Cell Transendothelial Migration Research Articles

Epac activation reduces trans-endothelial migration of undifferentiated neuroblastoma cells and cellular differentiation with a CDK inhibitor further enhances Epac effect.

Neuroblastoma (NB) is the most common solid extracranial neoplasm found in children and is derived from primitive sympathoadrenal neural precursor. The disease accounts for 15% of all cancer deaths in children. The mortality rate is high in patients presenting with a metastatic tumour even with extensive treatments. This signifies the need for further research towards the development of new additional therapies that can combat not only tumour growth but metastasis, especially amongst the high-risk groups. During metastasis, primary tumour cells become migratory and travel towards a capillary within the tumour. They then degrade the matrix surrounding the pericytes and endothelial cells traversing the endothelial barrier twice to establish a secondary. This led to the hypothesis that modulation of the endothelial cell junctional stability could have an influence on tumour metastasis. To test this hypothesis, agents that modulate endothelial permeability on NB cell line migration and invasion were assessed in vitro in a tissue culture model. The cAMP agonist and its antagonists were found to have no obvious effect on both SK-N-BE2C and SK-N-AS migration, invasion and proliferation. Next, NB cells were cocultured with HDMEC cells and live cell imaging was used to assess the effect of an Epac agonist on trans-endothelial cell migration of NB cells. Epac1 agonist remarkably reduced the trans-endothelial migration of both SK-N-BE2C and SK-N-AS cells. These results demonstrate that an Epac1 agonist may perhaps serve as an adjuvant to currently existing therapies for the high-risk NB patients.

Tumour cell-released autophagosomes promote lung metastasis by upregulating PD-L1 expression in pulmonary vascular endothelial cells in breast cancer.

Establishing an immunosuppressive premetastatic niche (PMN) in distant organs is crucial for breast cancer metastasis. Vascular endothelial cells (VECs) act as barriers to transendothelial cell migration. However, the immune functions of PMNs remain unclear. Tumour cell-released autophagosomes (TRAPs) are critical modulators of antitumour immune responses. Herein, we investigated the mechanism through which TRAPs modulate the immune function of pulmonary VECs in lung PMN in breast cancer. Immortalised mouse pulmonary microvascular endothelial cells were incubated with TRAPs in vitro. RNA sequencing, flow cytometry, and western blotting were employed to assess immunosuppressive function and mechanism. In vivo, TRAP-trained and autophagy-deficient tumour mice were used to detect immunosuppression, and high-mobility group box 1 (HMGB1)-deficient TRAP-trained and TLR4 knockout mice were utilised to investigate the underlying mechanisms of pulmonary VECs. Additionally, the efficacy of anti-programmed cell death ligand-1 (PD-L1) immunotherapy was evaluated in early tumour-bearing mice. HMGB1 on TRAPs surfaces stimulated VECs to upregulate PD-L1 via a TLR4-MyD88-p38/STAT3 signalling cascade that depended on the cytoskeletal movement of VECs. Importantly, PD-L1 on TRAP-induced VECs can inhibit T cell function, promote lung PMN immunosuppression, and result in more pronounced lung metastasis. Treatment with anti-PD-L1 reduces lung metastasis in early stage tumour-bearing mice. These findings revealed a novel role and mechanism of TRAP-induced immunosuppression of pulmonary VECs in lung PMN. TRAPs and their surface HMGB1 are important therapeutic targets for reversing immunosuppression, providing a new theoretical basis for the treatment of early stage breast cancer using an anti-PD-L1 antibody.

Zedoarondiol inhibits monocyte adhesion and expression of VCAM and ICAM in endothelial cells induced by oxidative stress

Zedoarondiol, a newly discovered compound derived from the roots of zedoary turmeric, a traditional Chinese herb, has demonstrated potential in reducing inflammation of the vascular endothelium and safeguarding it from harm. Nonetheless, the precise mechanism underlying these effects remains to be elucidated. In this study, we established a model of HUVEC injury induced by hydrogen peroxide. We observed whether Zedoarondiol could reduce the adhesion and transendothelial migration (TEM) of inflammatory cells by inhibiting the expression of VCAM-1 and ICAM-1 in HUVECs. The research findings indicate that utilising Zedoarondiol resulted in a significant reduction in the adhesion rate of THP1 cells to HUVECs, leading to a more condensed cytoskeletal structure. Moreover, Zedoarondiol demonstrated a decrease in NF-κBβ-Ser536 phosphorylation levels in H2O2-stimulated human umbilical vein endothelial cells, resulting in a hindered capacity to bind to target genes like ICAM-1 and VCAM-1, This findings may provide a new pharmacological basis and scientific evidence for Zedoarondiol to slow the atherosclerosis progression by maintaining endothelial function.

Tumor-induced endothelial RhoA activation mediates tumor cell transendothelial migration and metastasis.

The endothelial barrier plays an active role in transendothelial tumor cell migration during metastasis, however, the endothelial regulatory elements of this step remain obscure. Here we show that endothelial RhoA activation is a determining factor during this process. Breast tumor cell-induced endothelial RhoA activation is the combined outcome of paracrine IL-8-dependent and cell-to-cell contact β 1 integrin-mediated mechanisms, with elements of this pathway correlating with clinical data. Endothelial-specific RhoA blockade or in vivo deficiency inhibited the transendothelial migration and metastatic potential of human breast tumor and three murine syngeneic tumor cell lines, similar to the pharmacological blockade of the downstream RhoA pathway. These findings highlight endothelial RhoA as a potent, universal target in the tumor microenvironment for anti-metastatic treatment of solid tumors.

Combination of JAKi and HDACi Exerts Antiangiogenic Potential in Cutaneous T-Cell Lymphoma.

Angiogenesis plays a pivotal role in the growth and metastasis of tumors, including the development and progression of cutaneous lymphomas. The chick embryo CAM model has been utilized in various studies to assess the growth rate, angiogenic potential, and metastatic capability of different tumor types and malignant cell lines. However, the precise mechanisms of angiogenesis in CTCL and the influence of Ruxolitinib or Resminostat on angiogenesis in hematological malignancies and solid tumors are not well understood. Recent in vitro and in vivo data have demonstrated the synergistic inhibition of tumorigenesis and metastasis in experimental models of CTCL when using the combination of Resminostat (HDACi) with Ruxolitinib (JAKi). The present work aims to elucidate the effects of this combination on the tumor microenvironment's vascular components. We investigated the effects of Ruxolitinib (JAKi) in combination with Resminostat (HDACi) treatment in transendothelial migration of CTCL cells (106 MyLa and SeAx) by using a transwell-based transendothelial migration assay and tumor angiogenesis in vivo. We used the CTCL chick embryo CAM model with xenografted tumors derived from implanted MyLa and SeAx cells and administered topically 15 μM ruxolitinib and 5 μM Resminostat every two days during a 5-day period. JAKi and HDACi inhibited CTCL cell transendothelial migration by 75% and 82% (p < 0.05) in both CTCL engrafted cells (MyLa and SeAx, respectively) compared to the untreated group. Moreover, the combination of ruxolitinib with resminostat blocked angiogenesis by significantly reducing the number of blood vessel formation by 49% and 34% in both MyLa and SeAx, respectively (p < 0.05), indicating that the proposed combination exerted significant anti-angiogenic effects in the CAM CTCL model. Overall, these data provide valuable insights into potential therapeutic strategies targeting angiogenesis in CTCL, paving the way for more effective treatment approaches in the future.

Impact of oral melatonin supplementation on urine and serum melatonin concentrations and quality-of-life measures in persons with relapsing multiple sclerosis

IntroductionMelatonin is an antioxidant and anti-inflammatory agent that modulates the immune system by scavenging free radicals, reducing the upregulation of pro-inflammatory cytokines, and reducing transendothelial cell migration. Therefore, melatonin may play a role in regulating multiple sclerosis (MS) disease activity. However, little is known about how melatonin supplementation effects individuals with MS. ObjectiveDetermine if there was a dose-dependent elevation in urine and serum melatonin concentrations. Determine if melatonin supplementation had an impact on patient reported outcomes. MethodsThis was a randomized, dose-blinded exploratory study. Adults (age 18–65) with relapsing forms of multiple sclerosis (RMS) treated with a stable dose of oral disease modifying therapy for at least 6 months were randomized into melatonin 3 mg or 5 mg daily. Urinary and serum melatonin levels and modified fatigue impact scale (MFIS), multiple sclerosis impact scale (MSIS-29), and Pittsburgh sleep quality index (PSQI), patient determined disease steps (PDDS) and performance scales (PS) were measured at baseline, 3, 6, and 12 months. Urinary and serum melatonin analyses was performed to estimate mean concentrations and their differences between treatment arms over time by a repeated measures linear mixed model. The model included treatment, assessment time, and treatment × time interaction. ResultsThirty patients, randomized 1:1, were analyzed in an intent to treat population. Twenty-three completed the study. The repeated measures linear mixed model analysis of all timepoints revealed higher melatonin concentrations in patients on 5 mg compared to 3 mg melatonin for both urinary 6-SMT (p = 0.03) and serum melatonin (p = 0.04). MFIS, MSIS-29, PSQI, and PDSS-PS scores did not significantly change from baseline to month 12. No significant differences in these measures were seen between the two doses. Five patients stopped melatonin (three on 5 mg and two on 3 mg) due to adverse events, including one patient who developed focal spongiotic dermatitis. One patient experienced three consecutive serious adverse events that were unrelated to melatonin supplementation. ConclusionsThe 5 mg melatonin supplementation group had higher concentrations of urinary 6-SMT and serum melatonin compared to the 3 mg group over 12 months of treatment. There was a correlation between 6-SMT and serum melatonin concentrations. This suggests that measuring serum melatonin is a reliable alternative to measuring urinary 6-SMT. However, no differences in clinical benefit between the two dosage groups were demonstrated in the patient reported outcomes. Trial Registration NumberNCT03498131

Apelin modulates inflammation and leukocyte recruitment in experimental autoimmune encephalomyelitis

Demyelination due to autoreactive T cells and inflammation in the central nervous system are principal features of multiple sclerosis (MS), a chronic and highly disabling human disease affecting brain and spinal cord. Here, we show that treatment with apelin, a secreted peptide ligand for the G protein-coupled receptor APJ/Aplnr, is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Apelin reduces immune cell entry into the brain, delays the onset and reduces the severity of EAE. Apelin affects the trafficking of leukocytes through the lung by modulating the expression of cell adhesion molecules that mediate leukocyte recruitment. In addition, apelin induces the internalization and desensitization of its receptor in endothelial cells (ECs). Accordingly, protection against EAE major outcomes of apelin treatment are phenocopied by loss of APJ/Aplnr function, achieved by EC-specific gene inactivation in mice or knockdown experiments in cultured primary endothelial cells. Our findings highlight the importance of the lung-brain axis in neuroinflammation and indicate that apelin targets the transendothelial migration of immune cells into the lung during acute inflammation.

Double-faced CX3CL1 enhances lymphangiogenesis-dependent metastasis in an aggressive subclone of oral squamous cell carcinoma.

Because cancer cells have a genetically unstable nature, they give rise to genetically different variant subclones inside a single tumor. Understanding cancer heterogeneity and subclone characteristics is crucial for developing more efficacious therapies. Oral squamous cell carcinoma (OSCC) is characterized by high heterogeneity and plasticity. On the other hand, CX3C motif ligand 1 (CX3CL1) is a double-faced chemokine with anti- and pro-tumor functions. Our study reported that CX3CL1 functioned differently in tumors with different cancer phenotypes, both in vivo and in vitro. Mouse OSCC 1 (MOC1) and MOC2 cells responded similarly to CX3CL1 in vitro. However, in vivo, CX3CL1 increased keratinization in indolent MOC1 cancer, while CX3CL1 promoted cervical lymphatic metastasis in aggressive MOC2 cancer. These outcomes were due to double-faced CX3CL1 effects on different immune microenvironments indolent and aggressive cancer created. Furthermore, we established that CX3CL1 promoted cancer metastasis via the lymphatic pathway by stimulating lymphangiogenesis and transendothelial migration of lymph-circulating tumor cells. CX3CL1 enrichment in lymphatic metastasis tissues was observed in aggressive murine and human cell lines. OSCC patient samples with CX3CL1 enrichment exhibited a strong correlation with lower overall survival rates and higher recurrence and distant metastasis rates. In conclusion, CX3CL1 is a pivotal factor that stimulates the metastasis of aggressive cancer subclones within the heterogeneous tumors to metastasize, and our study demonstrates the prognostic value of CX3CL1 enrichment in long-term monitoring in OSCC.

Fuyuan decoction prevents nasopharyngeal carcinoma metastasis by inhibiting circulating tumor cells/ endothelial cells interplay and enhancing anti-cancer immune response.

Distant metastasis is a major cause of treatment failure in cancer patients and a key challenge to improving cancer care today. We hypothesized that enhancing anti-cancer immune response and inhibiting circulating tumor cells (CTCs) adhesion and transendothelial migration through synergistic multi-target approaches may effectively prevent cancer metastasis. "Fuyuan Decoction" (FYD) is a traditional Chinese medicine compound that is widely used to prevent postoperative metastasis in cancer patients, but its underlying mechanism remains unclear. In this work, we systematically elucidated the underlying molecular mechanism by which FYD prevents cancer metastasis through multi-compound and multi-target synergies in vitro and in vivo. FYD significantly prevented cancer metastasis at non-cytotoxic concentrations by suppressing the adhesion of CTCs to endothelial cells and their subsequent transendothelial migration, as well as enhancing anti-cancer immune response. Mechanistically, FYD interrupts adhesion of CTCs to vascular endothelium by inhibiting TNF-α-induced CAMs expression via regulation of the NF-κB signaling pathway in endothelial cells. FYD inhibits invasion and migration of CTCs by suppressing EMT, PI3K/AKT and FAK signaling pathways. Moreover, FYD enhances the anti-cancer immune response by significantly increasing the population of Tc and NK cells in the peripheral immune system. In addition, the chemical composition of FYD was determined by UPLC-HRMS, and the results indicated that multiple compounds in FYD prevents cancer metastasis through multi-target synergistic treatment. This study provides a modern medical basis for the application of FYD in the prevention of cancer metastasis, and suggesting that multi-drug and multi-target synergistic therapy may be one of the most effective ways to prevent cancer metastasis.

IGSF3 is a homophilic cell adhesion molecule that drives lung metastasis of melanoma by promoting adhesion to vascular endothelium.

The immunoglobulin superfamily (IgSF) is one of the largest families of cell-surface molecules involved in various cell-cell interactions, including cancer-stromal interactions. In this study, we undertook a comprehensive RT-PCR-based screening for IgSF molecules that promote experimental lung metastasis in mice. By comparing the expression of 325 genes encoding cell-surface IgSF molecules between mouse melanoma B16 cells and its highly metastatic subline, B16F10 cells, we found that expression of the immunoglobulin superfamily member 3 gene (Igsf3) was significantly enhanced in B16F10 cells than in B16 cells. Knockdown of Igsf3 in B16F10 cells significantly reduced lung metastasis following intravenous injection into C57BL/6 mice. IGSF3 promoted adhesion of B16F10 cells to vascular endothelial cells and functioned as a homophilic cell adhesion molecule between B16F10 cells and vascular endothelial cells. Notably, the knockdown of IGSF3 in either B16F10 cells or vascular endothelial cells suppressed the transendothelial migration of B16F10 cells. Moreover, IGSF3 knockdown suppressed the extravasation of B16F10 cells into the lungs after intravenous injection. These results suggest that IGSF3 promotes the metastatic potential of B16F10 cells in the lungs by facilitating their adhesion to vascular endothelial cells.

Cellular adhesion molecules in drug-naïve and previously medicated patients with schizophrenia-spectrum disorders

BackgroundEndothelial inflammation may be involved in the pathogenesis of schizophrenia, and cellular adhesion molecules (CAMs) on endothelial cells may facilitate leukocyte binding and transendothelial migration of cells and inflammatory factors. The aim of the present study was to assess levels of soluble cellular adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, mucosal addressin cell adhesion molecule (MADCAM), junctional adhesion molecule (JAM-A) and neural cadherin (N-CAD) in patients with schizophrenia compared to healthy controls. MethodsThe study population consists of 138 patients with schizophrenia-spectrum disorder, of whom 54 were drug-naïve, compared to 317 general population controls. The potential confounders age, gender, smoking and body mass index (BMI) were adjusted for in linear regression models. ResultsThe total patient group showed significantly higher levels of ICAM-1 (p < 0.001) and VCAM-1 (p < 0.001) compared to controls. Previously medicated patients showed higher ICAM-1 levels compared to drug-naïve patients (p = 0.042) and controls (p < 0.001), and elevated VCAM-1 levels compared to controls (p < 0.001). Drug-naive patients had elevated levels of VCAM-1 (p = 0.031) compared to controls. ConclusionsIn our study, patients with schizophrenia – including the drug-naïve – have higher levels of soluble CAMs compared to healthy controls. These findings suggest activation of the endothelial system as in inflammation.

Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

Abstract 1549: Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer

Abstract Background: CD146, an adhesion molecule involved in inflammatory regulation, is linked to increased aggressiveness in Triple Negative Breast Cancer (TNBC). Yet, the molecular mechanisms of CD1146-driven TNBC metastasis in an inflammatory context remain elusive. We synthesized CD146 nanobody (anti-CD146 #112-2), which degraded CD146 protein specifically, inhibiting IL-6-mediated JAK/STAT3 activation and suppressing TNBC metastasis. This study aims to elucidate the mechanisms of CD146 degradation by 112-2 and the role of CD146 in mediating inflammatory responses leading to TNBC metastasis. Methods: CD146 knockout and overxpression cell models were created using CRISPR/Cas9 or lentiviral techniques. Lung metastasis was evaluated by injecting luciferase-labeled cells into the tail veins of Balb/c-nu mice, monitored with the IVIS Kinetic system. The impact of CD146 on TNBC metastasis in an inflammatory environment was explored through cell-adhesion assays and trans-endothelial migration assays using CD146 knockout models and HUVEC cells. Transwell assays assessed the influence of IL-6 and 112-2 on TNBC. CD146 mutant vectors with specific truncations were generated to confirm the binding sites between CD146 and 112-2. ELISA and immunofluorescence assays examined CD146 protein internalization. CD146 degradation by 112-2 was confirmed using proteasome inhibitor MG132 and lysosome inhibitor Bafilomycin A1, along with western blotting and immunofluorescence experiments. Results: Stimulation of TNBC cells with IL-6 increased CD146 expression and tumor metastasis. Knocking down CD146 significantly reduced IL-6 secretion by TNBC cells, leading to reduced tumor-endothelial cell adhesion and trans-endothelial migration. CD146 knockout or 112-2 treatment inhibited IL-6-mediated JAK/STAT3 activation and tumor metastasis. Experiments with CD146 mutants and molecular docking identified the specific 112-2 binding site at leucine positions 331-332 of CD146 protein. 112-2 treatment led to CD146 protein internalization and cleavage, resulting in an 80 kDa fragment recognized by both 112-2 and C-terminus-specific CD146 antibodies, ultimately inhibiting the activation of IL-6/JAK/STAT3 signaling. Notably, the inhibition of proteasome or lysosome activity failed to reverse the 112-2-induced reduction in CD146 protein levels. However, inhibiting lysosomal acidification resulted in significant co-localization of CD146 with lysosomes, suggesting a potential role of lysosomes in 112-2-mediated cleavage of CD146. Conclusion: CD146 promotes TNBC metastasis by modulating IL-6/JAK/STAT3 activation. The nanobody anti-CD146 #112-2 specifically binds to CD146 extracellular domain induces the proteolysis of CD146 protein, thereby inhibiting the tumor-promoting effect of IL-6. Citation Format: Kaiyuan Huang, Hexing Sun, Chengyu Wu, Danping Lin, Jinling Chen, Yanhan Chen, Xinqiang Fang, Shuzhao Chen, Yuanke Liang, Haoyu Lin. Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer [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 1549.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

OP30 Endothelial cell-mediated smooth muscle hyperplasia in Crohn’s disease intestinal strictures: Caveolin 1 as a potential therapeutic target

Abstract Background Intestinal stricture is a severe and common complication of Crohn’s disease (CD). Current therapies offer limited success and surgery is often needed. Smooth muscle hyperplasia and hypertrophy play a key role in CD stricture development. This study explores the transcriptomic features of fibrostenotic CD, focusing on the endothelial contribution to smooth muscle changes within intestinal strictures. Methods 9 CD patients undergoing surgery for fibrotic strictures were prospectively enrolled. Dissected samples from the stricture and the adjacent proximal and distal non-strictured areas were obtained from fresh specimens collected directly from the operating theatre. RNA was extracted from all samples for bulk-transcriptomic analysis. Differentially expressed genes (adj. p-value &amp;lt;0.05) were identified and pathway enrichment analysis pinpointed relevant pathways. Subsequently, single-cell RNA sequencing (scRNAseq) analysis was performed using the 10x Chromium Controller® platform. A total of 31.195 cells were sequenced and bioinformatic analysis revealed 13 distinct clusters based on the expression of key lineage target genes. To better characterise the nature of clusters expressing stromal like markers and positioned closely in the UMAP plot, a further subclustering was performed. This resulted in 5 groups, including plasma cells, endothelial cells, fibroblasts, epithelial cells and dendritic like cells. Results Bulk-transcriptomic analysis identified 81 genes differentially expressed in strictures compared to both proximal and distal regions. Notably, genes associated with leukocyte and macrophage recruitment and chemotaxis, namely Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), C-C motif Chemokine Ligand 2 (CCL2), and Endothelin Receptor Type B (EDNRB), resulted overexpressed in strictures. scRNAseq identified a distinct subset of endothelial cells (CD34+, PECAM1+). Caveolin 1 (CAV1), a gene encoding for a structural protein predominantly expressed in smooth muscle cell plasma membrane invaginations, was exclusively up-regulated in endothelial cells of ileal stricture samples. Previous research linked CAV1 to smooth muscle proliferation and fibrosis in various organs. Conclusion This study highlights the pivotal role of endothelial cells in CD strictures. The transcriptomic analysis shows the involvement of inflammatory targets featured genes involved in transendothelial immune cell migration. Furthermore, the up-regulation of CAV1 in endothelial cells within stricture segments suggests a mechanistic connection between endothelial cells and smooth muscle hyperplasia in intestinal strictures. CAV1 holds promise as a potential therapeutic target in fibrostenotic CD.

CD177 drives the transendothelial migration of Treg cells enriched in human colorectal cancer.

Regulatory T (Treg) cells regulate immunity in autoimmune diseases and cancers. However, immunotherapies that target tumor-infiltrating Treg cells often induce unwanted immune responses and tissue inflammation. Our research focussed on exploring the expression pattern of CD177 in tumor-infiltrating Treg cells with the aim of identifying a potential target that can enhance immunotherapy effectiveness. Single-cell RNA sequencing (scRNA-seq) data and survival data were obtained from public databases. Twenty-one colorectal cancer patient samples, including fresh tumor tissues, peritumoral tissues and peripheral blood mononuclear cells (PBMCs), were analysed using flow cytometry. The transendothelial activity of CD177+ Treg cells was substantiated using in vitro experiments. ScRNA-seq and flow cytometry results indicated that CD177 was exclusively expressed in intratumoral Treg cells. CD177+ Treg cells exhibited greater activation status and expressed elevated Treg cell canonical markers and immune checkpoint molecules than CD177- Treg cells. We further discovered that both intratumoral CD177+ Treg cells and CD177-overexpressing induced Treg (iTreg) cells had lower levels of PD-1 than their CD177- counterparts. Moreover, CD177 overexpression significantly enhanced the transendothelial migration of Treg cells in vitro. These results demonstrated that Treg cells with higher CD177 levels exhibited an enhanced activation status and transendothelial migration capacity. Our findings suggest that CD177 may serve as an immunotherapeutic target and that overexpression of CD177 may improve the efficacy of chimeric antigen receptor T (CAR-T) cell therapy.

The role of endothelial junctions in the regulation of the extravasation of tumor cells. A historical reappraisal.

Endothelial cells lining the vessel wall are connected by adherent, tight and gap junctions. Adherent junctions are common to all endothelial cells, whereas tight and gap junctions graduate within different vascular segments. Endothelial cell-cell junctions sustain vascular homeostasis and to control the transendothelial migration of inflammatory cells. Tumor cells need to weaken endothelial cell-cell junctions to penetrate the endothelial barrier and transendothelial migration and metastasis of tumor cells are tightly controlled by endothelial cell-cell junctions.

Unraveling the Action Mechanism of Tubeimoside-1 against Tumor Microvessels via Network Pharmacology and Experimental Validation.

Objective: Tubeimoside-1 (TBMS1) is a plant-derived triterpenoid saponin that exhibits pharmacological properties and anti-tumor effects, but the anti-tumor microvessels of action of TBMS1 remains to be completely elucidated. This study aims to verify the effect of TBMS1 on tumor microvessels and its underlying mechanism. Methods: A SKOV3 xenografted mouse model were constructed to evaluate the anti-tumor microvessels of TBMS1 in vivo, followed by function assays to verify the effects of TBMS1 on the proliferation, cell cycle, migration, and tubule formation of vascular endothelial cells in vitro. Next, based on network pharmacology, the drug/disease-target protein-protein interaction (PPI) networks, biological functions and gene enrichment analyses were performed to predict the underlying mechanism. Finally, molecules and pathways associated with tumor trans-endothelial migration were identified. Results: TBMS1 treatment effectively reduced tumor microvessel density in ovarian cancer model and inhibited the proliferation, cell cycle, migration, and induced apoptosis of vascular endothelial cells in vitro. Network pharmacological data suggested that tumor cell adhesion and trans-endothelial migration may participate in antiangiogenic effects of TBMS1. By endothelial adhesion and permeability assay, we identified that tumor adhesion and the permeability of endothelial monolayers were reduced by TBMS1. Furthermore, adhesion protein (VCAM-1and ICAM-1) and tight junction (TJ) proteins (VE-cadhsion, ZO-1 and claudin-5) were found to be regulated. Finally, Akt, Erk1/2, Stat3 and NF-κB signaling were decreased by TBMS1 treatment. Conclusion: To sum up, our findings strongly suggest that clinical application of TBSM1 may serve as a vasoactive drug treatment to suppress tumor progression.

Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

Leukocyte recruitment from blood to tissue is a process that occurs at the level of capillary vessels during both physiological and pathological conditions. This process is also relevant for evaluating novel adoptive cell therapies, in which the trafficking of therapeutic cells such as chimeric antigen receptor (CAR)-T cells throughout the capillaries of solid tumors is important. Local variations in blood flow, mural cell concentration, and tissue stiffness contribute to the regulation of capillary vascular permeability and leukocyte trafficking throughout the capillary microvasculature. We developed a platform to mimic a biologically functional human arteriole-venule microcirculation system consisting of pericytes (PCs) and arterial and venous primary endothelial cells (ECs) embedded within a hydrogel, which self-assembles into a perfusable, heterogeneous microvasculature. Our device shows a preferential association of PCs with arterial ECs that drives the flow-dependent formation of microvasculature networks. We show that PCs stimulate basement membrane matrix synthesis, which affects both vessel diameter and permeability in a manner correlating with the ratio of ECs to PCs. Moreover, we demonstrate that hydrogel concentration can affect capillary morphology but has no observed effect on vascular permeability. The biological function of our capillary network was demonstrated using an inflammation model, where significantly higher expression of cytokines, chemokines, and adhesion molecules was observed after tumor necrosis factor-alpha (TNF-α) treatment. Accordingly, T cell adherence and transendothelial migration were significantly increased in the immune-activated state. Taken together, our platform allows the generation of a perfusable microvasculature that recapitulates the structure and function of an in vivo capillary bed that can be used as a model for developing potential immunotherapies.

Cerebral microvascular endothelial cell-derived extracellular vesicles regulate blood − brain barrier function

Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.