Integrin Activation Research Articles

Radiotheranostic landscape: A review of clinical and preclinical development.

Radiotheranostics combines diagnostic imaging with targeted radionuclide therapy, representing a transformative approach in precision oncology.Landmark approvals of Lutathera®and Pluvicto®havecatalyzed significant advancements in this field, driving research into novel radionuclides, targetingstrategies, and clinical applications. This review evaluates the evolving clinical and preclinicallandscape of radiotheranostics, highlighting advancements, emerging trends, and persistent challengesin radionuclide therapy. A comprehensive analysis was performed, encompassing active clinical trials as of December 2024, sourced from ClinicalTrials.gov and TheranosticTrials.org. Preclinical developments were evaluated through a review of recent literature, focusing on innovations in radionuclide production, targeting molecules, and radiochemistry. In reviewing the clinical landscape, agents targeting somatostatin receptors (SSTR) and prostate-specific membrane antigen (PSMA) still dominate the field, but new targets such as fibroblast activation protein (FAP), integrins, and gastrin-releasing peptide receptors (GRPR) are gaining traction in both clinical and preclinical development. While small molecules and peptides remain the most common radionuclide carriers, antibody-based carriers including bispecific antibodies, immunoglobin-derived antigen-binding fragments, and antibody-mimetic proteins are on the rise due to their specificity and adaptability. Innovations in radioligand design are driving a shift from agonists to antagonists, accompanied by the development of modified peptides with enhanced pharmacokinetics and tumor-targeting properties. Next-generation therapeutic radionuclides, such as the beta-emitter terbium-161 and alpha-emitters actinium-225 and lead-212, are under investigation to complement or replace lutetium-177, addressing the need for improved efficacy and reduced toxicity. Paired isotopic radionuclides are gaining popularity for their ability to optimize imaging and therapeutic dosimetry as they offer near-identical specificity, biodistribution, and metabolism. Additionally, radiohybrid systems represent an innovative approach to chelating chemically distinct radionuclide pairs within a single molecule, further enhancing flexibility in radiotheranostic design. Radiotheranostics has transformed cancer care through its precision and adaptability, but challenges in radionuclide production, regulatory frameworks, and workforce training hinder broader adoption. Advances in isotopic pairing, next-generation radionuclides, and radiohybrid systems in preclinical and clinical settings hold promise to overcome these barriers. Collaborative efforts among academia, industry, and regulatory bodies are critical to accelerating innovation and optimizing clinical outcomes.

Antiplatelet Effects of DMPC-Based Synthetic High-Density Lipoproteins: Exploring Particle Structure and Noncholesterol Efflux Mechanisms.

Platelet activation is a key factor in the development of cardiovascular diseases. High-density lipoprotein (HDL) is known for its cardioprotective activities including antithrombotic actions. While HDL mimetics have been explored for their potential to regulate thrombosis, their influence on platelet activity remains unclear. This study explores the capacity of synthetic HDL (sHDL) to modulate platelet function and investigates the underlying mechanisms. We examined the effects of sHDL, formulated with various ApoA1 mimetic peptides (18A, 5A, and 22A) and full-length ApoA1 protein, all complexed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), on platelet function. DMPC-based sHDL demonstrated pronounced antiplatelet effects across all formulations. Comparison with DMPC micelles showed that all sHDL molecules were more effective, highlighting the crucial role of the protein-phospholipid complex in reducing platelet reactivity. Further analysis revealed that DMPC sHDL dose-dependently inhibited various platelet functions, including aggregation, integrin activation, α-granule secretion, protein kinase C (PKC) activation, and platelet spreading. Mechanistic studies demonstrated that DMPC sHDL's antiplatelet effects are not entirely dependent on cholesterol efflux, despite effectively reducing total platelet cholesterol. Furthermore, sHDL's activity was found to be independent of scavenger receptor BI (SR-BI). Notably, inhibition of the CD36 receptor markedly attenuated sHDL's antiplatelet activity and uptake, suggesting a novel mechanism distinct from that of native HDL. In summary, DMPC sHDL modulates platelet function through a synergistic action between protein and phospholipid components, primarily via CD36 receptor engagement. These insights pave the way for novel antiplatelet therapies utilizing sHDL's distinct properties.

Nexinhib20 inhibits JFC1-mediated mobilization of a subset of CD11b/CD18+ vesicles decreasing integrin avidity, but does not inhibit Rac1.

Regulated sequential exocytosis of neutrophil granules is essential in orchestrating the innate immune response, while uncontrolled secretion causes inflammation. We developed and characterized Nexinhib20, a small-molecule inhibitor that targets azurophilic granule exocytosis in neutrophils by blocking the interaction between the small GTPase Rab27a and its effector JFC1. Its therapeutic potential has been demonstrated in several pre-clinical models of inflammatory disease. Here, using neutrophils from Jfc1-KO mice, we show that JFC1 regulates the mobilization of a small subpopulation of CD11b+ granules. Nexinhib20 inhibits the mobilization of β2-integrins from a subset of CD11b+ granules to the plasma membrane in human and mouse neutrophils. The putative impact of Nexinhib20 on integrin activation is caused by decreased avidity, secondary to its effect on β2-integrin mobilization. CD11b mobilization and integrin activation were unaffected by pharmacological inhibition or activation of Rac1. Using quantitative 3-dimensional enhanced resolution microscopy, we show that neutrophil activation induces the recruitment of JFC1 to CD11b+ granules. Nexinhib20 decreased the localization of JFC1 at CD11b+ granules without affecting the association of Rac1 with CD11b. Nexinhib20 inhibits JFC1 recruitment but not endogenous Rac1 activation in living cells. Using orthogonal analyses of Rac1 activity consisting of a sensitive, time-resolved FRET, Rac1-PAK1-binding assay, and endogenous Rac1-GTP examination, we show that Nexinhib20 does not interfere with Rac1 activation. Instead, we confirmed its molecular mode of action to the inhibition of the Rab27a-JFC1 binding axis. Thus, Nexinhib20 limits β2- integrin mobilization to the cell surface, decreasing avidity, affecting active integrin availability, in a JFC1-dependent but Rac1-independent manner.

Integrin Trafficking, Fibronectin Architecture, and Glomerular Injury upon AdipoR1 Depletion.

Deficiency of adiponectin and its downstream signaling may contribute to the pathogenesis of kidney injury in type 2 diabetes. Adiponectin activates intracellular signaling via adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2), but the role of AdipoR-mediated signaling in glomerular injury in type 2 diabetes remains unknown. The expression of AdipoR1 in the kidneys of people with type 2 diabetes and the expression of podocyte proteins or injury markers in the kidneys of AdipoR1-knockout (AdipoR1-KO) mice and immortalized AdipoR1-deficient human podocytes were investigated by immunohistochemistry and immunoblotting. The functional role of AdipoR1 was studied in AdipoR1-deficient podocytes by performing assays for apoptosis, cytokine secretion, mechanical stress, adhesion, and endocytic trafficking. Glomerular AdipoR1 expression was lower in type 2 diabetes and associated kidney disease, correlating with higher BMI and podocyte loss. Male AdipoR1-KO mice showed typical signs of early diabetic kidney disease, including albuminuria, glomerular structural abnormalities, and lower expression of central podocyte proteins; females were less affected. Podocyte apoptosis increased in female and male AdipoR1-KO mice and excessive podocyte loss, potentially due to detachment, was detected in males. AdipoR1 deficiency impaired the YAP-mediated mechanoresponse and induced accumulation of the extracellular matrix (ECM) protein fibronectin in the glomeruli in vivo, and podocytes in vitro. Functionally, AdipoR1 deficiency impaired endocytosis of the ECM receptor active integrin β1, disturbed focal adhesion turnover, and remodulated podocyte-derived ECM, thereby reducing podocyte adhesion. AdipoR1 deficiency in mice resulted in the development of kidney injury predominantly in males. Mechanistically, AdipoR1 loss in podocytes impaired endocytosis of active integrin β1, which plausibly compromised focal adhesion dynamics, disturbed fibronectin matrix turnover, and hindered podocyte adhesion.

Platelet activation and signaling in thrombus formation.

In thrombosis and hemostasis, the formation of a platelet-fibrin thrombus or clot is a highly controlled process that varies, depending on the pathological context. Major signaling pathways in platelets are well established. However, studies with genetically modified mice have identified the contribution of hundreds of additional platelet-expressed proteins in arterial thrombus formation and bleeding. Using phenotype information of 540 mouse genes, involved in arterial thrombosis and hemostasis, we review current insights into established and novel platelet signaling mechanisms. We discuss pathways involved in platelet adhesion, shape change, integrin activation, intracellular vesicle trafficking and protein processing, granule secretion, aggregate formation and procoagulant activity. Specific attention is paid to the signaling routes used by ITAM-linked, ITIM-linked and G protein-coupled receptors, as well as downstream events feeding into GTPase regulation and protein kinase activation. We further summarize known alterations in platelet responses under conditions of venous, inflammatory and infection-dependent thrombosis, taking into account interactions of platelets with the endothelium, leukocytes and red blood cells. Understanding the genes and proteins involved in platelet signaling in the context of hemostasis, thrombosis and inflammation may lead to improved therapies to prevent and treat thrombotic disorders.

Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3.

Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase, Rap1-GTP, and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate talin interaction with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first by releasing Rap1-GTP from its sequestration by SHANK3 and second by serving as the relevant Rap1 effector for integrin activation.

Paxillin and Kindlin: Research Progress and Biological Functions

Paxillin and kindlin are essential regulatory proteins involved in cell adhesion, migration, and signal transduction. Paxillin influences cytoskeletal dynamics by interacting with multiple signaling proteins, while kindlin regulates integrin activation, affecting adhesion and motility. This review examines the structures and functions of these proteins, focusing on their roles in cancer progression, immune response, and therapeutic potential. The cooperation between paxillin and kindlin in integrin activation and focal adhesion dynamics offers valuable insights into tumor metastasis, immune function, and tissue repair.

P0528 Leukocyte Adhesive Function as Potential Predictive Marker for Treatment Response to Vedolizumab Among Bio-naive Patients with Crohn’s Disease

Abstract Background Vedolizumab (VDZ) is approved for the treatment of Crohn’s diseases (CD), but around 60% of patients fail to respond. Predicting response to VDZ, especially prior to treatment, is desirable but remains challenging. In this study, we investigated if α4β7 and α4β1 integrin activity may be used to predict response to VDZ. Methods Both α4β7 and α4β1 integrin activity were assessed using Leukocyte adhesive function assay (LAFA). LAFA uses microfluidic techniques to mimic blood circulation in vitro. Blood samples were fluorescently labelled and then pulled into microfluidic channels precoated with either MAdCAM-1 or VCAM-1 as substrates. Interactions between leukocytes and substrates was recorded and analysed by cell tracking software. Cell kinetic parameters were calculated and used for the assessment of α4β7 and α4β1 integrin activity. Mn2+ was applied as activator to assess the full potential of integrin activity. Bio-naive CD patients were recruited and all received VDZ as treatment. Treatment response was assessed at week 14 by endoscopy. Patients were classified as primary loss of response to VDZ if the simplified endoscopic score for CD at week 14 failed to decrease by 25% compared to baseline. Blood samples were collected at weeks 0 (prior to VDZ infusion), 2, 6 and 14 and then applied for LAFA. The differences in α4β7 and α4β1 integrin activity between VDZ responders and non-responders were prospectively assessed and a prediction model was subsequently established. Results A total of 27 patients were recruited and received VDZ. Twenty patients were classified as responders and seven as non-responders. No difference was found in α4β7 integrin activity on CD4 and CD8 cells between responders and non-responders, either before or after VDZ treatment. However, a lower α4β1 integrin activity was found on CD4 and CD8 cells among VDZ non-responders at week 0 compared with responders in the presence of Mn2+. Using the best LAFA-derived markers, a model to predict VDZ non-responders at week 0 was developed, in which a sensitivity of 0.90 and a specificity of 0.73 were achieved, a substantially better performance than the existing prediction tool CDST. In addition, α4β1 integrin activity on CD4 and CD8 cells among non-responders was gradually enhanced following subsequent VDZ treatment, which was not detected in responders (figure), implying a possible mechanism underlining primary loss of response to VDZ. Conclusion Patients with a low α4β1 integrin activity at week 0 which can be enhanced after VDZ, is likely to be primary non-responders to VDZ. Integrin activity measured by LAFA prior to treatment may be used as new diagnostic tool for the prediction VDZ response in bio-naïve CD patients. References Dulai PS, Boland BS, Singh S, et al. Development and Validation of a Scoring System to Predict Outcomes of Vedolizumab Treatment in Patients With Crohn’s Disease. Gastroenterology. 2018;155(3):687-695.e10. doi:10.1053/j.gastro.2018.05.039

P0086 The role of enteric glial cells in patients with ulcerative colitis.

Abstract Background Enteric glial cells (EGCs) have traditionally been regarded as supportive cells within the enteric nervous system. However, recent studies indicate that, beyond their involvement in neural circuit formation, EGCs possess antigen-presenting capabilities and actively contribute to mucosal defense. In the mouse myenteric plexus, inflammation-inducing stimuli have been shown to trigger the expression of MHC class II on glial cells, which subsequently influences the differentiation of helper T-cell and regulatory T-cell subtypes, thereby maintaining immune homeostasis under inflammatory conditions. Nevertheless, research on EGCs in humans remains limited. Thus, this study aimed to elucidate the function and involvement of EGCs in the pathogenesis of ulcerative colitis (UC). Methods Normal colonic mucosa was obtained from histologically and macroscopically intact areas of patients with colorectal cancer (n=6). Colonic mucosa from UC patients, diagnosed based on established clinical, bacteriological, radiologic, and endoscopic criteria, was also obtained from surgically resected specimens (n=7). Lamina propria mononuclear cells were isolated through enzymatic digestion, and mesenchymal stromal fractions (CD31-CD45-EPCAM-CD90+), containing glial cells, were further isolated using flow cytometry for single-cell RNA sequencing (scRNA-seq) analysis and T cell differentiation assay. Results We identified a specific EGC population (Cluster 3: PDPN+ HLA-DR high) with high antigen-presenting capacity, which was selectively elevated at inflamed sites in UC. scRNA-seq analysis, transcription factor activity analysis, and gene-enriched pathway analysis indicated that IFNγ-STAT1 signaling activation within Cluster 3 enhances MHC class II expression. To assess T-cell effects, naive CD4+ T-cells were co-cultured with PDPN+ HLA-DR high cells isolated from UC samples. This co-culture induced differentiation into IL-10+ IL-17A+ CD4+ T-cells, with a positive correlation observed between the proportions of IL-10+ IL-17A+ CD4+ T-cells and PDPN+ HLA-DR high cells. Bulk RNA sequencing and qPCR analysis further demonstrated that PDPN+ HLA-DR high cells highly express the IL-27 subunit EBI3 and the TGF-β activator integrin αvβ8. To evaluate clinical relevance, we also examined the correlation between PDPN+ HLA-DR high cell numbers and clinical factors in 45 UC patients. Immunofluorescence staining for the glial marker CDH2 and HLA-DR revealed a negative correlation between CDH2+ HLA-DR+ cell numbers and preoperative Mayo endoscopic scores. Conclusion PDPN+ HLA-DR high glial cells are increased in inflamed areas of UC patients, suggesting a role in anti-inflammatory processes and the maintenance of immune homeostasis.

CFTR dictates monocyte adhesion by facilitating integrin clustering but not activation

Monocytes are critical in controlling tissue infections and inflammation. Monocyte dysfunction contributes to the inflammatory pathogenesis of cystic fibrosis (CF) caused by CF transmembrane conductance regulator (CFTR) mutations, making CF a clinically relevant disease model for studying the contribution of monocytes to inflammation. Although CF monocytes exhibited adhesion defects, the precise mechanism is unclear. Herein, superresolution microscopy showed that an integrin clustering but not an integrin activation defect determines the adhesion defect in CFTR-deficient monocytes, challenging the existing paradigm emphasizing an integrin activation defect in CF patient monocytes. We further found that the clustering defect is accompanied by defects in CORO1A membrane recruitment, actin cortex formation, and CORO1A engagement with integrins. Complementing canonical studies of leukocyte adhesion focusing on integrin activation, we highlight the importance of integrin clustering in cell adhesion and report that integrin clustering and activation are distinctly regulated, warranting further investigation for selective targeting in therapeutic strategy design involving leukocyte-dependent inflammation.

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%-40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

Experimental study of the effects of benz(a)pyrene and Chlorella growth factor in vivo associated with cellular clusters of innate and adaptive immunity

Benz(a)pyrene is a first hazard class aromatic compound which exerts carcinogenic, mutagenic, hematotoxic, and immunotropic effects. The study of cell clusters influenced in vivo by exogenous haptens using laboratory mice as a model object allows us to expand knowledge about the mechanisms and features of immunotropic effects induced by benzo(a)pyrene. The experimental series involved 12 female outbred white mice. The concentration of benzo(a)pyrene for a single dose was 6 μg/L, To neutralize its effects, the Chlorella growth factor (CGF) concentrate was used as an adaptogen, which was consistently administered over 4 weeks at a dose of 60-70 mlns/mL of living cells. Both substances were administered via oral tube in a volume of 1 mL. The study of cell phagocytosis was carried out by the method of V.N. Kaplin. Analysis of the total number of leukocytes and lymphocytes was carried out using a hematology analyzer. The study of cell differentiation clusters was carried out using flow cytometry. Microsoft® Office Excel and Statistica 6.0 programs were used for statistical evaluation. Results: subchronic intoxication with benzo(a) pyrene under the in vivo experimental conditions led to significant modification of cellular immunity, exhibiting a phenotype of deficient innate cellular immunity factors, and imbalance of adaptive cellular immunity with a predominant inhibition of apoptotic activity (CD95+) and modeling of integrin- and VEGFmediated events (CD11a, CD309). Sequential intake of the natural modifier (CGF) was characterized by normalization of phagocytic activity and alleviation of benzo(a)pyrene-induced effects. Conclusions of the study are limited by small number of experimental sample, The in vivo experiment have shown an imbalanced pattern of nonspecific and adaptive cellular immunity, associated with granulocyte changes caused by benzo(a)pyrene along with disturbed control of cell proliferation alleviated by CGF. The intake of the CGF complex led to optimization of immune system as shown by a number of indexes, along with positively modified immunotropic effects of benzo(a)pyrene with cancelled suppression of granulocytic and integrin-associated lymphoid lineages, which provides, mostly, phagocytosis protection, as well as antiapoptotic and integrin-mimetic effects.

Nanoscopy reveals integrin clustering reliant on kindlin-3 but not talin-1

Neutrophils are the most abundant leukocytes in human blood, and their recruitment is essential for innate immunity and inflammatory responses. The initial and critical step of neutrophil recruitment is their adhesion to vascular endothelium, which depends on G protein-coupled receptor (GPCR) triggered integrin inside-out signaling that induces β2 integrin activation and clustering on neutrophils. Kindlin-3 and talin-1 are essential regulators for the inside-out signaling induced β2 integrin activation. However, their contribution in the inside-out signaling induced β2 integrin clustering is unclear because conventional assays on integrin clustering are usually performed on adhered cells, where integrin-ligand binding concomitantly induces integrin outside-in signaling. We used flow cytometry and quantitative super-resolution stochastic optical reconstruction microscopy (STORM) to quantify β2 integrin activation and clustering, respectively, in kindlin-3 and talin-1 knockout leukocytes. We also tested whether wildtype or Pleckstrin homology (PH) domain deleted kindlin-3 can rescue the kindlin-3 knockout phenotypes. GPCR-triggered inside-out signaling alone can induce β2 integrin clustering. As expected, both kindlin-3 and talin-1 knockout decreases integrin activation. Interestingly, only kindlin-3 but not talin-1 contributes to integrin clustering in the scenario of inside-out-signaling, wherein a critical role of the PH domain of kindlin-3 was highlighted. Since talin was known to facilitate integrin clustering in outside-in-signaling-involved cells, our finding provides a paradigm shift by suggesting that the molecular mechanisms of integrin clustering upon inside-out signaling and outside-in signaling are different. Our data also contradict the conventional assumption that integrin activation and clustering are tightly inter-connected by showing separated regulation of the two during inside-out signaling. Our study provides a new mechanism that shows kindlin-3 regulates β2 integrin clustering and suggests that integrin clustering should be assessed independently, aside from integrin activation, when studying leukocyte adhesion in inflammatory diseases.

PEGylation of indium phosphide quantum dots prevents quantum dot mediated platelet activation.

Quantum dots (QDs) are semiconducting inorganic nanocrystals, that have garnered interest in biological and medical spheres due, to their potential benefits in biomedical imaging and drug-delivery systems. Indium phosphide QDs shelled with zinc sulphide (InP/ZnS) are viewed as more biocompatible than previous heavy metal based QDs. However, little is known about how InP/ZnS QDs affect a key blood cell, the platelet. Understanding how platelets interact with QDs is critical as unwanted activation can lead to pathological thrombus formation. Herein, we demonstrate PEGylation of InP/ZnS QDs coated with lipoic acid (QD-LA) or coated with penicillamine (QD-Pen) surface ligands induced markedly less platelet aggregation, platelet-QD interactions, integrin activation, alpha granule secretion and restored platelet spreading in washed platelets in comparison to their non-PEGylated counterparts. Furthermore, in whole blood, PEGylation of QDs reduced the number of QDs in the thrombus, thereby helping to minimise the chance of dysfunctional thrombus formation. Overall, we show that QD PEGylation is important to help prevent QD mediated platelet activation. In combination with the most biocompatible coating, PEGylation markedly reduced platelet activation, widening the concentrations at which QDs were viable for development as potential drug delivery or imaging agents.

Integrins in the kidney - beyond the matrix.

The development and proper functioning of the kidney is dependent on the interaction of kidney cells with the surrounding extracellular matrix (ECM). These interactions are mediated by heterodimeric membrane-bound receptors called integrins, which bind to the ECM via their extracellular domain and via their cytoplasmic tail to intracellular adaptor proteins, to assemble large macromolecular adhesion complexes. These interactions enable integrins to control cellular functions such as intracellular signalling and organization of the actin cytoskeleton and are therefore crucial to organ function. The different nephron segments and the collecting duct system have unique morphologies, functions and ECM environments and are thus equipped with unique sets of integrins with distinct specificities for the ECM with which they interact. These cell-type-specific functions are facilitated by specific intracellular integrin binding proteins, which are critical in determining the integrin activation status, ligand-binding affinity and the type of ECM signals that are relayed to the intracellular structures. The spatiotemporal expression of integrins and their specific interactions with binding partners underlie the proper development, function and repair processes of the kidney. This Review summarizes our current understanding of how integrins, their binding partners and the actin cytoskeleton regulate kidney development, physiology and pathology.

Extracellular Vesicles from a Novel Chordoma Cell Line, ARF-8, Promote Tumorigenic Microenvironmental Changes When Incubated with the Parental Cells and with Human Osteoblasts.

Chordomas are rare, generally slow-growing spinal tumors that nonetheless exhibit progressive characteristics over time, leading to malignant phenotypes and high recurrence rates, despite maximal therapeutic interventions. The tumors are notoriously resistant to therapies and are often located in regions that complicate achieving gross total resections. Cell lines from these tumors are rare as well. We cultured a new chordoma cell line (ARF-8) derived from an extensive clival chordoma that extended back to the cervical spine. We characterized the ARF-8 cellular and extracellular vesicle (EV) proteomes, as well as the impacts of ARF-8 EVs on the proteomes and secretomes of recipient cells (both ARF-8 and human osteoblasts) in autocrine and paracrine settings. Our proteomic analyses suggested roles for transforming growth factor beta (TGFB/TGFβ), cell-matrix interactions involving the epithelial-to-mesenchymal transition (EMT), and cell-extracellular matrix interactions in cell migration, consistent with a migratory/metastatic tumor phenotype. We demonstrated that ARF-8 tumor cell migration was dependent on general (arginine-glycine-aspartic acid [RGD]-based) integrin activity and that ARF-8 EVs could promote such migration. ARF-8 EVs also prompted proteomic/secretomic changes in human osteoblast cells, again with indications that cell-cell and cell-extracellular matrix interactions would be activated. All the characteristics typically associated with chordomas as cancers-migration and invasion, therapeutic resistance, metastatic potential-can be driven by tumor EVs. Overall, ARF-8 EVs promoted predicted tumorigenic phenotypes in recipient cells and suggested novel therapeutic targets for chordomas.

An in-silico study reveals that a C-terminal fragment of the adhesion protein Fibulin7 (Fbln7-C) regulates the activation of integrin α5β1 through dynamics of VWA and the hybrid domain in the β1 subunit

A C-terminal fragment of the adhesion protein Fibulin7 (Fbln7-C) binds to monocytes and neutrophils via integrin α5β1, regulating their adhesion and immunological functions through Erk and STAT signaling pathways. It also inhibits cell binding, spreading, and migration on fibronectin. However, the precise structural components of Fbln7-C that interact with various domains of integrin α5β1 and contribute to its regulatory effects are not entirely understood. This study investigated the structural dynamics of Fibulin7 fragments and the mechanisms by which Fbln7-C regulates α5β1 integrin activation using protein modeling, protein-protein docking, molecular dynamics simulation (MDS), and binding free energy calculations. An energy-minimized model of α5β1 integrin, Fibulin7 full length (Fbln7-FL), and Fbln7-C was developed and validated using 100 ns MDS. Additionally, protein-protein docking was used to confirm Fbln7-C’s better integrin binding ability over Fbln7-FL. A 500 ns MDS on the docked Fbln7-C integrin complex revealed the regulatory effects of Fbln7-C on arginine-glycine-aspartic acid (RGD) bound integrin α5β1. The simulation studies showed that Fbln7-C’s attachment to activated α5β1 integrin increased the distance between the RGD and its interacting residues on both integrin subunits, shifting the RGD ligand from its original binding position and inactivating the integrin. Further analysis using free energy landscape (FEL), principal component analysis (PCA), and binding energy calculation validated the alteration in α5β1 integrin’s structural dynamics following Fbln7-C binding. This could relate to obstruction in the outward swing of the integrin’s hybrid domain and result in the low-affinity, inactive headpiece conformation of the α5β1 integrin.

Human Disabled-2 regulates thromboxane A2 signaling for efficient hemostasis in thrombocytopenia

Understanding platelet protein functions facilitates better assessment of platelet disorders. Megakaryocyte lineage-restricted human Disabled-2 knock-in (hDAB2-KI) mice are generated to delineate the functions of hDab2, a regulator of platelet function, in the control of bleeding associated with thrombocytopenia. Here we show that hDab2-KI mice with thrombocytopenia display decreased bleeding time when compared to the control mice. hDab2 augments thromboxane A2 (TxA2) mimetic U46619- but not other agonists-stimulated granule secretion, integrin activation, and aggregation at a lower platelet concentration in vitro. Binding of hDab2 to phosphatidic acid (PA) facilitates formation of the PA-hDab2-AKT complex leading to an increase in U46619-stimulated AKT-Ser473 phosphorylation and the first wave of ADP/ATP release. Consistent with these findings, hDab2 expression in platelets from patients with immune thrombocytopenic purpura is positively correlated with U46619-stimulated ATP release, which in turn inversely correlated with their bleeding tendency. hDab2 appears crucial in regulating bleeding severity associated with thrombocytopenia by a functional interplay with ADP/ATP release underlying TxA2 signaling.

CD47 and thrombospondin-1 contribute to immune evasion by Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative anaerobic bacterium linked to periodontal disease. Remarkably, P. gingivalis thrives in an inflamed environment rich in activated neutrophils. Toll-like receptor 2 (TLR2) recognition is required for P. gingivalis to evade innate immune killing; however, the mechanisms through which P. gingivalis uncouples host inflammation from bactericidal activity are only partially known. Since integrin activation and alternative signaling are implicated in P. gingivalis TLR2-mediated immune escape, we explored the role of CD47, a widely expressed integrin-associated protein known to suppress phagocytosis and implicated as an interacting partner with other innate immune receptors. We found that CD47 associates with TLR2, and blocking CD47 leads to decreased intracellular P. gingivalis survival in macrophages in a manner dependent on the bacterial major fimbria. In vivo, CD47 knock-out mice cleared P. gingivalis more efficiently than wild-type mice. Next, we found increased expression and secretion of the CD47 ligand thrombospondin-1 (TSP-1) following P. gingivalis infection. Secreted TSP-1 broadly protected P. gingivalis and other periodontitis-associated bacterial species from neutrophil bactericidal activity. Therefore, CD47-TLR2 cosignaling in response to P. gingivalis induces TSP-1 that in turn suppresses neutrophil activity, an effect that can explain how species such as P. gingivalis survive in an inflamed environment and cause dysbiosis.

ANGI-14. DRUGGABLE GENOME CRISPR SCREEN IN 3D HYDROGEL DEVICES REVEALS TARGETABLE KINASES AND PHOSPHATASES IMPLICATED IN CYTOSKELETAL REORGANIZATION IN INVADING GBM CELLS

Abstract The invasive nature of glioblastoma cells into adjacent brain tissues is a key factor in its unfavorable prognosis. To decipher the mechanisms driving glioblastoma invasion, we developed microdissectable biomimetic 3D hydrogel invasion devices containing hyaluronic acid hydrogels decorated with integrin-binding peptides (RGD) and crosslinked with protease-cleavable crosslinkers. The ability to disassemble these devices and interrogate cells in the core vs. invasive fraction allows for high throughput screens. We thus performed a screen in GBM43 cells expressing CRISPR/Cas9 and a 13,250 gRNA library targeting the 2550 genes in the druggable human genome. Of seven initial hits from this screen, only two could be validated by single gene CRISPR and use of a drug to target the hit - ACP1 (also known as LMW-PTP) and Aurora Kinase B (AURKB). Integration of ChIP-seq and RNA-seq revealed no plausible transcriptional targets of ACP1 and AURKB that could drive invasion. Proximity labeling with the TurboID system identified CTTN (cortactin), an actin binding protein whose phosphorylation is integral to the formation of lamellipodia and invadopodia, as phosphorylated by AURKB and Ephrin A2 (EPHA2), whose dephosphorylation controls cell morphology, adhesion, migration and invasion by modifying the organization of the actin cytoskeleton and influencing the activities of integrins and intercellular adhesion molecules, as dephosphorylated by ACP1. These findings underscore the delicate balance between kinase and phosphatase activities in regulating the actin cytoskeleton broadly and at the cell’s front or “leading edge,” offering new therapeutic targets to disrupt the invasion that is a hallmark of glioblastoma.