Integrin Research Articles

Abstract 4266: Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation

Abstract Lung cancer is the leading cause of cancer related deaths worldwide, with the most prevalent subtype being non-small cell lung cancer (NSCLC). As only a subset of patients exhibits durable clinical responses to current treatments, including targeted therapy and immunotherapy, new treatment options are needed. Integrins (ITGs) are a diverse class of heterodimeric, transmembrane receptors that bind extracellularly to the extracellular matrix (ECM) and intracellularly to the cytoskeleton to integrate signals bi-directionally between the extracellular environment and cell interior. One of 18 α subunits is paired with one of 6 β subunits to create the 24 known αβ ITGs in mammals. ITGs are expressed at low levels in normal tissue and can be upregulated in cancer cells in response to stress signals in the tumor microenvironment (TME). Altered expression of ITGs is associated with enhanced cell proliferation, metastasis, and immune evasion. Targeting of the diverse ITG family members by inhibitors not specific to individual isoforms has obscured understanding of potentially distinct functions of each heterodimer. Through data mining of a genome wide CRISPR screening publicly available at the DepMap portal, we have identified a novel cancer dependency to ITG αvβ5 in approximately 40% of NSCLC cell lines. Using small molecule ITG αv inhibitors, we have mirrored sensitivity of NSCLC cell lines to their ITG αvβ5 CRISPR scores, suggesting that these inhibitors function specifically through ITG αvβ5 in cell lines. RNA sequencing analysis following αvβ5 inhibition in human NSCLC cell lines revealed downregulation of YAP and E2F1 target genes with alterations in gene ontologies associated with cell cycling and cell death in sensitive, but not resistant, lines. This is consistent with the known function of YAP driving gene transcription promoting cell proliferation and survival, and of E2F1 being implicated in regulating the proliferation and self-renewal of cancer stem cell (CSC). Additional changes to immune processes, specifically perturbations to interferon (IFN) pathways, were identified in response to ITG αvβ5 inhibition, suggesting that ITG αvβ5 may play a role in host anti-tumor immune responses. Pharmacological inhibition of ITG αvβ5 also led to a reduction of tumor growth in NSCLC murine models accompanied with increased CD8 T cell activation. These findings support ITG αvβ5 as a unique target of cancer dependency in NSCLC whose inhibition promotes anti-tumor responses through both tumor intrinsic and host immune regulatory mechanisms. Citation Format: Bitta P. Kahangi, William P. Crosson, Ji-Ann Lee, Camelia Dumitras, Tianhao Zhang, Ramin Salehi-Rad, Linh M. Tran, Michael Palazzolo, Dylan Conklin, Steven M. Dubinett, Bin Liu. Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4266.

Identification of circRNA-miRNA-mRNA Network Regulated by Hsp90 in Human Melanoma A375 Cells.

Melanoma is the deadliest form of skin cancer. Heat shock protein 90 (Hsp90) is highly expressed in human melanoma. Hsp90 inhibitors can suppress the growth of human melanoma A375 cells; however, the underlying mechanism remains unclear. A375 cells were treated with SNX-2112, an Hsp90 inhibitor, for 48 h, and wholetranscriptome sequencing was performed. A total of 2,528 differentially expressed genes were identified, including 895 upregulated and 1,633 downregulated genes. Pathway enrichment analyses of differentially expressed mRNAs identified the extracellular matrix (ECM)-receptor interaction pathway as the most significantly enriched pathway. The ECM receptor family mainly comprises integrins (ITGs) and collagens (COLs), wherein ITGs function as the major cell receptors for COLs. 19 upregulated miRNAs were found to interact with 6 downregulated ITG genes and 8 upregulated miRNAs were found to interact with 3 downregulated COL genes. 9 differentially expressed circRNAs in SNX-2112- treated A375 cells were identified as targets of the ITG- and COL-related miRNAs. Based on the differentially expressed circRNAs, miRNAs, and mRNAs, ITGs- and COL-based circRNAmiRNA- mRNA regulatory networks were mapped, revealing a novel regulatory mechanism of Hsp90-regulated melanoma. Targeting the ITG-COL network is a promising approach to the treatment of melanoma.

Genome-wide characterization of integrin (ITG) gene family and their expression profiling in half-smooth tongue sole (Cynoglossus semilaevis) upon Vibrio anguillarum infection

Integrins (ITGs) are transmembrane heterodimer receptors with ITGα subunit and ITGβ subunit, participating in various physiological processes, including immunity. At present, systematic research on ITGs in teleost is scarce, especially in half-smooth tongue sole (Cynoglossus semilaevis). In this study, a set of 28 ITG genes in half-smooth tongue sole have been identified and characterized. The phylogenetic analysis showed that ITGα and ITGβ subunits were respectively classified into five and two clusters, consistent with previous studies. The selection pressure analysis indicated that most of ITG genes were under purifying selection, except for ITGα11b and ITGαL with positive selection. The expression profiles of eight selected ITG genes, including ITGα1, ITGα5, ITGα8, ITGα11, ITGβ1, ITGβ2, ITGβ3, and ITGβ8, were analyzed in healthy tissues and after infection with Vibrio anguillarum, revealed their implications in immune response. The study provided a comprehensive characterization and expression analysis of ITG genes in half-smooth tongue sole, setting a solid foundation for further functional studies and promising potential in disease control.

Integrin-based prognostic model predicts survival, immunotherapy response, and drug sensitivity in gastric cancer

BackgroundExtracellular matrix (ECM) acts as a physical barrier to tumors, resulting in the lysis or delay of drug delivery. Integrins (ITGs) are essential for tumor cell-ECM interactions. Thus, we established a novel prognostic model to predict overall survival, immunotherapy benefits, and therapeutic agents in gastric cancer (GC) based on ITGs-related ECM landscape. MethodsUsing the TCGA-STAD dataset, we studied the genetic and transcriptional changes of ITGs. We used a merged cohort for ITGs survival analysis and determined molecular pattern clusters using consensus unsupervised clustering methodology. We confirmed the distinct ECM landscape between constructed clusters by performing gene set variation and Kaplan-Meier analysis. We utilized prognostic differentially expressed genes between clusters to develop a prognostic model utilizing logistic least absolute shrinkage and selection operator cox regression analysis, followed by stepwise multivariate Cox analysis in the training dataset. The model was validated by receiver operating characteristic curves and Kaplan-Meier analysis in the testing dataset and seven validation datasets. We compared our model to 35 previously published models. To analyze immune infiltration, we used multiple algorithms, which were further confirmed by single-cell RNA-sequencing and fluorescent multiplex immunohistochemistry. We explored tumor mutation burden (TMB), microsatellite instability-high (MSI-H) grade, immunotherapy response, chemotherapy sensitivity, and clinical significance between the low-risk and high-risk groups. Finally, we assessed the risk score in five reported molecular subtypes. ResultsThe two ITGs-related clusters were identified, and their ECM landscapes were distinct. The prognostic model was constructed and had shown stable performance in internal and external validation. In addition, our model outperformed 35 previously published models. High-risk patients had a bad prognostic ECM landscape, high stromal cell inflammation, a lower TMB, a lower MSI-H grade, a worse tumor stage, a worse response to immunotherapy, and less sensitivity to chemotherapy. In five reported molecular subtypes, the worse subtypes showed a higher risk score. ConclusionsThe prognostic model could be an effective and promising tool for predicting prognosis and therapy response in GC patients.

N-TELOPEPTIDE DERIVED FROM TYPE II COLLAGEN UPREGULATES EXPRESSION OF BETA1 INTEGRIN AND MMPS IN CARTILAGE OF PATIENTS RECEIVING HIP OR KNEE ARTHROPLASTY

Previous studies showed that telo-peptides degraded from type II collagen, a type of collagen fragments, could induce cartilage damage in bovine stifle joints. We aim to investigate the role of integrins (ITGs) and matrix metalloproteinases (MMPs) in collagen fragment-induced human cartilage damage that is usually observed in osteoarthritis (OA). We hypothesized that N-telopeptide (NT) derived from type II collagen could up-regulate the expression of β1 integrin (ITGB1) and then MMPs that may lead to osteoarthritic cartilage damage.Human chondrocytes were isolated from femoral head or tibial plateau of patients receiving arthroplasty (N = 24). Primary chondrocyte cultures were either treated with 30 µM NT, or 30 µM scrambled NT (SN), or PBS, or left untreated for 24 hrs. Total proteins and RNAs were extracted for examination of expression of ITGB1 and MMPs-3&13 with Western blotting and quantitative real-time PCR.Compared to untreated or PBS treated chondrocytes, NT-treated chondrocytes expressed significantly higher levels of ITGB1 and MMPs-3&-13. However, SN also up-regulated expression of ITGB1 and MMP-13.ITGB1 and MMPs-3&-13 might mediate the catalytic effect of NT, a type of collagen fragments, on human cartilage damage that is a hallmark of OA.

Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

The low survival rate of mesenchymal stem cells (MSCs) caused by anoikis, a form of apoptosis, limits the therapeutic efficacy of MSCs. As a proapoptotic molecule, mammalian Ste20-like kinase 1 (Mst1) can increase the production of reactive oxygen species (ROS), thereby promoting anoikis. Recently, we found that Mst1 inhibition could protect mouse bone marrow MSCs (mBMSCs) from H2O2-induced cell apoptosis by inducing autophagy and reducing ROS production. However, the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear. To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs. Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA (shRNA) adenovirus transfection. Integrin (ITGs) were tested by flow cytometry. Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA, respectively. The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays. The levels of the anoikis-related proteins ITGα5, ITGβ1, and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I, Beclin1 and p62 were detected by Western blotting. In isolated mBMSCs, Mst1 expression was upregulated, and Mst1 inhibition significantly reduced cell apoptosis, induced autophagy and decreased ROS levels. Mechanistically, we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4, ITGαv, or ITGβ3 expression. Moreover, autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis. Mst1 inhibition ameliorated autophagy formation, increased ITGα5β1 expression, and decreased the excessive production of ROS, thereby reducing cell apoptosis in isolated mBMSCs. Based on these results, Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.

Aberrant glycosylation of α3 integrins as diagnostic markers in epithelial ovarian cancer

BackgroundGlycans are strongly involved in stability and function of integrins (ITG) and tetraspanin protein CD63 and their respective interaction partners as they are dysregulated in the tumorigenic processes. Glycosylation changes is a universal phenomenon of cancer cells. In this study, glycosylation changes in epithelial ovarian cancer (EOC) are explored using tetraspanin and integrin molecules. MethodsITG and CD63 were immobilized from 10 EOC and 5 benign ovarian cyst fluid on microtiter wells and traced with 3 glycan binding proteins (STn, WGA, UEA) conjugated on europium nanoparticles. Total protein measurements (ITG & CD63 immunoassays) were also performed. The most promising glycovariant candidates identified were then clinically evaluated on the whole cohort of 77 ovarian cyst fluids. Additional testing was performed in ascites fluid samples of liver cirrhosis (n = 2) and EOC (n = 4). ResultsSialylated Tn antibody based glycovariants of ITGα3 (ITGα3STn) and CD63 (CD63STn) performed better than corresponding protein epitope-based immunoassays, ITGα3IA and CD63IA respectively. Combined ITGα3 based assays (ITGα3IA + ITGα3STn) detected 49 out of 55 malignant & borderline cases without detecting any of the 22 benign and healthy cysts. ConclusionOur findings indicate the potential diagnostic application of ITGα3STn along with total ITGα3IA, which could help reduce the unnecessary surgeries. The results encourage studying further the potential use of these novel assays to detect EOC at earlier clinical stages.

M2-like macrophage-derived exosomes facilitate metastasis in non-small-cell lung cancer by delivering integrin αVβ3.

Metastasis is the most prevalent cause of cancer deaths, and immunological components of the tumor microenvironment, especially tumor-associated macrophages (TAMs), play a vital role in cancer metastasis. However, the underlying mechanisms of TAMs on non-small-cell lung cancer (NSCLC) metastasis remain largely unexplored. Herein, we demonstrated that M2-like TAMs facilitate the migration and invasion of cancer cells in vitro and in vivo through intercellular delivery of M2-like macrophage-derived exosomes (M2-exos). Importantly, we found that M2-exos had considerably higher levels of integrin (ITG) αV and β3. The impact of M2-like macrophage-mediated invasion and migration of NSCLC cells was clearly decreased when ITG αVβ3 was blocked. Mechanistically, exosomal ITG αVβ3 produced from M2-like macrophages successfully triggered the focal adhesion kinase signaling pathway in recipient cells, boosting the migratory and invasive abilities of NSCLC cells. Clinically, we found that metastatic NSCLC patients had greater ITG αV and β3 expression, which was associated with a worse prognosis. This study reveals a novel mechanism by which M2-exos significantly increased NSCLC cell migration and invasion by delivering integrin αVβ3. Exosomal ITG αVβ3 can be used as a potential prognostic marker, and blocking ITG αVβ3 could be a viable treatment option for preventing tumor metastasis.

An altered extracellular matrix-integrin interface contributes to Huntington's disease-associated CNS dysfunction in glial and vascular cells.

Astrocytes and brain endothelial cells are components of the neurovascular unit that comprises the blood-brain barrier (BBB) and their dysfunction contributes to pathogenesis in Huntington's disease (HD). Defining the contribution of these cells to disease can inform cell-type-specific effects and uncover new disease-modifying therapeutic targets. These cells express integrin (ITG) adhesion receptors that anchor the cells to the extracellular matrix (ECM) to maintain the integrity of the BBB. We used HD patient-derived induced pluripotent stem cell (iPSC) modeling to study the ECM-ITG interface in astrocytes and brain microvascular endothelial cells and found ECM-ITG dysregulation in human iPSC-derived cells that may contribute to the dysfunction of the BBB in HD. This disruption has functional consequences since reducing ITG expression in glia in an HD Drosophila model suppressed disease-associated CNS dysfunction. Since ITGs can be targeted therapeutically and manipulating ITG signaling prevents neurodegeneration in other diseases, defining the role of ITGs in HD may provide a novel strategy of intervention to slow CNS pathophysiology to treat HD.

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Pentoxifylline and berberine mitigate diclofenac-induced acute nephrotoxicity in male rats via modulation of inflammation and oxidative stress

Nephrotoxicity (NT) is a renal-specific situation caused by different toxins and drugs like non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs like diclofenac (DCF) lead to glomerular dysfunction. Pentoxifylline (PTX) and berberine (BER) have antioxidant and anti-inflammatory properties. Thus, the objective of the present study was to investigate the ameliorative effect of PTX, BER and their combination against DCF-mediated acute NT. Induction of acute NT was done via DCF injection (150mg/kg I.P, for 6 days) in rats. PTX 200mg/kg, BER 200mg/kg and their combination were administrated for 6 days prior to DCF injection and concurrently with DCF for additional 6 days. Acute NT was evaluated biochemically and histopathologically by measuring blood urea (BU), serum creatinine (SCr), kidney injury molecule-1(KIM-1), integrin (ITG), and vitronectin (VTN), interleukin (IL)-18, Neutrophil gelatinase-associated lipocalin (NGAL), glomerular filtration rate (GFR), superoxide dismutase (SOD) and glutathione (GSH) and malondialdehyde (MDA) with the scoring of histopathological alterations. PTX, BER and their combination significantly (P<0.05) attenuated biochemical and histopathological changes in DCF-mediated acute NT by amelioration of BU, SCr, KIM-1, ITG, VTN, IL-18, NGAL, GFR, SOD, GSH, MDA and scoring of histopathological alterations. The combined effects of PTX and BER produced more significant effects (P<0.05) than either PTX or BER when used alone against DCF-induced acute NT. In conclusion, BER and BTX were found to have potential renoprotective effects against DCF-induced NT in rats by inhibiting inflammatory reactions and oxidative stress.

Protocadherin B9 Is Associated with Human Esophageal Squamous Cell Carcinoma Progression

Introduction: Esophageal cancer is the sixth leading cause of cancer-related death worldwide. However, molecular targeted therapy and novel therapeutic targets are needed for esophageal squamous cell cancer (ESCC). In a previous study, we reported that protocadherin (PCDH) B9 plays an important role in several cancers. Therefore, in this study, we examined the clinical significance of PCDHB9 expression in ESCC. Methods: PCDHB9 expression was examined using immunohistochemistry in 128 cases and using quantitative reverse transcription-polymerase chain reaction in 16 cases of ESCC. PCDHB9 function in ESCC cells was examined using RNA interference. Results: High PCDHB9 expression was identified in 5 of 16 (31.3%). In total, 51 (40%) ESCC cases showed strong PCDHB9 expression, whereas nonneoplastic mucosa rarely showed its expression. High PCDHB9 expression was significantly associated with T classification, N grade, and stage in ESCC. In ESCC cell lines, PCDHB9 knockdown affected cell growth, migration, and adhesion. Further, the expression of integrin (ITG) A3, ITGA4, ITGA5, ITGB1, ITGB6, vimentin, snail family transcriptional repressor 1, and cadherin 2 (NCAD) was significantly reduced and cadherin 1 was significantly increased in PCDHB9 knockdown ESCC cells. Conclusion: These results suggest that PCDHB9 plays a tumor-promoting role and is a potential biomarker and therapeutic target in ESCC.

Lung adenocarcinoma-specific three-integrin signature contributes to poor outcomes by metastasis and immune escape pathways.

ABSTRACTBackground:Inhibitors targeting integrins (ITGs) are applied as a novel strategy for cancers including lung cancer; however, the heterogeneity of ITG subunits might explain why ITG-targeted inhibitors only show limited efficacy for a small group of lung cancer patients.Materials and methods:RNA-Seq data of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients were obtained from the TCGA database. Cox regression analysis was performed to construct the prognostic signature and generate the nomogram combined with pathologic stages (pStage). GEO datasets were used for verification. The related biological functions were analyzed by Gene Set Enrichment Analysis (GSEA) software and the TIMER database.Results:By Cox regression analysis of 30 ITG subunits, ITG subunit alpha 5 (ITGA5), ITG subunit alpha 6 (ITGA6), and ITG subunit alpha L (ITGAL) were identified as the prognostic factors in LUAD, which were included in the construction of a LUAD-specific 3-ITG signature. Following the calculation of risk score (RS) of each patient based on 3-ITG signature, patients with high RS in LUAD were found to exhibit worse prognosis, especially in early stage. Nomogram combined with RS and pStage could predict the prognosis of LUAD patients accurately. Mechanism exploration by GSEA showed that metastasis-related microenvironmental pathways were significantly enriched in the high-RS group. An elevated expression of ITGA5 was mainly associated with the promotion of cell migration and invasion, while the high expression of ITGAL had a strong positive correlation with the capability of recognizing and killing cancer cells.Conclusions:Three-ITG signature could improve the prediction ability combined with pStage in LUAD and might contribute to poor prognosis by metastasis and immune escape-related pathways.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

301P Integrins in different subsets of circulating tumor cells in breast cancer

Circulating tumor cells (CTCs) are released from tumor tissue and provide tumor progression. The presence of stem and epithelial-mesenchymal transition (EMT) features in CTCs makes population extremely heterogenous and determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. Integrins (ITGs), adhesion molecules, can determine organotropic metastasis when expressed on exosomes, as was shown in a study conducted by Hoshino A. et al.

Expression of integrins β1, α4 and cell adhesion molecule ICAM-1 in the presence of sodium deoxyribonucleate with ferrum complex (DNA-Na-Fe) by MT-4 cells transformed by human T-lymphotropic virus type 1 (Retroviridae: Orthoretrovirinae: Deltaretrovirus: Human T-lymphotropic virus type 1)

The important role of integrins (IG) in the initiation and development of cancer processes makes these structures convenient targets for the development of immunomodulatory therapeutic drugs that have an effect directly on these molecules. Among the latter, IG β1, α4 and cell adhesion receptor ICAM-1 (intercellular adhesion molecule 1) are of particular interest. Immunomodulators are capable of changing the IG activity through non-specific mechanisms, which, however, in some cases can cause a decrease in the protective functions of the immune system and health deterioration.The aim of the study was to determine the effect on the levels of cellular expression and the nature of IG metabolism of the drug sodium deoxyribonucleate with ferrum complex, DNA-Na-Fe, which is having been used in the Russian Federation as an immunomodulatory agent, but whose action has not been studied in details so far. We used 2 variants of the neoplastic CD4+ T-lymphocyte cell line transformed with human T-lymphotropic virus type 1 (HTLV-1) of the Retroviridae family, MT-4 (MT-4/1 and MT-4/2). The indicated variants were characterized by different levels of expression of the protein activation markers CD28 and CD38. After cell culture in the presence of 500 μg/ml DNA-Na-Fe, the expression levels of IG β1 (CD29), α4 (CD49d), and ICAM-1 (CD54) were studied by flow cytometry. The cells of the both lines contained many membrane proteins CD29+ (90.4% ± 4.5) and CD54+ (97.9% ± 1.4), while small percentage of cells contained protein CD49d+ (1.9% ± 1.0). No changes in the expression of the studied proteins were observed in the presence of the drug. The levels of IG β1, α4 and ICAM-1 expression may serve as one of the phenotypic characteristics of MT-4 cells. The obtained data are of great importance because the peculiarities of CD4+ T-lymphocytes transformation and their metabolism during HTLV-1 infection have not been sufficiently studied so far. The results of this work may be helpful in determining the pathogenesis of HTLV-1-induced diseases, some types of malignancies, and in searching for new specific pharmacological agents, including molecularly targeted ones. The results of the study will help to expand the existing knowledge on the markers of MT-4 cell line.

CIB1 Regulates Hemostasis through Integrin Outside-in Signaling

On circulating platelets, integrin αIIbβ3, the fibrinogen receptor, is in a low affinity state unable to bind soluble ligands. Agonist-dependent inside-out signaling is necessary for activation of the integrin αIIbβ3 in order to bind ligands such as fibrinogen (Fg). Ligand binding induces a cascade of signaling that culminates in platelet aggregation, leading to hemostatic plug formation and clot retraction. Although it is well known that signaling through integrin αIIbβ3 is necessary, the mechanism of clot retraction is not well understood. Previously, we have identified a calcium- and integrin-binding protein 1 (CIB1) that specifically interacts with integrin αIIbβ3. One study implicated CIB1 as an endogenous inhibitor of integrin αIIbβ3 activation, while another study suggested CIB1 to be an activator of inside-out signaling. Using a novel technique to inhibit interaction of CIB1 with integrin in vivo, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Definitive proof for the role of CIB1 in platelets remained absent, however. Here, using Cib1 null platelets, we show for the first time that CIB1 is a key regulator of hemostasis. Genetic ablation of CIB1 had no effect on agonist-induced platelet aggregation, excluding the role for CIB1 in inside-out signaling. However, deletion of the Cib1 gene significantly increased mouse bleeding times. More than 50% of the mice also showed a rebleeding phenotype, suggesting a defect in clot stability. Incomplete and significantly delayed clot retraction was observed in Cib1 null platelets compared to wildtype littermates. The absence of CIB1 also affects the ability of platelets to spread on immobilized Fg. Platelet adhesion and subsequent filopodia formation was not affected by the absence of CIB1. Platelet spreading can be restored by addition of exogenous ADP, suggesting that the defect is not in the ability of the platelet to spread. These in vivo and in vitro results clearly show that CIB1 regulates hemostasis by supporting transition of platelets from filopodial stage to fully spread morphology during outside-in signaling through integrin αIIbβ3.

Behavior of Primary Human Oral Keratinocytes Grown on Invisalign® SmartTrack® Material

Orthodontic clear aligner treatment is gaining tremendous popularity. The world market leader is Align Technology® and its product Invisalign®. Although numerous patients are treated with Invisalign® aligners, only little is known about the cellular effects of aligner material on oral epithelial cells. In the present study, we aimed to investigate the effects of SmartTrack® clear aligner material on directly cultured primary human oral keratinocytes (HOKs). Cell morphology and behavior were investigated by scanning electron microscopy and bright field microscopy. Aligner effects on viability were detected by cell-counting-kit (CCK)-8 and live/dead staining. Gene expression of several inflammatory and barrier proteins was assessed by qPCR. Cells cultured on tissue culture plastic served as control. Cell proliferation/viability was significantly lower in cells cultured on aligner material (p &lt; 0.05) in comparison to control. Live/dead staining did not reveal an increase in the number of dead cells on aligner surfaces. After two and seven days of incubation, interleukin (IL)-6 expression decreased, and IL-8 expression increased in HOKs cultured on aligner surfaces. The expression of intercellular adhesion molecule 1 (ICAM-1) significantly decreased after seven days. Gene expression of epithelial barrier markers showed that integrin (ITG)-α6 significantly decreased after two and seven days. A significant decrease in ITG-β4 and E-cadherin expression levels compared to control could only be seen after seven days. We did not find any cytotoxic effect, but alterations in the cell’s barrier functions and inflammatory reaction were obvious. Clinical studies are required to give further insights into clinical reactions on the underlying aligner material of this quickly expanding orthodontic appliance.

Chimeric antigen receptor T-cell therapy for multiple myeloma

CD19 chimeric antigen receptor (CAR) T cell therapy is effective in B cell leukemia and lymphoma. Many researchers are attempting to develop CAR T cells for different types of cancer. B cell maturation antigen (BCMA) is shown to be a promising target for multiple myeloma (MM). However, the treatment for MM remains challenging, and many researchers are trying to identify better target molecules for MM treatment. We have reported that activated integrin β7 is an excellent target for CAR T cells against MM and are currently performing a clinical trial.

RGD-binding integrins mediated phagocytosis involved in the entry of Edwardsiella tarda into mudskipper MO/MФ

The versatile fish pathogen Edwardsiella tarda is an intracellular pathogen with the ability to invade and replicate in host phagocytes. However, the mechanism mediating the uptake of E. tarda in fish monocytes/macrophages (MO/MΦ) is not yet understood. Generating mudskipper kidney-derived MO/MФ transcriptomic resources from mudskipper challenged by E. tarda is crucial for understanding the molecular mechanisms underlying the mudskipper invasion process. In the present study, a total of 1185 up-regulated and 885 down-regulated differentially expressed genes (DEGs) were identified using RNA-seq. Enrichment and pathway analysis of DEGs revealed the centrality of the phagosome and regulation of actin cytoskeleton pathways in pathogen entry. The progress of phagosome formation was observed by transmission electron microscopy. Eight conserved integrin (ITG) subunit genes, belonging to the phagocytic receptors, were found in the transcriptomic sequence data. Additionally, quantitative real-time PCR showed that the mRNA expressions of most ITG subunit genes were related to the different infection times of E. tarda and the different bacterial pathogens. Further assays demonstrated that phagocytosis of FITC-labeled E. tarda by mudskipper MO/MФ was significantly reduced by the tetrapeptide Asp-Gly-Arg-Ser (RGDS). In summary, phagocytosis is one of the entry pathways into mudskipper MO/MΦ, and RGD-binding ITGs are involved in the phagosome formation process.