Activated Leukocyte Cell Adhesion Molecule Expression Research Articles

MiRNA-192-5p-targeted activated leukocyte cell adhesion molecule improved inflammatory injury of neonatal necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a common gastrointestinal emergency in neonates. MiRNA-192-5p was found associated with ulcerative colitis (UC) progression, also with aberrant expression in intestinal cancer tissue. However, the effects of miRNA-192-5p on NEC have not been reported. Based on the bioinformatics analysis of the GEO dataset, miR-192-5p was identified as the differentially expressed miRNA in NEC, and activated leukocyte cell adhesion molecule (ALCAM) was predicted as its target. After that, in vitro, rat intestinal epithelial cell-6 (IEC-6) were stimulated with LPS to construct a cell model of NEC. IEC-6 cells were transfected with miRNA-192-5p mimics, miRNA-192-5p inhibitors, or miRNA-192-5p inhibitors + sh-ALCAM, and relevant negative control. In vivo, SD rats were treated with artificial feeding, hypoxic reoxygenation, cold stimulation, and LPS gavage to induce NEC, followed by injection of agomiR-NC or agomiRNA-192-5p. Then effects of miRNA-192-5p on NEC model IEC-6 cell viability, apoptosis, ALCAM expression, Interleukin (IL)-1β and IL-6 levels, intestinal injury, intestinal permeability were detected. MiRNA-192-5p expression was downregulated in NEC IEC-6 cells, whose overexpression increased IEC-6 cell viability. MiRNA-192-5p inhibitors increased IL-1β, IL-6 levels and promoted IEC-6 cell apoptosis. MiRNA-192-5p targeting of ALCAM decreased ALCAM expression, IL-1β, and IL-6 levels. AgomiRNA-192-5p decreased ALCAM, IL-1β, and IL-6 levels in intestinal tissue and pathological damage and increased miRNA-192-5p levels. MiR-192-5p protects against intestinal injury by inhibiting ALCAM-mediated inflammation and intestinal epithelial cells, which would provide a new idea for NEC treatment.

Activated Leukocyte Cell Adhesion Molecule Regulates the Expression of Interleukin-33 in RSV Induced Airway Inflammation by Regulating MAPK Signaling Pathways.

The respiratory syncytial virus (RSV) is a common respiratory virus that causes acute lower respiratory tract infectious diseases, particularly in young children and older individuals. Activated leukocyte cell adhesion molecule (ALCAM) is a membrane glycoprotein expressed in various cell types, including epithelial cells, and is associated with inflammatory responses and various cancers. However, the precise role of ALCAM in RSV-induced airway inflammation remains unclear, and our study aimed to explore this gap in the literature. C57BL/6 wild-type, ALCAM knockout mice and airway epithelial cells were infected with RSV and the expression of ALCAM and inflammatory cytokines were measured. We also conducted further experiments using Anti-ALCAM antibody and recombinant ALCAM in airway epithelial cells. The expression levels of ALCAM and inflammatory cytokines increased in both RSV-infected mice and airway epithelial cells. Interestingly, IL-33 expression was significantly reduced in ALCAM-knockdown cells compared to control cells following RSV infection. Anti-ALCAM antibody treatment also reduced IL-33 expression following RSV infection. Furthermore, the phosphorylation of ERK1/2, p38, and JNK was diminished in ALCAM-knockdown cells compared to control cells following RSV infection. Notably, in the control cells, inhibition of these pathways significantly decreased the expression of IL-33. In vivo study also confirmed a reduction in inflammation induced by RSV infection in ALCAM deficient mice compared to wild-type mice. These findings demonstrate that ALCAM contributes to RSV-induced airway inflammation at least partly by influencing IL-33 expression through mitogen-activated protein kinase signaling pathways. These results suggest that targeting ALCAM could be a potential therapeutic strategy for alleviating IL-33-associated lung diseases.

Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM's T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM-CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM-ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.

Activated Leukocyte Cell Adhesion Molecule (ALCAM), a Potential ‘Seed’ and ‘Soil’ Receptor in the Peritoneal Metastasis of Gastrointestinal Cancers

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a cell-cell adhesion protein conferring heterotypic and homotypic interactions between cells of the same type and different types. It is aberrantly expressed in various cancer types and has been shown to be a regulator of cancer metastasis. In the present study, we investigated potential roles of ALCAM in the peritoneal transcoelomic metastasis in gastrointestinal cancers, a metastatic type commonly occurred in gastro-intestinal and gynaecological malignancies and resulting in poor clinical outcomes. Specifically, we studied whether ALCAM acts as both a 'seed' receptor in these tumour cells and a 'soil' receptor in peritoneal mesothelial cells during cancer metastasis. Gastric cancer and pancreatic cancer tissues with or without peritoneal metastasis were compared for their levels of ALCAM expression. The impact of ALCAM expression in these tumours was also correlated to the patients' clinical outcomes, namely peritoneal metastasis-free survival. In addition, cancer cells of gastric and pancreatic origins were used to create cell models with decreased or increased levels of ALCAM expression by genetic knocking down or overexpression, respectively. Human peritoneal mesothelial cells were also genetically transfected to generate cell models with different profiles of ALCAM expression. These cell models were used in the tumour-mesothelial interaction assay to assess if and how the interaction was influenced by ALCAM. Both gastric and pancreatic tumour tissues from patients who developed peritoneal metastases had higher levels of ALCAM transcript than those without. Patients who had tumours with high levels of ALCAM had a much shorter peritoneal metastasis free survival compared with those who had low ALCAM expression (p = 0.006). ALCAM knockdown of the mesothelial cell line MET5A rendered the cells with reduced interaction with both gastric cancer cells and pancreatic cancer cells. Likewise, levels of ALCAM in both human gastric and pancreatic cancer cells were also a determining factor for their adhesiveness to mesothelial cells, a process that was likely to be triggered the phosphorylation of the SRC kinase. A soluble ALCAM (sALCAM) was found to be able to inhibit the adhesiveness between cancer cells and mesothelial cells, mechanistically behaving like a SRC kinase inhibitor. ALCAM is an indicator of peritoneal metastasis in both gastric and pancreatic cancer patients. It acts as not only a potential peritoneal 'soil' receptor of tumour seeding but also a 'soil' receptor in peritoneal mesothelial cells during cancer metastasis. These findings have an important therapeutic implication for treating peritoneal transcoelomic metastases.

Expression of ALCAM in Clinical Colon Cancer and Relationship With Patients' Treatment Responses.

Activated leukocyte cell adhesion molecule (ALCAM) plays an important role in cancer via its homotypical and heterotypical interactions with ALCAM or other proteins and can also mediate cell-cell interactions. The present study investigated the expression of ALCAM in relation to epithelial-to-mesenchymal transition (EMT) markers and its downstream signal proteins including Ezrin-Moesin-Radixin (ERM), in clinical colon cancer and in the progression of the disease. Expression of ALCAM was determined in a clinical colon cancer cohort and assessed against the clinical pathological factors and outcome, together with the expression patterns of the ERM family and EMT markers. ALCAM protein was detected using immunohistochemistry. Cell line models, with ALCAM knock-down and over-expression, were established and used to test cells' responses to drugs. Tumours from patients who had distant metastasis and died of colon cancer had low levels of ALCAM. Dukes B and C tumours also had lower ALCAM expression than Dukes A tumours. Patients with high levels of ALCAM had a significantly longer overall and disease-free survival than those with lower ALCAM levels (p=0.040 and p=0.044). ALCAM is not only significantly correlated with SNAI1 and TWIST, also positively correlated with SNAI2. ALCAM enhanced the adhesiveness of colorectal cancer, an effect inhibited by both sALCAM and SRC inhibitors. Finally, high ALCAM expression rendered cells resistant, especially to 5-fluorouracil. Reduced expression of ALCAM in colon cancer is an indicator of disease progression and a poor prognostic indicator for patient's survival. However, ALCAM can enhance the adhesion ability of cancer cells and render them resistant to chemotherapy drugs.

Intrahepatic activated leukocyte cell adhesion molecule induces CD6highCD4+ T cell infiltration in autoimmune hepatitis.

Background and objectivesAutoimmune hepatitis (AIH) is characterized by the expansion and accumulation of pathogenic T cells in liver. Although CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) are involved in the evolution of multiple inflammatory diseases, their roles in the pathogenesis of AIH remain unknown. Herein, we aimed to investigate ALCAM-CD6 axis in AIH development.MethodsImmunohistochemistry was performed to examine hepatic expression of CD6 and ALCAM. The concentration of serum ALCAM was evaluated by ELISA. The phenotypes of liver infiltrating T cells were determined by flow cytometry. Primary human CD4+ T cells were used for functional studies.ResultsOur data showed that patients with AIH exhibited significantly higher expression of CD6 in the liver as compared to primary biliary cholangitis (PBC), chronic hepatitis B (CHB), non-alcoholic liver disease (NAFLD), and healthy controls (HC). In addition, hepatic CD6 expression was strongly correlated with disease severity of AIH. CD6 was mainly expressed on CD4+ T cells in the liver and intrahepatic CD6highCD4+ T cells demonstrated stronger proinflammatory response and proliferation features than CD6low counterparts in both AIH and HC. ALCAM, the ligand of CD6, was highly expressed in the hepatocytes of AIH and serum ALCAM was strongly associated with clinical indices of AIH. Interestingly, close spatial location between CD6+CD4+ T cells and ALCAM+ hepatocytes was observed. Finally, we found that CD6highCD4+ T cells showed enhanced capacity of trans-endothelial migration in vitro, which could be promoted by recombinant ALCAM.ConclusionsOur study found that ALCAM-CD6 axis was upregulated in the AIH liver, suggesting a potential target for alleviating AIH.

Elevated ALCAM Expression Associated with Endotypes and Postoperative Recurrence in Chronic Rhinosinusitis with Nasal Polyps.

BackgroundChronic rhinosinusitis with polyps (CRSwNP) is characterized by high heterogeneity and postoperative recurrence rate. This study aimed to explore the clinical significance of activated leukocyte cell adhesion molecule (ALCAM) in endotyping CRSwNP and predicting its recurrence.MethodsWe recruited 120 CRSwNP patients including 70 non-eosinophilic CRSwNP (neCRSwNP) and 50 eosinophilic CRSwNP (eCRSwNP) patients, and 40 healthy controls (HCs). Serum and tissue samples were collected. Serum ALCAM levels were detected by enzyme-linked immunosorbent assay (ELISA), and tissue ALCAM expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting (WB) and immunohistochemistry (IHC). The predictive values of ALCAM expression for CRSwNP endotypes and postoperative recurrence were assessed.ResultsThe serum levels of ALCAM were significantly increased in CRSwNP patients in comparison with HCs and were correlated with the peripheral eosinophil count, tissue eosinophil counts, and percentage. Multivariate analysis and receiver operating characteristic (ROC) curve highlighted that serum ALCAM levels were associated with CRSwNP endotypes. Tissue ALCAM expression was significantly enhanced in CRSwNP patients, especially in eCRSwNP patients. At the end of the study, 110 patients completed the follow-up schedule, 78 patients were categorized into the non-recurrent group, and the other 32 patients were included in the recurrent group. The serum ALCAM levels were elevated in the recurrent group compared with the non-recurrent group, and ALCAM expression in the tissue was significantly elevated. The ROC curve exhibited a high predictive ability of serum ALCAM in predicting postoperative recurrence. Logistic regression and Kaplan–Meier curves demonstrated that serum ALCAM was an independent risk factor for postoperative recurrence.ConclusionThis is the first report suggesting that ALCAM expression was upregulated and associated with mucosal eosinophil infiltration and CRSwNP recurrence. Serum ALCAM could be a promising biomarker for distinguishing endotypes and predicting postoperative recurrence in CRwNP patients.

The CD6/ALCAM pathway promotes lupus nephritis via T cell-mediated responses.

T cells are central to the pathogenesis of lupus nephritis (LN), a common complication of systemic lupus erythematosus (SLE). CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM), are involved in T cell activation and trafficking. Previously, we showed that soluble ALCAM is increased in urine (uALCAM) of patients with LN, suggesting that this pathway contributes to disease. To investigate, uALCAM was examined in 1038 patients with SLE and LN from 5 ethnically diverse cohorts; CD6 and ALCAM expression was assessed in LN kidney cells; and disease contribution was tested via antibody blockade of CD6 in murine models of SLE and acute glomerulonephritis. Extended cohort analysis offered resounding validation of uALCAM as a biomarker that distinguishes active renal involvement in SLE, irrespective of ethnicity. ALCAM was expressed by renal structural cells whereas CD6 expression was exclusive to T cells, with elevated numbers of CD6+ and ALCAM+ cells in patients with LN. CD6 blockade in models of spontaneous lupus and immune-complex glomerulonephritis revealed significant decreases in immune cells, inflammatory markers, and disease measures. Our data demonstrate the contribution of the CD6/ALCAM pathway to LN and SLE, supporting its use as a disease biomarker and therapeutic target.

ALCAM-EGFR interaction regulates myelomagenesis

AbstractMultiple myeloma, a plasma cell malignancy in the bone marrow, remains largely incurable with currently available therapeutics. In this study, we discovered that the activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR), and regulated myelomagenesis. ALCAM was a negative regulator of myeloma clonogenicity. ALCAM expression was positively correlated with patients' survival. ALCAM-knockdown myeloma cells displayed enhanced colony formation in the presence of bone marrow stromal cells (BMSCs). BMSCs supported myeloma colony formation by secreted epidermal growth factor (EGF), which bound with its receptor (EGFR) on myeloma cells and activated Mek/Erk cell signaling, PI3K/Akt cell signaling, and hedgehog pathway. ALCAM could also bind with EGFR, block EGF from binding to EGFR, and abolish EGFR-initiated cell signaling. Hence, our study identifies ALCAM as a novel negative regulator of myeloma pathogenesis.

Abstract PS17-48: Potential role of activated leukocyte cell adhesion molecule (ALCAM) in hepatocyte growth factor (HGF) signalling in vascular endothelial cells and implications in breast cancer

Abstract Background. Activated Leukocyte Cell Adhesion Molecule, ALCAM (also known as CD166) is a member of the immunoglobulin superfamily and a cell surface adhesion molecule involved in heterophilic and homophilic interactions during cell-cell adhesion in epithelial cells, cancer cells and endothelial cells. ALCAM appears to be linked to tumour progression in a number of cancers including breast cancer, though there are conflicting reports as to its precise prognostic implications and significance. We have previously reported that ALCAM has a tumour suppressive role in breast cancer and is inversely correlated with clinical outcome (1) and interestingly bone metastasis of breast cancer (2). We have also reported that ALCAM has a diverse role in other cell types including keratinocytes and endothelial cells. In the present study, we investigated the association between ALCAM and hepatocyte growth factor (HGF)/cMET, a signalling pathway established as a key promoter of cancer progression, influencing both the aggressive nature of cancer cells and acting as an angiogenic and lymphangiogenic factor, in breast cancer and endothelial cells. Methods. The expression pattern and correlation of ALCAM, HGF and cMET in human breast cancer were deduced from a clinical breast cancer cohort. ALCAM manipulated HECV cell lines, previously established in our laboratories, were used in conjunction with recombinant human HGF and cMET inhibitors to assess cellular functions and the implications of this relationship at a cellular level. Results. We compared the expression of ALCAM with that of HGF and cMET within the breast cancer cohort and found that ALCAM/CD166 showed a highly significant negative correlation with the HGF receptor cMET (r=-0.17, p<0.0001) and a weak negative correlation with HGF, although this was not significant. ALCAM was not found to correlate with its heterophilic partner CD6. In previously generated HECV models, ALCAM manipulation showed a marked influence on cellular function and responsiveness to HGF treatment. Discussion. ALCAM’s role in breast cancer progression and related mechanisms is complex. Our current data suggests this may involve an interaction with the cMET/HGF signalling pathway and may also impact the endothelial component within the tumour microenvironment to influence disease progression. Reference1 King JA, Ofori-Acquah SF, Stevens T, et al. Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator. Breast Cancer Res. 2004;6:R478-87. Reference2 Davies SR, Dent C, Watkins G et al. Expression of the cell to cell adhesion molecule, ALCAM, in breast cancer patients and the potential link with skeletal metastasis. Oncol Rep. 2008;19:555-61. Citation Format: Andrew James Sanders, David Guo Jiang, Jianyuan Zeng, Robert Edward Mansel, Eleri Davies, Amber Xinyu Li, Keith Gordon Harding, Wen Guo Jiang. Potential role of activated leukocyte cell adhesion molecule (ALCAM) in hepatocyte growth factor (HGF) signalling in vascular endothelial cells and implications in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-48.

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion.

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.

A novel HDGF-ALCAM axis promotes the metastasis of Ewing sarcoma via regulating the GTPases signaling pathway.

Ewing sarcoma (ES) is a type of highly aggressive pediatric tumor in bones and soft tissues and its metastatic spread remains the most powerful predictor of poor outcome. We previously identified that the transcription factor hepatoma-derived growth factor (HDGF) promotes ES tumorigenesis. However, the mechanisms underlying ES metastasis remain unclear. Here, we show that HDGF drives ES metastasis in vitro and in vivo, and HDGF reduces metastasis-free survival (MFS) in two independent large cohorts of human ES patients. Integrative analyses of HDGF ChIP-seq and gene expression profiling in ES cells reveal that HDGF regulates multiple metastasis-associated genes, among which activated leukocyte cell adhesion molecule (ALCAM) emerges as a major HDGF target and a novel metastasis-suppressor in ES. HDGF down-regulates ALCAM, induces expression and activation of the downstream effectors Rho-GTPase Rac1 and Cdc42, and promotes actin cytoskeleton remodeling and cell-matrix adhesion. In addition, repression of ALCAM and activation of Rac1 and Cdc42 are required for the pro-metastatic functions of HDGF in vitro. Moreover, analyses in murine models with ES tumor orthotopic implantation and experimental metastasis, as well as in human ES samples, demonstrate the associations among HDGF, ALCAM, and GTPases expression levels. Furthermore, high HDGF/low ALCAM expression define a subgroup of patients harboring the worst MFS. These findings suggest that the HDGF/ALCAM/GTPases axis represents a promising therapeutic target for limiting ES metastasis.

A novel molecular magnetic resonance imaging agent targeting activated leukocyte cell adhesion molecule as demonstrated in mouse brain metastasis models

Molecular magnetic resonance imaging (MRI) allows visualization of biological processes at the molecular level. Upregulation of endothelial ALCAM (activated leukocyte cell adhesion molecule) is a key element for leukocyte recruitment in neurological disease. The aim of this study, therefore, was to develop a novel molecular MRI contrast agent, by conjugating anti-ALCAM antibodies to microparticles of iron oxide (MPIO), for detection of endothelial ALCAM expression in vivo. Binding specificity of ALCAM-MPIO was demonstrated in vitro under static and flow conditions. Subsequently, in a proof-of-concept study, mouse models of brain metastasis were induced by intracardial injection of brain-tropic human breast carcinoma, lung adenocarcinoma or melanoma cells to upregulate endothelial ALCAM. At selected time-points, mice were injected intravenously with ALCAM-MPIO, and ALCAM-MPIO induced hypointensities were observed on T2*-weighted images in all three models. Post-gadolinium MRI confirmed an intact blood-brain barrier, indicating endoluminal binding. Correlation between endothelial ALCAM expression and ALCAM-MPIO binding was confirmed histologically. Statistical analysis indicated high sensitivity (80–90%) and specificity (79–83%) for detection of endothelial ALCAM in vivo with ALCAM-MPIO. Given reports of endothelial ALCAM upregulation in numerous neurological diseases, this advance in our ability to image ALCAM in vivo may yield substantial improvements for both diagnosis and targeted therapy.

Amelioration of skin and kidney disease in a spontaneous murine lupus model via CD6 modulation

Abstract Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that can affect multiple organ systems, including the kidneys, skin, and brain. T cells are an important mediator in this end organ damage. CD6 is a co-stimulatory receptor, predominantly expressed on T cells, which binds with activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. This signaling pathway is vital for T cell activation, proliferation, differentiation and trafficking. We found increased expression of both CD6 and ALCAM in the kidneys of MRL/lpr mice (a spontaneous model of SLE) versus healthy control B6 mice. In a separate experiment, female MRL/lpr mice were aged to 9–10 weeks of age, at which point we began treating with either anti-CD6 antibody (60 ug/dose, intraperitoneally twice per week), isotype control (60 ug/dose, twice per week), or cyclophosphamide (25 mg/kg, once per week). We also included a no treatment group and a group of MRL/MpJ mice, a congenic healthy control strain. Mice treated with anti-CD6 show lower levels of proteinuria and BUN (p&amp;lt;0.05), improved survival rates, and decreased renal pathology compared to isotype control mice. Flow cytometry revealed decreased numbers of activated and effector T cells within the kidneys of anti-CD6 treated mice compared to isotype control mice. While there was no difference in anti-DNA levels, anti-CD6 treatment significant improved the spontaneous skin lesions associated with disease progression. Overall, these results indicate that targeting CD6-ALCAM interactions may have promising therapeutic potential within the context of different end organ pathologies within lupus.

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium.

BackgroundBrain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC.MethodsImmunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice.ResultsALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors.ConclusionsOur findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM.Key Points1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC.2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium.3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

Anti-CD6 Monoclonal Antibody Itolizumab Efficiently Inhibits T Cell Proliferation after in Vitro TCR Stimulation in the Setting of Acute Graft Versus Host Disease

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon&gt;CD8&gt;Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

Expression of melanoma cell adhesion molecule-1 (MCAM-1) in natalizumab-treated multiple sclerosis

The objectives were to study the expression of very late antigen (VLA)-4, melanoma cell adhesion molecule-1 (MCAM-1) and activated leukocyte cell adhesion molecule (ALCAM) on CD4+ T cells during natalizumab treatment and to investigate the association with disease activity.We find that subgroups of autoreactive T cells are retained in peripheral blood, in particular MOG-reactive CD4+ T cells expressing MCAM-1. The expression of MCAM-1 or ALCAM on CD4+ T cells was, however, not clearly associated with disease activity (clinical or MRI) during natalizumab treatment. We confirm upregulation of MCAM-1 on CD4+ T cells during natalizumab treatment while VLA-4 is downregulated.

Decreased ALCAM expression and promoter hypermethylation is associated with preeclampsia.

Preeclampsia (PE) is a major obstetrical complication that results in maternal and fetal morbidity and mortality. Aberrant epigenetic modifications are widely involved in the pathogenesis of PE. Previously, the activated leukocyte cell adhesion molecule (ALCAM) was reported to be required for blastocyst implantation but has not been described in the context of pathological pregnancy. This study explored the expression of ALCAM and its methylation levels in the placentas and peripheral venous blood of patients with PE from a Chinese Han population. The mRNA and protein expression levels of ALCAM were downregulated in the PE placentas compared with the control placentas (P < 0.05). The methylation rate of the ALCAM gene promoter was considerably elevated in the placentas (P = 0.003, odds ratio (OR) = 0.264, 95% confidence interval (95% CI) [0.108-0.647], cases n = 47, controls n = 53) and peripheral blood (P = 0.007, OR = 0.455, 95% CI [0.256-0.806], cases n = 100, controls n = 100) of the PE patients compared with those of the normotensive women, suggesting a negative relationship between ALCAM methylation and gene transcription. Moreover, the transcriptional expression of ALCAM was dramatically increased by demethylating treatment in trophoblastic cells. ALCAM is expected to be involved in the pathogenesis of PE through methylation regulation.

Abstract 1114: Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases

Abstract Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related death with still a 5-year survival rate of &amp;lt;10%. Every second patient with advanced NSCLC will develop brain metastases and in 50% of those the brain is the only site of tumor relapse (oligo-metastasis). Cancer cells need to have specific properties to access and colonize the brain microenvironment leading to the question which metastatic factors foster brain metastasis formation. Different cell adhesion molecules (CAMs) have been shown to be involved in the process of metastasis in different tumor entities. The activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein of the immunoglobulin superfamily and involved in the maintenance of cell-cell contacts and has a possible role in the “homing” of metastatic cells in distant organs. ALCAM is also involved in the leukocyte transmigration across the blood-brain barrier. Here we therefore investigated the role of ALCAM in NSCLC brain metastases formation. Our studies on patient material revealed that the ALCAM protein expression in brain metastases (n=71) is significantly increased (50.7% positive, p=0.023) compared to the primary tumor (21.7% positive, n=47). Analysis of matched pairs of primary tumors and brain metastases confirmed this observation, as in 33% a de novo ALCAM expression in the matching brain metastases could be observed. Additionally, patients with a strong ALCAM expression in either their primary tumors or brain metastases showed a significantly shortened overall survival (p=0.017 and p=0.035 respectively). In vitro analysis of the effect of ALCAM knock out in the H460 lung cancer cell line showed no effect on proliferation, migration or colony-forming behavior. However, cell adhesion was severely hampered in the knock out cells. Currently in vivo mice experiments are running. Together these findings indicate an important role for ALCAM in brain metastasis formation. Citation Format: Desiree Loreth, Justine Münsterberg, Cecile Maire, Stefan Werner, Monja Gandrass, Christian Bernreuther, Jolanthe Kropidlowski, Manfred Westphal, Stefan Steurer, Katrin Lamszus, Markus Glatzel, Klaus Pantel, Harriet Wikman. Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases [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 1114.

Modulation of cell adhesion and migration through regulation of the immunoglobulin superfamily member ALCAM/CD166.

In epithelial-derived cancers, altered regulation of cell-cell adhesion facilitates the disruption of tissue cohesion that is central to the progression to malignant disease. Although numerous intercellular adhesion molecules participate in epithelial adhesion, the immunoglobulin superfamily (IgSF) member activated leukocyte cell adhesion molecule (ALCAM), has emerged from multiple independent studies as a central contributor to tumor progression. ALCAM is an archetypal member of the IgSF with conventional organization of five Ig-like domains involved in homo- and heterotypic adhesions. Like many IgSF members, ALCAM is broadly expressed and involved in cellular adhesion across many cellular processes. While the redundancy of intercellular adhesion molecules (CAMs) could diminish the impact of any single CAM, consistent correlation between ALCAM expression and patient outcome for multiple cancers underscores its role in tumor progression. Unlike most oncogenes and tumor suppressors, ALCAM is neither mutated nor amplified or deleted. Experimental disruption of ALCAM-mediated adhesions implies that this IgSF member contributes to tumor progression through dynamic turnover of the protein at the cell surface. Since ALCAM is not frequently altered at the gene level, it appears to promote malignant behavior through regulation of its availability rather than its specific activity. These observations help explain its heterogeneous expression within malignant disease and the drastic changes in protein levels across tumor progression. To reveal how ALCAM contributes to tumor progression, we review regulation of its gene expression, alternative splicing, targeted proteolysis, binding partners, and surface shedding within the context of cancer. Studying ALCAM regulation has led to a novel understanding of the fine-tuning of cell adhesive state through the utilization of otherwise normal regulatory processes, which thereby enable tumor cell invasion and metastasis.