Cell Transendothelial Migration Research Articles

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

SummaryThe migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.

A Potent Leukocyte Transmigration Blocker: GT-73 Showed a Protective Effect against LPS-Induced ARDS in Mice.

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Contribution of Heparan Sulphate Binding in CCL21-Mediated Migration of Breast Cancer Cells.

Simple SummaryBreast cancer is a leading cause of cancer-related deaths worldwide, predominantly caused by metastasis. Chemokine receptor CCR7 and its ligand CCL21 are implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon binding to their specific chemokine receptors and negatively charged molecules on the cell surface (heparan sulphate). The role of heparan sulphate in CCR7-mediated lymph node metastasis was investigated by creating a non-heparan sulphate binding mutant chemokine CCL21. Mutant-CCL21 was tested in vitro in a range of assays, including cell migration, calcium flux and surface plasmon resonance spectroscopy. Mutant-CCL21 induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of breast cancer cells. A murine model was used to assess the potential of mutant-CCL21 to prevent lymph node metastasis in vivo. Lymph node metastasis was significantly reduced by the administration of mutant-CCL21 compared to the control. Targeting chemokine–heparan sulphate interactions may be a promising approach to inhibit chemokine activity and metastasis.Chemokine receptor CCR7 is implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon their binding to both cell-surface heparan sulphate (HS) and to their specific receptors; thus, the role of HS in CCR7-mediated lymph node metastasis was investigated by creating a non-HS binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ103–134) induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of PBMCs (p < 0.001) and 4T1-Luc cells (p < 0.01). Furthermore, the effect of heparin and HS on the chemotactic properties of wild-type (WT) and mut-CCL21 was examined. Interestingly, heparin and HS completely inhibit the chemotaxis mediated by WT-CCL21 at 250 and 500 µg/mL, whereas minimal effect was seen with mut-CCL21. This difference could potentially be attributed to reduced HS binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind HS compared to WT-CCL21. A murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20 µg mut-CCL21. Mice were imaged weekly with IVIS and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p = 0.0148), suggesting the potential to target chemokine binding to HS as a therapeutic option.

Exosomal miR-375-3p breaks vascular barrier and promotes small cell lung cancer metastasis by targeting claudin-1.

BackgroundHigh incidence of metastasis is the main cause of death for small cell lung cancer (SCLC), with its underlying molecular mechanisms remain unclear. Exosomal miRNAs are important regulators in metastatic processes of various tumors, but their specific role in SCLC metastasis is unknown.MethodsSmall RNA sequencing followed by qRT-PCR verification was used to screen the potential exosomal miRNAs that might mediate SCLC metastasis. SCLC-cell-secreted exosomes were labeled followed by incubating with vascular endothelial cells to evaluate exosome-mediated communication between SCLC cells and vascular endothelial cells. In vitro permeability assay and transendothelial migration assay were applied to investigate the function of exosomal miRNA on vascular endothelial cells. In vivo permeability assay and mouse lung colonization assay were used to verify the effects of exosomal miRNA on vascular barriers and SCLC metastasis in vivo. Proteomics technology, dual-luciferase reporter system together with rescue assays were conducted to excavate the downstream pathways of miRNA.ResultsCompared with 57 healthy volunteers and 46 non-small cell lung cancer patients, we identified that the level of exosomal miR-375-3p in 126 SCLC patients was obviously higher and was positively correlated with patient TNM stages. In vitro functional experiments found that SCLC-cell-secreted exosomal miR-375-3p could increase the permeability of vascular endothelial cells and facilitate the transendothelial migration of SCLC cells. In vivo, miR-375-3p-enriched exosomes also destroyed the barrier structure of lung, liver and brain tissues of mice, leaded to an increased blood vessel permeability and finally promoted SCLC metastasis. Mechanistically, SCLC-cell-secreted exosomal miR-375-3p was transferred to vascular endothelial cells. The internalized miR-375-3p broke the tight junction of vascular endothelial cells by directedly binding to the 3’UTR of tight junction protein claudin-1 and negatively regulating its expression. Overexpressing claudin-1 in vascular endothelial cells could rescue the broken vascular barriers induced by miR-375-3p.ConclusionsOur findings underline the crucial roles of exosomal miRNA-375-3p in regulating vascular endothelial barrier integrity and SCLC metastasis. miRNA-375-3p has a great potential to be a novel biomarker monitoring metastasis and guiding clinical therapeutics of SCLC patients.

Behind Brain Metastases Formation: Cellular and Molecular Alterations and Blood-Brain Barrier Disruption.

Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells’ (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood–brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC–BMEC interaction compromised BBB integrity, as revealed by junctional proteins (β-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. β4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.

Endothelial STING controls T cell transmigration in an IFNI-dependent manner.

The stimulator of IFN genes (STING) protein senses cyclic dinucleotides released in response to double-stranded DNA and functions as an adaptor molecule for type I IFN (IFNI) signaling by activating IFNI-stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING–/– mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared with control EC, whereas T cell adhesion was not impaired. STING–/– T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell–expressed molecules ICAM1 and VCAM1, compared with WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α–stimulated STING–/– EC, and genetic loss or pharmacologic antagonisms of IFNI receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC-STING during T cell TEM that is dependent on the ISG CXCL10 and on IFNI/IFNAR signaling.

Reduced Lamin A/C Does Not Facilitate Cancer Cell Transendothelial Migration but Compromises Lung Metastasis.

Simple SummaryThe nucleus is the largest and stiffest organelle of tumor cells. Cancer metastasis depends on the ability of cancer cells circulating in the blood to exit blood vessels and survive in target organs. The roles of the shell (lamina) of the nucleus in cancer cell migration and survival in distinct organs of metastasis are still unclear. A-type lamins are key lamina components that increase nuclear stiffness and reduce squeezing capacity through highly rigid barriers. We addressed how reduced expression of A-lamins (lamin A/C) affects cancer cell survival and crossing of endothelial barriers and lung capillaries and found that reduced lamin A/C expression impairs cancer growth in spheroids and restricts cancer metastasis to lungs without improving cancer cell squeezing and extravasation from lung vessels, the key platform for cancer entry into lungs.The mechanisms by which the nuclear lamina of tumor cells influences tumor growth and migration are highly disputed. Lamin A and its variant lamin C are key lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C in two prototypic metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, and reduced heterochromatin content. Surprisingly, both lamin A/C knockdown cells grew poorly in 3D spheroids within soft agar, and lamin A/C deficient cells derived from spheroids transcribed lower levels of the growth regulator Yap1. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown and their metastasis in lungs was even dramatically reduced. Our results are the first indication that reduced lamin A/C content in distinct types of highly metastatic cancer cells does not elevate their transendothelial migration (TEM) capacity and diapedesis through lung vessels but can compromise lung metastasis at a post extravasation level.

Deficiency of ITGAM Attenuates Experimental Abdominal Aortic Aneurysm in Mice.

BackgroundIntegrin αM (CD11b), which is encoded by the Integrin Subunit Alpha M (ITGAM) gene, is not only a surface marker of monocytes but also an essential adhesion molecule. In this study, we investigated the effect of CD11b on experimental abdominal aortic aneurysm and the potential underlying mechanisms.Methods and ResultsThe incidence of abdominal aortic aneurysm was not significantly lower in ITGAM(‐/‐) mice than in control mice. Nevertheless, knockout of CD11b reduced the maximum abdominal aortic diameter, macrophage infiltration, matrix metalloproteinase‐9 expression, and elastin and collagen degradation. Additionally, lower expression of IL‐6 was found in both the peripheral blood and abdominal aortas of ITGAM(‐/‐) mice, indicating a biological correlation between CD11b and the inflammatory response in abdominal aortic aneurysm. In vitro, the number of ITGAM(‐/‐) bone marrow–derived macrophages (BMDMs) that adhered to endothelial cells was significantly lower than the number of wild‐type BMDMs. Moreover, the CD11b monoclonal antibody and CD11b agonist leukadherin‐1 decreased and increased the number of adherent wild‐type BMDMs, respectively. Through RNA sequencing, genes associated with leukocyte transendothelial migration were found to be downregulated in ITGAM(‐/‐) BMDMs. Furthermore, immunoprecipitation–mass spectrometry analysis predicted that the Akt pathway might be responsible for the impaired transmigratory ability of ITGAM(‐/‐) BMDMs. The reduced activation of Akt was then confirmed, and the Akt agonist SC79 partially rescued the transendothelial migratory function of ITGAM(‐/‐) BMDMs.ConclusionsCD11b might promote the development and progression of abdominal aortic aneurysm by mediating the endothelial cells adhesion and transendothelial migration of circulating monocytes/macrophages.

Multi-Arm PEG/Peptidomimetic Conjugate Inhibitors of DR6/APP Interaction Block Hematogenous Tumor Cell Extravasation.

The binding of amyloid precursor protein (APP) expressed on tumor cells to death receptor 6 (DR6) could initiate the necroptosis pathway, which leads to necroptotic cell death of vascular endothelial cells (ECs) and results in tumor cells (TCs) extravasation and metastasis. This study reports the first inhibitor of DR6/APP interaction as a novel class of anti‐hematogenous metastatic agent. By rationally utilizing three combined strategies including selection based on phage display library, d‐retro‐inverso modification, and multiple conjugation of screened peptidomimetic with 4‐arm PEG, the polymer–peptidomimetic conjugate PEG‐tAHP‐DRI (tetra‐(D‐retro‐inverso isomer of AHP‐12) substitued 4‐arm PEG5k) is obtained as the most promising agent with the strongest binding potency (K D = 51.12 × 10−9 m) and excellent pharmacokinetic properties. Importantly, PEG‐tAHP‐DRI provides efficient protection against TC‐induced ECs necroptosis both in vitro and in vivo. Moreover, this ligand exhibits prominent anti‐hematogenous metastatic activity in serval different metastatic mouse models (B16F10, 4T1, CT26, and spontaneous lung metastasis of 4T1 orthotopic tumor model) and displays no apparent detrimental effects in preliminary safety evaluation. Collectively, this study demonstrates the feasibility of exploiting DR6/APP interaction to regulate hematogenous tumor cells transendothelial migration and provides PEG‐tAHP‐DRI as a novel and promising inhibitor of DR6/APP interaction for developments of anti‐hematogenous metastatic therapies.

Abstract PO015: JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells

Abstract Though survival is improving for breast cancer patients, clinical outcomes remain poor for patients with aggressive molecular subtypes. Triple negative breast cancer (TNBC) is considered the most aggressive breast cancer subtype, and despite having a 90% five-year relative survival rate for localized disease, that number drops to as low as 11% for patients with distant TNBC metastasis. The poor clinical outcomes for TNBC patients are largely due to lack of clinically actionable molecular targets and pathways compared to other breast cancer subtypes. Given the lack of effective treatment strategies for advanced TNBC, there is an urgent and unmet need to understand the means through which tumor cells disseminate and metastasize to distant tissues in hopes of identifying new targets for therapeutic intervention. For tumor cells to metastasize, they acquire complex series of pathophysiological phenotypes, starting with invasion into surrounding tissue and intravasation into systemic lymphatic or blood vascular systems. Only a small fraction of tumor cells can survive in circulation, with a smaller fraction able to extravasate from vessels and establish distant metastases. While some cancer cells partially coopt the mechanisms that leukocytes use to extravasate, circulating TNBC cells also use distinct mechanisms to breach an endothelial barrier. However, the mechanism(s) of TNBC extravasation remains largely unknown, underlying the need to study further. High expression of Notch ligand JAGGED-1 (JAG1) is associated with increased metastasis and mortality in patients with breast cancer. While JAG1 enhances tumor angiogenesis and tumor growth, the means through which JAG1 promotes tumor cell dissemination is not clear. Our preliminary data supports a role for JAG1 as a key regulator of tumor cell interactions with the endothelium, controlling tumor cell adhesion, transendothelial migration (TEM), and transcription of critical paracrine-acting extravasation proteins, such as SPARC and ICAM1. Using both JAG-specific ligand traps as well as CRISPR/Cas9 mediated deletion of JAG1, we show that JAG1 promotes TEM in static transwell models, promotes primary tumor growth, and directs a tumor cell transcriptional program that upregulates cell surface interaction pathways distinct from global Notch inhibition. It is our central hypothesis that JAG1 promotes TNBC migration across the vascular endothelium, thereby allowing disseminated cancer cells to exit from circulation and enhancing TNBC metastasis. To define the JAG1-mediated elements of extravasation, we will examine five CRISPR/Cas9 JAG1 knockout lines in a cutting-edge flow system that recapitulates the vascular shear stress microenvironments where extravasation is thought to occur in vivo. We will evaluate multiple steps of extravasation including tumor cell binding, rolling, and TEM under various flow parameters. Moreover, we will study in vivo lung extravasation using a murine tail vein injection model and interrogate transcriptional candidates downstream of JAG1 as identified by our RNAseq data. Citation Format: Benjamin Gordon, Reyhaan A. Chaudhri, Tim Sargis, L.A. Naiche, Jan K. Kitajewski. JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO015.

Heparanase induces necroptosis of microvascular endothelial cells to promote the metastasis of hepatocellular carcinoma

Heparanase (HPSE) is a kind of multifunctional extracellular hydrolase, and related to metastasis of hepatocellular carcinoma (HCC). Endothelial necroptosis promotes the metastasis of cancer cells. It is not clear whether HPSE could mediate necroptosis of microvascular endothelial cells (MVECs) to promote HCC metastasis. Here we found HPSE expression was up-regulated in HCC tissues and its over-expression was correlated with multiple tumor foci, microvascular invasion, and poor outcome of HCC patients. Non-contact co-culture experiments showed high-expressed HPSE in HCC cells mediated the necroptosis of human umbilical vein endothelial cells (HUVECs) and elevated the expression levels of syndecan-1 (SDC-1) and tumor necrosis factor-α (TNF-α) in vitro. As a result of necroptosis, trans-endothelial migration (TEM) of HCC cells was increased. Conversely, both HPSE and SDC-1 knockdowns reversed necroptosis and decreased TNF-α expression level, while HPSE over-expression increased SDC-1 and TNF-α expression and aggravated necroptosis. Animal experiments found that the nude mice, intraperitoneally injected with HPSE high expressing HCC cells, had obvious necroptosis of MVECs and high intrahepatic metastasis rate, which could be relieved by inhibitor of necroptosis. Morever, HPSE elevated the expression levels of p38 mitogen-activated protein kinase (p38 MAPK) rather than nuclear factor kappa B in vitro. Our data suggest that HPSE induces necroptosis of MVECs to promote the metastasis of HCC by activating HPSE/SDC-1/TNF-α axis and p38 MAPK pathway.

A Novel Neoplastic Fusion Transcript, RAD51AP1-DYRK4, Confers Sensitivity to the MEK Inhibitor Trametinib in Aggressive Breast Cancers.

Luminal B breast tumors are more aggressive estrogen receptor-positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies. We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms. Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition. This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

Vertebral-specific activation of the CX3CL1/ICAM-1 signaling network mediates non-small-cell lung cancer spinal metastasis by engaging tumor cell-vertebral bone marrow endothelial cell interactions.

Rationale: The spine is one of the most common metastatic sites of non-small cell lung cancer (NSCLC), and NSCLC spinal metastasis results in serious consequences. Metastatic extravasation of disseminated cancer cells including increased invasiveness, adhesion and transendothelial migration is crucial for tumor metastasis. This study aimed to investigate the mechanisms underlying NSCLC spinal metastasis based on the C-X3-C motif chemokine ligand 1- (CX3CL1) and intercellular adhesion molecule-1- (ICAM-1) mediated signaling network.Methods: Immunohistochemistry, western blotting, and reverse transcription-quantitative PCR were conducted to detect the distribution of CX3CL1/ICAM-1 in different organs. Transwell, adhesion, and transendothelial migration assays were performed to evaluate the regulatory effects of CX3CL1/ICAM-1 on NSCLC cell invasion, adhesion, and transendothelial migration in vitro. A spontaneous spinal metastasis mouse model was established via injection of NSCLC cells into the left cardiac ventricle of NOD/SCID mice. The effects of CX3CL1/ICAM-1 on NSCLC spinal metastasis in vivo were validated using bioluminescent, micro-computerized tomography, immunohistochemistry and histological analyses.Results: CX3CL1 expression was specifically higher in vertebral bone compared with limb bones and lung tissue, and was associated with NSCLC spinal metastasis. Mechanically, vertebral bone marrow endothelial cells (VBMECs) enhanced NSCLC cell invasion via CX3CL1 signaling-mediated activation of the PI3K/AKT pathway. Furthermore, we found that VBMECs effectively induced ICAM-1-dependent NSCLC cell adhesion in coordination with platelets through the CX3CL1/ICAM-1/LFA-1 pathway. Meanwhile, CX3CL1 enhanced NSCLC cell transendothelial migration by increasing permeability of VBMECs via ICAM-1-dependent activation of the Src/GEF-H1 pathway. Interestingly, NSCLC cells were indicated to promote CX3CL1 secretion of VBMECs through MAPK14/ADMA17-dependent CX3CL1 release and NF-κB-dependent CX3CL1 synthesis. Based on these findings, we revealed a novel feedback cycle between circulating NSCLC cells and VBMECs mediated by CX3CL1/ICAM-1 signaling. Further disengagement of the CX3CL1/ICAM-1-mediated feedback cycle in vivo significantly restricted metastasis and prolonged mouse survival.Conclusions: Our results indicated a unique feedback cycle between circulating NSCLC cells and VBMECs mediated by CX3CL1/ICAM-1 signaling, which is necessary for NSCLC spinal metastasis. This work provides a new perspective for underlying the mechanisms of NSCLC spinal metastasis and indicates potential novel targets for the prevention of NSCLC spinal metastasis.

Phosphatidylserine-exposing tumor-derived microparticles exacerbate coagulation and cancer cell transendothelial migration in triple-negative breast cancer.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations.Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay.Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells.Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Platelet endothelial cell adhesion molecule-1 is a gatekeeper of neutrophil transendothelial migration in ischemic stroke

RationaleAdhesion molecules are key elements in stroke-induced brain injury by regulating the migration of effector immune cells from the circulation to the lesion site. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule highly expressed on endothelial cells and leukocytes, which controls the final steps of trans-endothelial migration. A functional role for PECAM-1 in post-ischemic brain injury has not yet been demonstrated. ObjectiveUsing genetic Pecam-1 depletion and PECAM-1 blockade using a neutralizing anti-PECAM-1 antibody, we evaluated the role of PECAM-1 mediated trans-endothelial immune cell migration for ischemic injury, delayed brain atrophy, and brain immune cell infiltrates. Trans-endothelial immune cell migration was furthermore evaluated in cultured human cerebral microvascular endothelial cells. Methods and resultsTransient middle cerebral artery occlusion (tMCAO) was induced in 10–12-week-old male Pecam-1−/− and Pecam-1+/+ wildtype mice. PECAM-1 levels increased in the ischemic brain tissue due to the infiltration of PECAM-1+ leukocytes. Using magnetic resonance imaging, we observed smaller infarct volume, less edema formation, and less brain atrophy in Pecam-1−/− compared with Pecam-1+/+ wildtype mice. The transmigration of leukocytes, specifical neutrophils, was selectively reduced by Pecam-1−/−, as shown by immune fluorescence and flow cytometry in vivo and transmigration assays in vitro. Importantly, inhibition with an anti-PECAM-1 antibody in wildtype mice decreased neutrophil brain influx and infarct. ConclusionPECAM-1 controls the trans-endothelial migration of neutrophils in a mouse model of ischemic stroke. Antibody blockade of PECAM-1 after stroke onset ameliorates stroke severity in mice, making PECAM-1 an interesting target to dampen post-stroke neuroinflammation, reduce ischemic brain injury, and enhance post-ischemic brain remodeling.

Thymoquinone inhibits IL-7-induced tumor progression and metastatic invasion in prostate cancer cells by attenuating matrix metalloproteinase activity and Akt/NF-κB signaling.

Interleukin (IL)-7 acts via the IL-7 receptor in metastatic tumor progression in prostate cancer (PC). The current study aimed to evaluate thymoquinone (Tq), an active constituent from Nigella sativa against IL-7-driven tumor progression and metastatic invasion in PC cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the proliferation of PC cells. Enzyme-linked immunosorbent assay was used to detect the expression of IL-7 and matrix metalloproteinases (MMPs). Tumor-cell transendothelial, scratch wound and cell scatter assays were performed to mimic metastasis. Western immunoblotting was used to measure the level of proteins. Tq effectively controlled the proliferation of DU-145, PC-3, and LNCaP cells with GI50 of 10.18, 12.40, and 16.78 µM, respectively. IL-7 and IL-7R were natively expressed in all PC types, while maximal expression was detected in DU-145. IL-7 promoted metastatic events, such as transendothelial migration, cell scatter, and cell invasion of DU-145 cells in a dose-dependent manner that was inhibited by Tq. Furthermore, Tq also downregulated p-Akt and NF-κB in DU-145 cells induced by IL-7 antibody and reduced the levels of MMP-3 and MMP-7 in these cells in a dose-dependent manner. Collectively, Tq has excellent efficacy in controlling tumor progression, migration, and invasion of DU-145 cells that were driven by the activation of MMPs through IL-7/Akt/NF-κB signaling.

The T cell CD6 receptor operates a multitask signalosome with opposite functions in T cell activation.

To determine the respective contribution of the LAT transmembrane adaptor and CD5 and CD6 transmembrane receptors to early TCR signal propagation, diversification, and termination, we describe a CRISPR/Cas9-based platform that uses primary mouse T cells and permits establishment of the composition of their LAT, CD5, and CD6 signalosomes in only 4 mo using quantitative mass spectrometry. We confirmed that positive and negative functions can be solely assigned to the LAT and CD5 signalosomes, respectively. In contrast, the TCR-inducible CD6 signalosome comprised both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation. Moreover, CD6 associated independently of TCR engagement to proteins that support its implication in inflammatory pathologies necessitating T cell transendothelial migration. The multifaceted role of CD6 unveiled here accounts for past difficulties in classifying it as a coinhibitor or costimulator. Congruent with our identification of UBASH3A within the CD6 signalosome and the view that CD6 constitutes a promising target for autoimmune disease treatment, single-nucleotide polymorphisms associated with human autoimmune diseases have been found in the Cd6 and Ubash3a genes.

Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells.

Simple SummaryMetastasis is one of the most devastating aspect of cancer progression and involves biochemical and physical interactions between cancer cells and surrounding microenvironment. In particular, cancer cells adhesion to endothelium and their subsequent transendothelial migration represent important steps in the metastatic process in target organs. In this study we characterized the functional role of Protein Disulfide Isomerase A1 (PDIA1) in breast cancer cells adhesion and transendothelial migration. We identified the full repertoire of protein disulfide isomerases in endothelial cells as well as in breast cancer cells. We provided insight into the mechanisms involved in cancer-endothelial cells interactions and suggested that PDIA1 regulates the adhesion and transendothelial migration of breast cancer cells by disulphide exchange involving most likely the activation of integrins. Our results suggest that the inhibition of extracellular PDIA1 or other PDIs represents an interesting target for anti-metastatic treatment. Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that β1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer–endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. Invivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.