Surface Intercellular Adhesion Molecule-1 Research Articles

Submicron immunoglobulin particles exhibit FcγRII-dependent toxicity linked to autophagy in TNFα-stimulated endothelial cells

In intravenous immunoglobulins (IVIG), and some other immunoglobulin products, protein particles have been implicated in adverse events. Role and mechanisms of immunoglobulin particles in vascular adverse effects of blood components and manufactured biologics have not been elucidated. We have developed a model of spherical silica microparticles (SiMPs) of distinct sizes 200–2000 nm coated with different IVIG- or albumin (HSA)-coronas and investigated their effects on cultured human umbilical vein endothelial cells (HUVEC). IVIG products (1–20 mg/mL), bare SiMPs or SiMPs with IVIG-corona, did not display significant toxicity to unstimulated HUVEC. In contrast, in TNFα-stimulated HUVEC, IVIG-SiMPs induced decrease of HUVEC viability compared to HSA-SiMPs, while no toxicity of soluble IVIG was observed. 200 nm IVIG-SiMPs after 24 h treatment further increased ICAM1 (intercellular adhesion molecule 1) and tissue factor surface expression, apoptosis, mammalian target of rapamacin (mTOR)-dependent activation of autophagy, and release of extracellular vesicles, positive for mitophagy markers. Toxic effects of IVIG-SiMPs were most prominent for 200 nm SiMPs and decreased with larger SiMP size. Using blocking antibodies, toxicity of IVIG-SiMPs was found dependent on FcγRII receptor expression on HUVEC, which increased after TNFα-stimulation. Similar results were observed with different IVIG products and research grade IgG preparations. In conclusion, submicron particles with immunoglobulin corona induced size-dependent toxicity in TNFα-stimulated HUVEC via FcγRII receptors, associated with apoptosis and mTOR-dependent activation of autophagy. Testing of IVIG toxicity in endothelial cells prestimulated with proinflammatory cytokines is relevant to clinical conditions. Our results warrant further studies on endothelial toxicity of sub-visible immunoglobulin particles.

LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins

Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.

Piperine from Black Pepper Decreased the Expression of Intercellular Adhesion Molecule-1 in Macrophages.

Macrophages are the main players involved in inflammation. Intercellular adhesion molecule-1 (ICAM-1) facilitates macrophage polarization prior to extravasation into inflamed tissue. Piperine, a natural product derived from black pepper, possesses useful biological and pharmacological activities. In the current study, the possible anti-inflammatory effect of piperine on the expression of ICAM-1 on J774.1 murine macrophage cell line was investigated. Lipopolysaccharide (LPS)-stimulated J774.1 cells were cultured in the presence of different concentrations of piperine to examine the changes in ICAM-1 expression by real-time PCR and flow cytometry. We found that piperine decreased ICAM-1 gene expression level from 2.4 ± 0.25 RFC (relative fold change) in LPS-only treated cells to 0.85 ± 0.525 RFC at 1µg/ml (p<0.05), 0.43 ± 0.27 RFC at 10µg/ml (p<0.01), and 0.26 ± 0.25 RFC at 20µg/ml (p<0.01). In flow cytometry, piperine at all concentrations significantly decreased ICAM-1 surface expressions (P<0.05). The geometric mean fluorescence intensity (g-MFI) in LPS-only treated cells (792 ± 57.3) decreased to 482±70 g-MFI at 20 μg/ml piperine. According to the results of this study, by decreasing the expression of ICAM-1, piperine has been suggested to reduce inflammation and have the potential to provide therapeutic benefits for immune-mediated diseases.

Abstract 2322: ICAM-1 targeting CAR T cell therapy for triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. TNBC patients do not respond to hormone receptor or HER2-targeted therapies owing to low expression of these cell surface biomarkers. The lacking of targeted therapy leaves conventional combination of surgery, chemotherapy and radiation therapy as the standard-of-care treatment options for TNBC. But even this combination usually fails to prevent disease recurrence. Intercellular adhesion molecule-1 (ICAM-1) was recently discovered to be upregulated in TNBC and could serve as an attractive molecular target (Guo et al., PNAS 2014). Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success against hematological malignancies, however there has been little success in the treatment of solid tumors. Here, we developed an ICAM-1 targeting CAR T cell-based immunotherapeutic strategy to redirect T cell to kill solid TNBC. Methods: Patient-derived xenograft (PDX) models of TNBC were utilized to determine ICAM-1 expression by immunohistochemistry. ICAM-1 surface protein expression in TNBC cell line MDA-MB-231 was measured by flow cytometry. Primary T cells (n = 4 donors) were isolated and transduced with ICAM-1 CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. In vitro killing ability was determined by co-incubation of GFP-Firefly Luciferase (GFP/Fluc) transduced target cells (HeLa, MDA-MB-231, and 293T) with transduced and non-transduced T cells. NSG mice bearing MDA-MB-231 xenografts were treated with CAR T cells to test the in vivo efficacy. Bioluminescence was used to quantify cell lysis in vitro and to track tumor growth in vivo. Results: 4 out of 8 TNBC PDX models (JAX, tumor markers: TER−/PR−/HER2−) showed strong IHC ICAM-1 staining. We validated by flow cytometry that ICAM-1 is highly expressed on TNBC cell line MDA-MB-231. These results provide further evidence supporting ICAM-1 as a molecular target for TNBC by CAR T cell therapy. Primary T cells were transduced to express ICAM-1 targeting CAR at approximately 50%. Co-incubation of CAR T cells or non-transduced T cells with ICAM-1 positive cell lines (HeLa and MDA-MB-231) or negative control cell line (293T) showed that CAR T cell-mediated killing was ICAM-1 expression dependent. After 48 hours, 70% of HeLa and 85% of MDA-MB-231 cells were specifically lysed at effector to target ratio of 5:1, while no obvious killing of 293T cells was observed. Additionally, non-transduced T cells produced little killing with less than 20% of target cell lysis. Ongoing in vivo studies will determine the efficacy of this ICAM-1 targeting CAR T cell against TNBC. Conclusions: We developed an ICAM-1 targeting CAR T cell with the ability to induce potent and specific killing of TNBC cells. Preclinical studies are being conducted to evaluate the feasibility of ICAM-1 specific CAR T cells as a potential therapy for patients with ICAM-1 positive TNBC. Citation Format: Yanping Yang, Yogindra Vedvyas, Jaclyn E. McCloskey, Irene M. Min, Moonsoo M. Jin. ICAM-1 targeting CAR T cell therapy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2322.

Biological function of intercellular adhesion molecule-1 gene in coronary heart disease and its mediated primary regulatory network: a literature based secondary analysis

To explore the biological function of intercellular adhesion molecule-1 (ICAM-1) gene in coronary heart disease (CHD) and to establish a primary regulatory network mediated by ICAM-1 gene. The databases were searched from January 1st 2006 to December 31st 2018, including Wanfang, CNKI, VIP, Google scholar and PubMed databases for published researches on clinical study of CHD gene ICAM-1. The experimental group was CHD patients, and the control group was healthy people. Meta-analysis was employed to analyze the relationship between ICAM-1 gene and CHD. Gene Ontology (GO) and KEGG Pathway database were employed to conduct function annotation and pathway analysis. A total of 8 papers were enrolled into this analysis, Meta-analysis showed that the level of ICAM-1 in the experimental group was significantly higher than that in the control group [mean difference (MD) = 15.29, 95% confidence interval (95%CI) = 10.95-19.62, P < 0.000 01], surface ICAM-1 was significantly related with CHD. The GO analysis results showed that ICAM-1 molecular function mainly involved virus receptor activity, receptor activity, transmembrane signal receptor activity, protein binding, etc.; cell components mainly involve plasma membrane integral components, immune synapses, plasma membrane, etc.; biological processes mainly involve the positive regulation of cell adhesion, leukocyte adhesion, leukocyte migration and leukocyte adhesion, etc. A primary CHD regulatory network mediated by ICAM-1 gene was established based on KEGG Pathway database, and ICAM-1 was mainly expressed in endothelial cells, and further regulated the occurrence and development of CHD by promoting the proliferation of leukocytes. ICAM-1 may regulate the occurrence and development of CHD by regulating the related biological processes of leukocytes. The successful establishment of the ICAM-1 mediated CHD regulatory network can lay a theoretical foundation for further exploring the specific regulatory role of ICAM-1 and other related genes in CHD occurrence.

Induction of osteoclast-like cell formation by leptin-induced soluble intercellular adhesion molecule secreted from cancer cells.

Background:Leptin is considered a tumorigenic adipokine, suggested to promote tumorigenesis and progression in many cancers. On the other hand, intercellular adhesion molecule-1 (ICAM-1) shows altered expression in a variety of benign and malignant diseases. Histologically, ICAM-1 expression is reported as proportional to cancer stage and considered as a potential diagnosis biomarker. The altered expressions of ICAM-1 and its soluble form in malignant diseases have gained interests in recent years.Material and methods:The expression of ICAM-1 and its regulatory signaling were examined by Western blot or flow cytometry. The effect of soluble ICAM-1 on osteoclast formation was investigated by tartrate-resistance acid phosphatase staining of RAW cells and tumor-induced osteolysis in vivo.Results:In our study, we found that leptin enhanced soluble ICAM-1 production but not surface ICAM-1 expression in lung and breast cancer cells, and this effect was regulated through leptin receptor (ObR), while silencing ObR abrogated leptin-induced soluble ICAM-1 expression. In addition, we revealed that leptin administration provoked the JAK1/2, STAT3, FAK, ERK, and GSK3αβ signaling cascade, leading to the elevation of ICAM-1 expression. Moreover, soluble ICAM-1 secreted by leptin-stimulated cancer cells synergize with the receptor activator of nuclear factor kappa-B ligand (RANKL) in inducing osteoclast formation. Soluble ICAM also enhanced tumor-induced osteolysis in vivo.Conclusion:These findings suggest that soluble ICAM-1 produced under leptin treatment enhances osteoclast formation and is involved in tumor-induced osteolysis.Leptin plays an important role in physiology in health and diseases. Leptin affects immune responses that may induce inflammation and carcinogenesis. Leptin is also considered as a tumorigenic adipokine suggested to promote tumorigenesis and progression in many cancers. On the other hand, intercellular adhesion molecule-1 (ICAM-1) shows altered expression in a variety of benign and malignant diseases. Histologically, ICAM-1 expression is reported to be proportional to cancer stage and considered as a potential diagnosis biomarker. It has been reported that soluble ICAM-1 allows tumor cells to escape from immune recognition and stimulates angiogenesis and tumor growth. The altered expressions of ICAM-1 and its soluble form in malignant diseases have gained interests in recent years. In our study, we found that leptin enhanced soluble ICAM-1 production but not surface ICAM-1 expression in lung and breast cancer cells, and this effect was regulated through leptin receptor (ObR), while silencing ObR abrogated leptin-induced soluble ICAM-1 expression. In addition, we revealed that leptin administration provoked the JAK1/2, STAT3, FAK, ERK, and GSK3αβ signaling cascade, leading to the elevation of ICAM-1 expression. Moreover, soluble ICAM-1 secreted by leptin-stimulated cancer cells synergize with receptor activator of nuclear factor-kappa B ligand in inducing osteoclast formation. Soluble ICAM also enhanced tumor-induced osteolysis in vivo. These findings suggest that soluble ICAM-1 produced under leptin treatment is possibly involved in lung and breast cancer bone metastasis.

Palmitoleic Acid has Stronger Anti‐Inflammatory Potential in Human Endothelial Cells Compared to Oleic and Palmitic Acids

Fatty acids (FAs) may affect endothelial cell (EC) function, influencing atherogenesis and inflammatory processes. Palmitoleic acid (POA) has been described as an anti-inflammatory FA. However, its effects on ECs are underexplored. This study compares the effects of POA with those of palmitic acid (PA) and oleic acid (OA) on EC inflammatory responses. EAHy926 cells (EC lineage) are exposed to PA, OA, or POA, and stimulated with tumor necrosis factor (TNF)-α. Associated with the FA's own incorporation, PA induces a twofold increase in arachidonic acid, while POA increases the amount of cis-vaccenic acid. PA, but not OA, enhances the production of IL-6 and IL-8 in response to TNF-α. In contrast, POA decreases production of monocyte chemotactic protein (MCP)-1, IL-6, and IL-8 compared to PA. TNF-α increases surface intercellular adhesion molecule-1 expression previously decreased by POA. TNF-α stimulation increases the expression of NFκB, cyclooxygenase (COX)-2, MCP-1, and IL-6 genes and reduces the expression of peroxisome proliferator-activated receptor (PPAR)-α gene. PA enhances the expression of MCP-1, IL-6, and COX-2 genes, while POA downregulates these genes, decreases expression of NFκB, and upregulates PPAR-α gene expression. POA has anti-inflammatory effects on ECs stimulated with TNF-α and may counter endothelial dysfunction.

Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%]FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. The supernatants [10%-40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of β-arrestin-1 with BLT2, protein kinase C (PKC)βI , and PKCδ and served as the first event in a two-event rodent model of ALI. Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.

HIV-1 Vpu Downmodulates ICAM-1 Expression, Resulting in Decreased Killing of Infected CD4 + T Cells by NK Cells

HIV-1 Vpu is known to alter the expression of numerous cell surface molecules. Given the ever-increasing list of Vpu targets identified to date, we undertook a proteomic screen to discover novel cell membrane proteins modulated by this viral protein. Plasma membrane proteome isolates from Vpu-inducible T cells were subjected to stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry analysis, and putative targets were validated by infection of primary CD4+ T cells. We report here that while intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 are upregulated by HIV-1 infection, expression of Vpu offsets this increase by downregulating these molecules from the cell surface. Specifically, we show that Vpu is sufficient to downregulate and deplete ICAM-1 in a manner requiring the Vpu transmembrane domain and a dual-serine (S52/S56) motif necessary for recruitment of the beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) component of the Skp, Cullin, F-box (SCFβ-TrCP) E3 ubiquitin ligase. Vpu interacts with ICAM-1 to induce its proteasomal degradation. Interestingly, the E3 ubiquitin ligase component β-TrCP-1 is dispensable for ICAM-1 surface downregulation yet is necessary for ICAM-1 degradation. Functionally, Vpu-mediated ICAM-1 downregulation lowers packaging of this adhesion molecule into virions, resulting in decreased infectivity. Importantly, while Vpu-mediated downregulation of ICAM-3 has a limited effect on the conjugation of NK cells to HIV-1-infected CD4+ T cells, downregulation of ICAM-1 by Vpu results in a reduced ability of NK cells to bind and kill infected T cells. Vpu-mediated ICAM-1 downregulation may therefore represent an evolutionary compromise in viral fitness by impeding the formation of cell-to-cell contacts between immune cells and infected T cells at the cost of decreased virion infectivity.IMPORTANCE The major barrier to eradicating HIV-1 infection is the establishment of treatment-resistant reservoirs early in infection. Vpu-mediated ICAM-1 downregulation may contribute to the evasion of cell-mediated immunity during acute infection to promote viral dissemination and the development of viral reservoirs. By aiding the immune system to clear infection prior to the development of reservoirs, novel treatments designed to disrupt Vpu-mediated ICAM-1 downregulation may be beneficial during acute infection or as a prophylactic treatment.

Alcohol Inhibits Organic Dust-induced ICAM-1 Expression on Bronchial Epithelial Cells.

Aims: Exposure to dusts/bioaerosols in concentrated animal feeding operations (CAFOs) results in inflammatory lung diseases in workers. Hog CAFOs dust extract (HDE) increases expression of intercellular adhesion molecule-1 (ICAM-1), neutrophil adhesion, and TNFα release in bronchial epithelial cells. Alcohol consumption is increasingly recognized to impair lung immunity. We hypothesized that alcohol impairs HDE-induced TNFα, ICAM-1 expression and neutrophil adhesion by directly inhibiting TNFα converting enzyme (TACE) activity.MethodsBronchial epithelial cells (BEAS-2B) and primary human bronchial epithelial cells were pretreated with ethanol (EtOH) or TACE inhibitor. ICAM-1 surface expression, TNFα release, and TACE activity were analyzed following HDE stimulation. The effect of alcohol and TACE inhibition on HDE-regulated epithelial cell/neutrophil adhesion interactions was investigated. Finally, utilizing an established animal model, C57BL/6 mice were fed ad libitum ethanol (20%) in drinking water for 8 wk followed by daily intranasal inhalation of HDE or saline during the final two weeks. Mice were sacrificed and lung sections immunostained for ICAM-1.ResultsPretreatment with alcohol or TACE inhibitor significantly decreased HDE-induced ICAM-1 expression and TNFα release. HDE augmented neutrophil adhesion to epithelial cells, which was decreased with alcohol (32% decrease) or TACE inhibitor (55% decrease) pretreatment. TACE activity increased following HDE exposure, but TACE activity was inhibited following alcohol pretreatment. Alcohol-fed mice demonstrated decreased HDE-induced airway epithelium ICAM-1 expression.ConclusionsAlcohol diminishes HDE-induced ICAM-1 expression, TNFα release, and neutrophil adhesion via inhibition of TACE activity. These results suggest that alcohol may be an important modulator of lung innate immune responses following CAFO exposure.

Peroxisome proliferator-activated receptor (PPAR) α and δ activators induce ICAM-1 expression in quiescent non stimulated endothelial cells.

BackgroundPeroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are implicated in the regulation of lipid and glucose homeostasis. PPAR agonists have been shown to control inflammatory processes, in part by inhibiting the expression of distinct proinflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1), IL-8, and intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is an important endothelial membrane receptor that facilitates the transmigration of leukocytes across the endothelium. To date, the influence of PPARα and δ activators on the expression of ICAM-1 in non-induced, quiescent endothelial cells has been unclear. Therefore, we examined the effects of various PPARα and δ agonists on the expression of ICAM-1 in non-stimulated primary human endothelial cells.ResultsWe found that PPARα and PPARδ agonists significantly induced ICAM-1 surface, intracellular protein, and mRNA expression in a time and concentration-dependent manner. The PPARδ induced ICAM-1 expression could be paralleled with a significantly increased T-cell adherence to the endothelial cells whereas PPARα failed to do so. Transcriptional activity studies using an ICAM-1 reporter gene constructs revealed that PPARδ, but not PPARα agonists induced gene expression by stimulating ICAM-1 promoter activity via an Sp1 transcription factor binding site and inhibit the binding of the transcription factors Sp1 and Sp3. Furthermore, we performed mRNA stability assays and found that PPARα and PPARδ agonists increased ICAM-1 mRNA stability.ConclusionTherefore, our data provide the first evidence that PPARα and PPARδ agonists induce ICAM-1 expression in non-stimulated endothelial cells via transcriptional and posttranscriptional mechanisms.

Biomimetic channel modeling local vascular dynamics of pro-inflammatory endothelial changes.

Endothelial cells form the inner lining of blood vessels and are exposed to various factors like hemodynamic conditions (shear stress, laminar, and turbulent flow), biochemical signals (cytokines), and communication with other cell types (smooth muscle cells, monocytes, platelets, etc.). Blood vessel functions are regulated by interactions among these factors. The occurrence of a pathological condition would lead to localized upregulation of cell adhesion molecules on the endothelial lining of the blood vessel. This process is promoted by circulating cytokines such as tumor necrosis factor-alpha, which leads to expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface among other molecules. ICAM-1 is critical in regulating endothelial cell layer dynamic integrity and cytoskeletal remodeling and also mediates direct cell-cell interactions as part of inflammatory responses and wound healing. In this study, we developed a biomimetic blood vessel model by culturing confluent, flow aligned, endothelial cells in a microfluidic platform, and performed real time in situ characterization of flow mediated localized pro-inflammatory endothelial activation. The model mimics the physiological phenomenon of cytokine activation of endothelium from the tissue side and studies the heterogeneity in localized surface ICAM-1 expression and F-actin arrangement. Fluorescent antibody coated particles were used as imaging probes for identifying endothelial cell surface ICAM-1 expression. The binding properties of particles were evaluated under flow for two different particle sizes and antibody coating densities. This allowed the investigation of spatial resolution and accessibility of ICAM-1 molecules expressed on the endothelial cells, along with their sensitivity in receptor-ligand recognition and binding. This work has developed an in vitro blood vessel model that can integrate various heterogeneous factors to effectively mimic a complex endothelial microenvironment and can be potentially applied for relevant blood vessel mechanobiology studies.

Abstract CT205: Intravenous delivery of a novel oncolytic immunotherapy agent, CAVATAK, in advanced cancer patients

Abstract Background: Coxsackievirus A21 (CVA21) is a naturally occurring “common cold” intercellular adhesion molecule-1 (ICAM-1)-targeted RNA virus. Surface ICAM-1 is up-regulated on a number of cancers including melanoma, non-small cell lung, bladder and prostate cancers. CAVATAK is a novel bio-selected formulation of CVA21, which displays potent oncolytic activity against in vitro cultures of cancer cells and in vivo xenografts of a number of cancers. In this phase I/II study advanced cancer patients received multiple intravenous (IV) doses of CAVATAK to assess treatment tolerance, levels of viral replication and viral-induced immune activation within the tumor micro-environment. Methods: The phase I/II STORM (Systemic Treatment Of Resistant Malignancies: NCT02043665) study is investigating the tolerance of multiple escalating IV doses of CVA21 in approximately 30 advanced cancer patients. In cohort 1 (n = 3), patients were infused with CVA21 at a dose of 1 × 108 TCID50, in cohort 2 patients (n = 3) were infused with CVA21 at a dose of 3 × 108 TCID50 and treatment of patients in Cohort 3 (n = 12-18) with CVA21 at a dose of 1 × 109 TCID50 has commenced. Tumor biopsies at 8 days following the initial CVA21 infusion are being monitored for levels of virus and markers of potential immune activation. Sequential serum samples are being analyzed for viral loads, kinetics of anti-CVA21 neutralizing antibody (nAb) development and immune system activation via relative serum levels of a panel of immune inflammatory cytokines /immune cell subsets. Results: To date, multiple CVA21 infusions of patients in Cohorts 1 and 2 have been generally well tolerated. Preliminary data indicate that the prolonged presence of serum CVA21 RNA in some, but not all, patients at times (up to 4 days post-infusion), when complete decay of the administered viral dose was expected; this may indicate possible viral replication within tumor. Such replication in pre-clinical xenograft models was potentially immunogenic, as evidenced by gene expression increases of CXCL-10 and PD-L1. Of particular interest was the finding of comparable kinetics of anti-CVA21 nAb development in patients receiving multiple infusions relative to those administered a single CVA21 infusion during a previous phase I dose-ranging study (NCT00636558). The interim data highlight a robust “multi-dosing-window” in the absence of significant levels of nAb for approximately 7 days post initial viral infusion. Conclusion: To date, multiple IV infusions in advanced cancer patients have been generally well tolerated. Initial serum viral load data indicate potential tumor-specific CVA21 replication in some patients. Overall, the preliminary data offer an exciting possibly that tumor targeting, infection and immune activation mediated by IV CVA21 may lead to increases in anti-tumor activity, particularly when in future used in combination with immune checkpoint blockade. Citation Format: Hardev Pandha, Kevin Harrington, Cristy Ralph, Alan Melcher, Darren R. Shafren. Intravenous delivery of a novel oncolytic immunotherapy agent, CAVATAK, in advanced cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT205. doi:10.1158/1538-7445.AM2015-CT205

Phase I/II storm study: Intravenous delivery of a novel oncolytic immunotherapy agent, Coxsackievirus A21, in advanced cancer patients

Meeting abstracts Coxsackievirus A21 (CVA21) is a naturally occurring “common cold” intercellular adhesion molecule-1 (ICAM-1)-targeted RNA virus. Surface ICAM-1 is up-regulated on a number of cancers including melanoma, non-small cell lung, bladder and prostate cancers. CAVATAK is a novel bio-

Butein suppresses ICAM-1 expression through the inhibition of IκBα and c-Jun phosphorylation in TNF-α- and PMA-treated HUVECs.

Butein (3,4,2',4'-tetrahydroxychalcone), a flavonoid derivative, has been reported to show several biological actions, including anti-inflammatory and anti-cancer. However, the possible molecular mechanisms involved are poorly understood. Treatment of human umbilical vein endothelial cells (HUVECs) with butein significantly inhibited cell surface intercellular adhesion molecule-1 (ICAM-1) expression, ICAM-1 protein synthesis, and mRNA expression induced by tumor necrotic factor-α (TNF-α) and/or phorbol 12-myristate 13-acetate (PMA). Electrophoretic mobility shift assay revealed that butein blocked activation of transcription factors, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), induced by TNF-α and PMA. Moreover, butein abolished TNF-α- and PMA-induced IκBα phosphorylation, which participates in NF-κB activation, and PMA-induced phosphorylation of c-Jun, a subunit composed of AP-1. In vitro, butein inhibited the phosphorylation of c-Jun, binding to GST beads, mediated by JNK isolated from PMA-treated cells. The inhibitory action of butein on the JNK-mediated in vitro c-Jun phosphorylation was abrogated in the presence of ATP. These results indicate that in HUVECs, butein suppresses the expression of ICAM-1 mRNA and protein through the inhibition of the activation of NF-κB and AP-1 induced by TNF-α and PMA, that the inhibitory action of butein on NF-κB activation results from the inhibition of IκBα phosphorylation by IκB kinase (IKK), and that the inactivation of PMA-activated AP-1 by butein is due to the blocking of JNK-mediated c-Jun phosphorylation through the inhibition of ATP binding.

Abstract 2939: CAVATAK-mediated oncolytic immunotherapy in advanced melanoma patients

Abstract Background: Coxsackievirus A21 (CVA21) is a naturally occurring “common cold” intercellular adhesion molecule-1 (ICAM-1) targeted RNA virus. Surface ICAM-1 is up-regulated on a number of cancers including melanoma, non-small cell lung, bladder, breast and prostate cancers. CAVATAK is a novel bio-selected formulation of CVA21, which displays potent oncolytic activity against in vitro cultures of cancer cells and in vivo xenografts of a number of cancers. In addition, in vivo tumor challenge studies in immune-competent mouse models have shown that CVA21 lysed tumor cells can induce a secondary systemic host-generated anti-tumor immune response. In this report, advanced melanoma patients were administered multiple intralesional injections of CAVATAK and monitored for markers of host anti-tumor activity on metastatic non-injected lesions. Methods: The Phase II CALM (CAVATAK in Late stage Melanoma) study is investigating the efficacy and safety of intratumoral CAVATAK in 54 advanced melanoma patients. Patient tumors are injected with up to 3 x 10e8 TCID50 CAVATAK on study days 1, 3, 5, 8 and 22 then every 3 weeks for a further 5 injections. CAVATAK-mediated immune anti-tumor activity was assessed by monitoring reductions in non-injected lesions (CT-scan and callipers) and assessing the relative serum levels of a panel of immune inflammatory cytokines. In addition, levels of serum CAVATAK and anti-CAVATAK neutralizing antibodies were assessed on multiple occasions prior to intralesional injections. Results: Intralesional CAVATAK injections resulted in significant responses in a number of melanoma lesions. However, in addition to melanoma tumor responses directly from CAVATAK-mediated oncolysis, a number of non-injected metatastic lesions displayed significant reductions or even complete destruction. Such anti-tumor activity in non-injected lesions is highly suggestive of being mediated via a host generated anti-tumor immune response. Such responses have occurred at times when no circulating infectious CAVATAK was detected in patient serum and in an environment of high levels of anti-CAVATAK neutralizing antibody. In further support of the generation of CAVATAK-mediated immune anti-tumor activity is the identification a possible novel serum cytokine signature of elevated levels of IL-8 and γ-IFN in treated patients linked to systemic tumor response. IL-8 is known to be secreted by activated macrophages, while γ-IFN is produced by stimulated cytotoxic T-cells / NK cells. Conclusion: With a number of patients in the Phase II CALM study displaying reductions in non-injected lesions, together with elevated serum levels of the immune inflammatory cytokines IL-8 and γ-IFN, at times with no detectable levels of circulating infectious CAVATAK, we conclude the generation of a targeted CAVATAK-mediated host anti-tumor response acting on metastatic melanoma lesions is highly likely. Clinical trial information: NCT01227551. Citation Format: Robert H. Andtbacka, Darren R. Shafren, Mark Grose, Len Post, Jeffrey Weisberg. CAVATAK-mediated oncolytic immunotherapy in advanced melanoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2939. doi:10.1158/1538-7445.AM2014-2939

ICAM-1 as a molecular target for triple negative breast cancer.

Triple negative breast cancers (TNBCs) have a high mortality rate owing to aggressive proliferation and metastasis and a lack of effective therapeutic options. Herein, we describe the overexpression of intercellular adhesion molecule-1 (ICAM-1) in human TNBC cell lines and tissues, and demonstrate that ICAM-1 is a potential molecular target and biomarker for TNBC therapy and diagnosis. We synthesized ICAM-1 antibody-conjugated iron oxide nanoparticles (ICAM-IONPs) as a magnetic resonance imaging (MRI) probe to evaluate tumor targeting. Quantitative analysis of ICAM-1 surface expression predicted the targeting capability of ICAM-IONPs to TNBC cells. MRI of the TNBC xenograft tumor after systemic administration of ICAM-IONPs, coupled with iron quantification and histology, demonstrated a significant and sustained MRI contrast enhancement and probe accumulation in tumors with ICAM-1 overexpression relative to control. Identification of ICAM-1 as a TNBC target and biomarker may lead to the development of a new strategy and platform for addressing a critical gap in TNBC patient care.

Effects of single and fractionated low-dose irradiation on vascular endothelial cells

ObjectiveAn increasing number of epidemiological studies suggest that chronic low-dose irradiation increases the risk of atherosclerosis. We evaluated and compared the in vitro biological effects of both single and fractionated low-doses of X-ray irradiation on endothelial cells. MethodsHuman umbilical vein endothelial cells (HUVECs) were irradiated with X-rays, with single doses of 0.125, 0.25 and 0.5 Gy or fractionated doses of 2 × 0.125 Gy and 2 × 0.25 Gy, with 24 h interfraction interval. Survival, apoptosis, reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activation, intercellular adhesion molecule-1 (ICAM-1) expression, HUVEC adhesiveness and DNA damage were investigated. ResultsWe did not observe any effect on viability and apoptosis. Both single and fractionated doses induced ROS generation, NF-κB activation, ICAM-1 protein expression and HUVEC adhesiveness, but only fractionated doses increase significantly ICAM-1 mRNA. The effects measured after fractionated dose result always higher than those induced by the single dose. Moreover, we observed that DNA double strand break (DSB), visualized with γ-H2AX foci, is dose-dependent and that the kinetics of γ-H2AX foci is not affected by fractionated doses. ConclusionsWe showed that single and fractionated low-dose irradiations with low energy X-rays do not affect cell viability and DNA repair. Interestingly, the greater increase of ICAM-1 surface exposure and endothelial adhesiveness observed after fractionated irradiation, suggests that fractionated low-doses may accelerate chronic vascular inflammation, from which the atherosclerotic process can arise.

Leptin-mediated regulation of ICAM-1 is Rho/ROCK dependent and enhances gastric cancer cell migration

Background:Our previous study indicates that leptin enhances gastric cancer (GC) invasion. However, the exact effect of leptin on GC metastasis and its underlying mechanism remain unclear. Intercellular adhesion molecule-1 (ICAM-1), a major molecule in stabilising cell–cell and cell–extracellular matrix interactions, is overexpressed and has crucial roles in tumour metastasis.Methods:Here, we investigated leptin and ICAM-1 expression in GC tissues. Furthermore, we characterised the influence of leptin on ICAM-1 expression in GC cells and elucidated the underlying mechanism.Results:Leptin and ICAM-1 were overexpressed in GC tissues, and a strong positive correlation was observed. They were also related with clinical stage or lymph node metastasis. Furthermore, leptin induced GC cell (AGS and MKN-45) migration by upregulating ICAM-1, and knockdown of ICAM-1 by small interference RNA (siRNA) blocked this process. Cell surface ICAM-1, as well as soluble ICAM-1 (sICAM-1), was also enhanced by leptin. Moreover, leptin increased ICAM-1 expression through Rho/ROCK pathway, which was attenuated by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated protein kinase (ROCK) (Y-27632).Conclusions:Our findings indicate that leptin enhances GC cell migration by increasing ICAM-1 through Rho/ROCK pathway, which might provide new insight into the significance of leptin in GC.

Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages

Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages