 602) via a direct protein‒protein interaction. A glycosylated statue of SEMA7A is necessary for its intra-cellular trafficking from the cytoplasm to the cytomembrane. Cytokine EGF triggers SEMA7A N-glycosylation through increasing the binding affinity of SEMA7A toward FUT8, whereas TGF-ß1 promotes abnormal glycosylation of SEMA7A via induction of epithelial-mesenchymal transition. Aberrant N-glycosylation of SEMA7A leads to the differentiation of CD8+ T cells along a trajectory toward an exhausted state, thus shaping an immunosuppressive microenvironment and being resistant immunogenic cell death. Deglycosylation of SEMA7A significantly improves the clinical outcome of EGFR-targeted and anti-PD-L1-based immunotherapy. Finally, we also define RBM4, a splice regulator, as a downstream effector of glycosylated SEMA7A and a pivotal mediator of PD-L1 alternative splicing. These findings suggest that targeting FUT8-SEMA7A axis might be a promising strategy for improving antitumor responses in head and neck squamous cell carcinoma patients.

Association of single nucleotide polymorphisms in ITLN1 gene with ischemic stroke risk in Xi'an population, Shaanxi province.

Background: Ischemic stroke (IS) is the main cause of death and adult disability. However, the pathogenesis of this complicated disease is unknown. The present study aimed to assess the relationship between ITLN1 single nucleotide polymorphisms (SNPs) and the susceptibility to IS in Xi'an population, Shaanxi province. Methods: In this study, we designed polymerase chain reaction (PCR) primers located at -3,308 bp upstream of the transcription initiation site within promoter region of the ITLN1 gene. The target fragment was amplified by PCR and identified by agarose gel electrophoresis. Sanger sequencing was then performed in the samples extracted from a cohort comprising 1,272 participants (636 controls and 636 cases), and the obtained sequences were compared with the reference sequences available on the National Center for Biotechnology Information (NCBI) website to detect SNPs in the ITLN1 gene promoter region. Logistic regression analysis was employed to assess the relationship between ITLN1 polymorphisms and IS risk, with adjustments for age and gender. Significant positive results were tested by false-positive report probability (FPRP) and false discovery rate (FDR). The interaction among noteworthy SNPs and their predictive relationship with IS risk were explored using the Multi-Factor Dimensionality Reduction (MDR) software. Results: The results of Sanger sequencing were compared with the reference sequences on the NCBI website, and we found 14 SNPs in ITLN1 gene promoter satisfied Hardy-Weinberg equilibrium (HWE). Logistic regression analysis showed that ITLN1 was associated with a decreased risk of IS (rs6427553: Homozygous C/C: adjusted OR: 0.69, 95% CI [0.48-0.97]; Log-additive: adjusted OR: 0.83, 95% CI [0.70-0.98]; rs7411035: Homozygous G/G: adjusted OR: 0.66, 95% CI [0.47-0.94]; Dominant G/T-G/G: adjusted OR: 0.78, 95% CI [0.62-0.98]; Log-additive: adjusted OR: 0.81, 95% CI [0.69-0.96]; rs4656958: Heterozygous G/A: adjusted OR: 0.74, 95% CI [0.59-0.94]; Homozygous A/A: adjusted OR: 0.51, 95% CI [0.31-0.84]; Dominant G/A-A/A: adjusted OR: 0.71, 95% CI [0.57-0.89]; Recessive A/A: adjusted OR: 0.59, 95% CI [0.36-0.96]; Log-additive: adjusted OR: 0.73, 95% CI [0.61-0.88]), especially in people aged less than 60 years and males. Conclusions: In short, our study revealed a correlation between ITLN1 variants (rs6427553, rs7411035 and rs4656958) and IS risk in Xi'an population, Shaanxi province, laying a foundation for ITLN1 gene as a potential biomarker for predicting susceptibility to IS.

Omentin associates with serum metabolite profiles indicating lower diabetes risk: KORA F4 Study.

INTRODUCTION: Circulating omentin levels have been positively associated with insulin sensitivity. Although a role for adiponectin in this relationship has been suggested, underlying mechanisms remain elusive. In order to reveal the relationship between omentin and systemic metabolism, this study aimed to investigate associations of serum concentrations of omentin and metabolites. RESEARCH DESIGN AND METHODS: This study is based on 1124 participants aged 61-82 years from the population-based KORA (Cooperative Health Research in the Region of Augsburg) F4 Study, for whom both serum omentin levels and metabolite concentration profiles were available. Associations were assessed with five multivariable regression models, which were stepwise adjusted for multiple potential confounders, including age, sex, body mass index, waist-to-hip ratio, lifestyle markers (physical activity, smoking behavior and alcohol consumption), serum adiponectin levels, high-density lipoprotein cholesterol, use of lipid-lowering or anti-inflammatory medication, history of myocardial infarction and stroke, homeostasis model assessment 2 of insulin resistance, diabetes status, and use of oral glucose-lowering medication and insulin. RESULTS: Omentin levels significantly associated with multiple metabolites including amino acids, acylcarnitines, and lipids (eg, sphingomyelins and phosphatidylcholines (PCs)). Positive associations for several PCs, such as diacyl (PC aa C32:1) and alkyl-alkyl (PC ae C32:2), were significant in models 1-4, whereas those with hydroxytetradecenoylcarnitine (C14:1-OH) were significant in all five models. Omentin concentrations were negatively associated with several metabolite ratios, such as the valine-to-PC ae C32:2 and the serine-to-PC ae C32:2 ratios in most models. CONCLUSIONS: Our results suggest that omentin may influence insulin sensitivity and diabetes risk by changing systemic lipid metabolism, but further mechanistic studies investigating effects of omentin on metabolism of insulin-sensitive tissues are needed.

Delivery of a BET protein degrader via a CEACAM6-targeted antibody-drug conjugate inhibits tumour growth in pancreatic cancer models.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

Matrix Metalloproteinases in the Periodontium-Vital in Tissue Turnover and Unfortunate in Periodontitis.

The extracellular matrix (ECM) is a complex non-cellular three-dimensional macromolecular network present within all tissues and organs, forming the foundation on which cells sit, and composed of proteins (such as collagen), glycosaminoglycans, proteoglycans, minerals, and water. The ECM provides a fundamental framework for the cellular constituents of tissue and biochemical support to surrounding cells. The ECM is a highly dynamic structure that is constantly being remodeled. Matrix metalloproteinases (MMPs) are among the most important proteolytic enzymes of the ECM and are capable of degrading all ECM molecules. MMPs play a relevant role in physiological as well as pathological processes; MMPs participate in embryogenesis, morphogenesis, wound healing, and tissue remodeling, and therefore, their impaired activity may result in several problems. MMP activity is also associated with chronic inflammation, tissue breakdown, fibrosis, and cancer invasion and metastasis. The periodontium is a unique anatomical site, composed of a variety of connective tissues, created by the ECM. During periodontitis, a chronic inflammation affecting the periodontium, increased presence and activity of MMPs is observed, resulting in irreversible losses of periodontal tissues. MMP expression and activity may be controlled in various ways, one of which is the inhibition of their activity by an endogenous group of tissue inhibitors of metalloproteinases (TIMPs), as well as reversion-inducing cysteine-rich protein with Kazal motifs (RECK).

The CD177 c.1291A Allele Leads to a Loss of Membrane Expression and Mimics a CD177-Null Phenotype.

CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3-5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described CD177 c.1291G>A variant on CD177 expression. Recombinant CD177 c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The CD177 c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that CD177 c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of CD177 c.1291A leads to the production of an unstable CD177 protein and an apparent "CD177-null" phenotype.

Exploring the molecular and immune-landscape of lung cancer associated with cystic airspaces.

To explore the molecular biological characteristics of lung cancer associated with cystic airspaces (LCCA) and its potential roles on prognosis. A total of 165 LCCAs and 201 non-LCCAs were enrolled in this study. Bulk RNA sequencing was implemented in eight LCCAs and nine non-LCCAs to explore the differentially expressed genes. TCGA data were used to analyze LCCA-specific genes that associated with overall survival (OS). The median age was 60 (IQR 53 to 65) years in LCCA cohort. We found LCCA were predominant in men and had less visceral pleura invasion (VPI) or lympho-vascular invasion (LVI). Moreover, LCCA presented with higher histological heterogeneity. Kaplan-Meier analysis showed that patients of age more than 60 and positive VPI had significantly less PFS in LCCA. Cox regression suggested that LCCA, micropapillary subtype proportion and VPI were the independent risk factors for PFS. LCCA had up-regulated pathways associated with EMT, angiogenesis and cell migration. In addition, LCCA displayed higher levels of immunosuppressor infiltration (M2 macrophages, CAFs and MDSCs) and distinct cell death and metabolic patterns. BCR/TCR repertoire analysis revealed less BCR richness, clonality and high-abundance shared clonotypes in LCCA. Finally, Cox regression analysis identified that four cystic-specific genes, KCNK3, NRN1, PARVB and TRHDE-AS1, were associated with OS of lung adenocarcinoma (LUAD). And cystic-specific risk scores (CSRSs) were calculated to construct a nomogram, which performance well. Our study for the first time indicated significantly distinct molecular biological and immune characteristics between LCCA and non-LCCA, which provide complementary prognostic values in early-stage non-small cell lung cancer (NSCLC).

ART1 knockdown decreases the IL-6-induced proliferation of colorectal cancer cells.

Colorectal cancer (CRC) is a worldwide health concern. Chronic inflammation is a risk factor for CRC, and interleukin-6 (IL-6) plays a pivotal role in this process. Arginine-specific mono-ADP-ribosyltransferase-1 (ART1) positively regulates inflammatory cytokines. ART1 knockdown reduces the level of glycoprotein 130 (gp130), a key transducer in the IL-6 signalling pathway. However, the relationship between ART1 and IL-6 and the resulting effects on IL-6-induced proliferation in CRC cells remain unclear. The aims of this study were to investigate the effects of ART1 knockdown on IL-6-induced cell proliferation in vitro and use an in vivo murine model to observe the growth of transplanted tumours. The results showed that compared with the control, ART1-sh cancer cells induced by IL-6 exhibited reduced viability, a lower rate of colony formation, less DNA synthesis, decreased protein levels of gp130, c-Myc, cyclin D1, Bcl-xL, and a reduced p-STAT3/STAT3 ratio (P < 0.05). Moreover, mice transplanted with ART1-sh CT26 cells that had high levels of IL-6 displayed tumours with smaller volumes (P < 0.05). ART1 and gp130 were colocalized in CT26, LoVo and HCT116 cells, and their expression was positively correlated in human CRC tissues. Overall, ART1 may serve as a promising regulatory factor for IL-6 signalling and a potential therapeutic target for human CRC.

CD109 identified in circulating proteomics mitigates postoperative recurrence in chronic rhinosinusitis with nasal polyps by suppressing TGF-ß1-induced epithelial-mesenchymal transition.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common inflammatory disorder with a high rate of recurrence. This study aimed to explore biomarkers for identifying patients with recurrent CRSwNP (rCRSwNP). METHODS: We recruited two independent cohorts. In the discovery cohort, rCRSwNP patients and non-recurrent CRSwNP (non-rCRSwNP) patients were recruited, and the serum proteomic profile was characterized. The top 5 upregulated and downregulated proteins were confirmed in the validation cohort by ELISA, WB, and qRT-PCR, and their predictive values for postoperative recurrence were assessed. In vitro, human nasal epithelial cells (HNEpCs) were employed to assess the ability of candidate proteins to induce epithelial-mesenchymal transition (EMT). RESULTS: Serum proteomics identified 53 different proteins, including 30 increased and 23 decreased, between the rCRSwNP and non-rCRSwNP groups. ELISA results revealed that serum levels of CD163 and TGF-ß1 were elevated, CD109 and PRDX2 were decreased in the rCRSwNP group compared to the non-rCRSwNP group, and serum CD163, TGF-ß1, and CD109 levels were proved to be associated with the risk of postoperative recurrence. In addition, qRT-PCR and WB revealed that tissue CD163, TGF-ß1, and CD109 expressions in rCRSwNP patients were enhanced compared to those non-rCRSwNP patients. Kaplan-Meier analysis showed that increased CD163 and TGF-ß1 expression and decreased CD109 expression are associated with the risk of recurrence in CRSwNP patients. Receiver operating characteristic curves showed that TGF-ß1 and CD109 had superior diagnostic performances for rCRSwNP. In vitro experiments showed that TGF-ß1 promoted EMT in HNEpCs, and overexpression of CD109 reversed this effect. Functional recovery experiments confirmed that CD109 could attenuate EMT in HNEpCs by inhibiting the TGF-ß1/Smad signaling pathway, attenuating EMT in epithelial cells. CONCLUSION: Our data suggested that TGF-ß1 and CD109 might serve as promising predictors of rCRSwNP. The TGF-ß1/Smad pathway was implicated in fostering EMT in epithelial cells, particularly those exhibiting low expression of CD109. Consequently, the absence of CD109 expression in epithelial cells could be a potential mechanism underlying rCRSwNP.

Phenotypic screen of sixty-eight colorectal cancer cell lines identifies CEACAM6 and CEACAM5 as markers of acid resistance.

Elevated cancer metabolism releases lactic acid and CO2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as "acid-sensitive"/"acid-resistant" versus "low metabolic acid production"/"high metabolic acid production." Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12. CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors.

Comparison of real-time quantitative PCR and two digital PCR platforms to detect copy number variation in FCGR3B.

The importance of structural genetic variants, such as copy number variations (CNVs), in modulating human disease is being increasingly recognized. Several clinical conditions require investigation of human neutrophil antigen (HNA-1), which is encoded by the Fc gamma receptor IIIb gene (FCGR3B), including suspicion of neutropenia, infections, and proactive testing of blood component donors to reduce the potential risk in transfusion. In this study, we compared real-time quantitative polymerase chain reaction (qPCR) with two digital PCR (dPCR) platforms, namely droplet digital PCR and an array-based platform, to determine copy numbers (CNs) in FCGR3B. We initially tested 400 anonymous blood donors with qPCR using a commercially available TaqMan probe assay (Applied Biosystems) on a Quant Studio 12 Flex. CNs was determined for all 400 tested individuals with CNs ranging from zero to four. Zero copies were detected in 0.2% (1/400), one copy was detected in 3.8% (15/400), two copies were detected in 87.8% (351/400), three copies were detected in 8.0% (32/400), and four copies were detected in 0.2% (1/400) of tested individuals. From this cohort, we selected 32 donors with CNs from zero to four for analyses with Digital Real-Time PCR (dPCR) using Lab on an array (LOAA) on an On-Point analyzer from Optolane Technologies Inc. and the Droplet Digital PCR (ddPCR) platform from Bio-Rad Laboratories. We compared the obtained CNs of FCGR3B on the three platforms and found full concordance between the CNs obtained. We therefore conclude that all three platforms can be used for quantification of CNs for FCGR3B, and although dPCR has some advantages over qPCR, it was not necessary for reliably estimating CNs of the FCGR3B gene.

CD109 Attenuates Bleomycin-induced Pulmonary Fibrosis by Inhibiting TGF-ß Signaling.

Pulmonary fibrosis is a fatal condition characterized by fibroblast and myofibroblast proliferation and collagen deposition. TGF-ß plays a pivotal role in the development of pulmonary fibrosis. Therefore, modulation of TGF-ß signaling is a promising therapeutic strategy for treating pulmonary fibrosis. To date, however, interventions targeting TGF-ß have not shown consistent efficacy. CD109 is a GPI-anchored glycoprotein that binds to TGF-ß receptor I and negatively regulates TGF-ß signaling. However, no studies have examined the role and therapeutic potential of CD109 in pulmonary fibrosis. The purpose of this study was to determine the role and therapeutic value of CD109 in bleomycin-induced pulmonary fibrosis. CD109-transgenic mice overexpressing CD109 exhibited significantly attenuated pulmonary fibrosis, preserved lung function, and reduced lung fibroblasts and myofibroblasts compared with wild-type (WT) mice. CD109-/- mice exhibited pulmonary fibrosis comparable to WT mice. CD109 expression was induced in variety types of cells, including lung fibroblasts and macrophages, upon bleomycin exposure. Recombinant CD109 protein inhibited TGF-ß signaling and significantly decreased ACTA2 expression in human fetal lung fibroblast cells in vitro. Administration of recombinant CD109 protein markedly reduced pulmonary fibrosis in bleomycin-treated WT mice in vivo. Our results suggest that CD109 is not essential for the development of pulmonary fibrosis, but excess CD109 protein can inhibit pulmonary fibrosis development, possibly through suppression of TGF-ß signaling. CD109 is a novel therapeutic candidate for treating pulmonary fibrosis.

Identification of CD160-TM as a tumor target on triple negative breast cancers: possible therapeutic applications.

BACKGROUND: Despite major therapeutic advances, triple-negative breast cancer (TNBC) still presents a worth prognosis than hormone receptors-positive breast cancers. One major issue relies in the molecular and mutational heterogeneity of TNBC subtypes that is reinforced by the absence of reliable tumor-antigen that could serve as a specific target to further promote efficient tumor cell recognition and depletion. CD160 is a receptor mainly expressed by NK lymphocytes and presenting two isoforms, namely the GPI-anchored form (CD160-GPI) and the transmembrane isoform (CD160-TM). While CD160-GPI is constitutively expressed on resting cells and involved in the generation of NK cells' cytotoxic activity, CD160-TM is neo-synthesized upon activation and promotes the amplification of NK cells' killing ability. METHODS: CD160 expression was assessed by immunohistochemistry (IHC) and flow cytometry on TNBC patient biopsies or cell lines, respectively. Antibody (Ab)-mediated tumor depletion was tested in vitro by performing antibody-dependent cell cytotoxicity (ADCC) and phagocytosis (ADCP) assays, and in vivo on a TNBC mouse model. RESULTS: Preliminary data obtained by IHC on TNBC patients' tumor biopsies revealed an unconventional expression of CD160 by TNBC tumor cells. By using a specific but conformation-dependent anti-CD160-TM Ab, we established that CD160-TM, but not CD160-GPI, was expressed by TNBC tumor cells. A conformation-independent anti-CD160-TM mAb (22B12; muIgG2a isotype) was generated and selected according to pre-defined specificity and functional criterions. In vitro functional assays demonstrated that ADCC and ADCP could be induced in the presence of 22B12, resulting in TNBC cell line apoptosis. The ability of 22B12 to exert an in vivo anti-tumor activity was also demonstrated on a TNBC murine model. CONCLUSIONS: Our data identify CD160-TM as a tumor marker for TNBC and provide a rational for the use of anti-CD160-TM antibodies as therapeutic tools in this tumor context.