Integrin Ligands Research Articles

Superposition of Substrate Deformation Fields Induced by Molecular Clutches Explains Cell Spatial Sensing of Ligands.

Cells can sense the physical properties of the extracellular matrices (ECMs), such as stiffness and ligand density, through cell adhesions to actively regulate their behaviors. Recent studies have shown that varying ligand spacing of ECMs can influence adhesion size, cell spreading, and even stem cell differentiation, indicating that cells have the spatial sensing ability of ECM ligands. However, the mechanism of the cells' spatial sensing remains unclear. In this study, we have developed a lattice-spring motor-clutch model by integrating cell membrane deformation, the talin unfolding mechanism, and the lattice spring for substrate ligand distribution to explore how the spatial distribution of integrin ligands and substrate stiffness influence cell spreading and adhesion dynamics. By applying the Gillespie algorithm, we found that large ligand spacing reduces the superposition effect of the substrate's displacement fields generated by pulling force from motor-clutch units, increasing the effective stiffness probed by the force-sensitive receptors; this finding explains a series of previous experiments. Furthermore, using the mean-field theory, we obtain the effective stiffness sensed by bound clutches analytically; our analysis shows that the bound clutch number and ligand spacing are the two key factors that affect the superposition effects of deformation fields and, hence, the effective stiffness. Overall, our study reveals the mechanism of cells' spatial sensing, i.e., ligand spacing changes the effective stiffness sensed by cells due to the superposition effect of deformation fields, which provides a physical clue for designing and developing biological materials that effectively control cell behavior and function.

Enhanced breast cancer cell targeting: RGD integrin ligand potentiates RWQWRWQWR’s cytotoxicity and inhibits migration

Enhanced breast cancer cell targeting: RGD integrin ligand potentiates RWQWRWQWR’s cytotoxicity and inhibits migration

CellPLATO - an unsupervised method for identifying cell behaviour in heterogeneous cell trajectory data.

Advances in imaging, segmentation and tracking have led to the routine generation of large and complex microscopy datasets. New tools are required to process this 'phenomics' type data. Here, we present 'Cell PLasticity Analysis Tool' (cellPLATO), a Python-based analysis software designed for measurement and classification of cell behaviours based on clustering features of cell morphology and motility. Used after segmentation and tracking, the tool extracts features from each cell per timepoint, using them to segregate cells into dimensionally reduced behavioural subtypes. Resultant cell tracks describe a 'behavioural ID' at each timepoint, and similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Here, we use cellPLATO to investigate the role of IL-15 in modulating human natural killer (NK) cell migration on ICAM-1 or VCAM-1. We find eight behavioural subsets of NK cells based on their shape and migration dynamics between single timepoints, and four trajectories based on sequences of these behaviours over time. Therefore, by using cellPLATO, we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration.

A multiscale dynamic model of cell–substrate interfaces

Cell–extracellular matrix (ECM) interactions play a pivotal role in many functions of cells, for example, sensing, signaling, migration, and gene expression. The spatial–temporal dynamic evolution of cell–substrate adhesions involves complicated mechano-bio-chemical coupling mechanisms of integrin, adaptor and signaling proteins, as well as the interplay between the cytoskeleton and ECM. In this paper, we establish a multiscale dynamic model of cell–substrate interfaces considering intermolecular force transmission pathways, i.e., intra- and extra-cellular bond dynamics, and mechanochemical coupling regulations. To illustrate its applications, this model is used to reproduce several adhesion-related experimental phenomena of cells, including substrate rigidity sensing, actin-assisted focal adhesion formation, and biphasic interface stress–actin speed relation. The results suggest that the cluster growth patterns are mediated by the intermolecular force transmission pathway and the actin flow speed, and that the integrin–ligand (IL) bond strengthening tends to inhibit the motion of clusters. We reveal that the monotonic-to-biphasic transition of force transmission and adhesion assembly is regulated by the bond rupture mechanosensitivity of integrin adhesion complexes (IACs). It is also demonstrated that this multiscale dynamic model can well describe the spatial–temporal evolutions of collective cell–substrate adhesions with the effects of actin flow and substrate deformation, and can also capture the morphological features of adhesion clusters and related interface force transmissions. This work provides a theoretical tool for studying mechano-biochemical mechanisms of cell–substrate interactions and for understanding various subcellular and cellular dynamic processes in, for example, tumors.

MAdCAM-1 co-stimulation combined with retinoic acid and TGF-β induces blood CD8+ T cells to adopt a gut CD101+ TRM phenotype

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that MAdCAM-1, a ligand for the gut homing receptor α4β7 integrin, in the presence of retinoic acid and TGF-β provide a costimulatory signal that induces blood CD8+ T cells to adopt a TRM -like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell surface markers, along with CD101. They also express CCR5, CCR9 and α4β7, three receptors associated with gut homing. A subset also express E-cadherin, a ligand for αEβ7. Fluorescent lifetime imaging indicated an αEβ7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into TRMs and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue specific immunotherapies.

Tissue Factor Maintains Lung Epithelial Barrier Integrity in Acute Lung Injury Through Cell Adhesion

Background. Acute Respiratory Distress Syndrome (ARDS) is a common cause of acute respiratory failure. While loss of lung epithelial barrier integrity is a pathologic mechanism of ARDS, little is known about the factors that regulate epithelial barrier integrity in the lung. Our previously published mouse studies showed that deletion of alveolar type II epithelial cell Tissue Factor (TF) caused impaired lung barrier integrity in models of Acute Lung Injury (ALI). TF is an integral membrane protein that both initiates the extrinsic coagulation cascade and serves several non-coagulant functions. Notably, TF promotes cell adhesion through interactions with cell surface integrin heterodimers, comprised of individual α and β integrin subunits. As such, we hypothesize that epithelial TF is necessary for maintaining lung barrier integrity and that its overexpression is protective in ALI through increased cell adhesion. Methods. To determine whether supraphysiologic overexpression of TF in the lung epithelium can enhance barrier integrity we created a novel transgenic mouse in which TF was inducibly overexpressed in the lung epithelium (TFEpi+) using a CMV-TetO promoter and crossed with SPC-rtTA59 mice. High alveolar epithelial TF expression compared to wild-type littermates (WT) was confirmed through immunohistochemistry and western blot analysis after one week of doxycycline in drinking water. To induce ALI, mice were intranasally infected with 2000 colony forming units of Klebsiella pneumoniae (KP) or PBS control. At 24-hours post infection, mice were euthanized, lung tissue was collected, and a bronchoalveolar lavage (BAL) was performed. BAL protein, clot time, and leukocyte influx were measured. Lung wet-to-dry weight ratios and bacterial burden were measured. To better understand TF regulation of β1 integrin function, TF-knockout A549 cells were created using CRISPR and cultured on specific β1 integrin ligands (Collagen I, IV, Laminin-511, and Fibronectin). On-cell western blots and crystal violet were used to assess cell surface β1 integrin expression and cell adhesion. Results. TFEpi+ mice showed higher lung TF protein expression (median 38031 [IQR 25033, 57948] vs 1801 [IQR 804.3, 2083] expression normalized to total protein; p=0.0016) compared to WT. TFEpi+ mice infected with KP showed lower BAL protein (mean 248.51 [SD 98.03] vs 366.3 [SD 159.8] μg/ml; p=0.0035) and lung wet-to-dry weight ratios (mean 4.27 [SD 0.37] vs 5.29 [SD 0.75]; p=0.0152) compared to WT. Bacterial burden and BAL inflammatory cell counts were higher, while BAL clot time was lower in infected WT mice compared to control. However, these outcomes did not differ between infected TFEpi+ and WT mice. Loss of TF in alveolar epithelial cells reduced cell surface β1 integrin expression and cell adhesion to Laminin-511. Conclusion: Alveolar epithelial TF overexpression is protective for maintaining lung barrier integrity independent of coagulation and inflammation. Further, cell adhesion studies suggest that this is potentially mediated through increased a6β1 epithelial cell adhesion. AHA Postdoctoral Fellowship, National Institute of Health Grants (HL150783, I01BX002288). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tissue-resident Lymphocytes Are Released During Hypothermic and Normothermic Machine Perfusion of Human Donor Kidneys.

Machine perfusion is the preferred preservation method for deceased donor kidneys. Perfusate fluid, which contains a complex mixture of components, offers potential insight into the organ's viability and function. This study explored immune cell release, particularly tissue-resident lymphocytes (TRLs), during donor kidney machine perfusion and its correlation with injury markers. Perfusate samples from hypothermic machine perfusion (HMP; n = 26) and normothermic machine perfusion (NMP; n = 16) of human donor kidneys were analyzed for TRLs using flow cytometry. Residency was defined by expressions of CD69, CD103, and CD49as. TRL release was quantified exclusively in NMP. Additionally, levels of cell-free DNA, neutrophil gelatinase-associated lipocalin, and soluble E-cadherin (sE-cadherin) were measured in NMP supernatants with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Both HMP and NMP samples contained a heterogeneous population of TRLs, including CD4 + tissue-resident memory T cells, CD8 + tissue-resident memory T cells, tissue-resident natural killer cells, tissue-resident natural killer T cells, and helper-like innate lymphoid cells. Median TRL proportions among total CD45 + lymphocytes were 0.89% (NMP) and 0.84% (HMP). TRL quantities in NMP did not correlate with donor characteristics, perfusion parameters, posttransplant outcomes, or cell-free DNA and neutrophil gelatinase-associated lipocalin concentrations. However, CD103 + TRL release positively correlated with the release of sE-cadherin, the ligand for the CD103 integrin. Human donor kidneys release TRLs during both HMP and NMP. The release of CD103 + TRLs was associated with the loss of their ligand sE-cadherin but not with general transplant injury biomarkers.

Abstract 4380: Role of kindlin-2 in prostate cancer progression and metastasis

Abstract Prostate cancer (PC) is the leading type of cancer in men accounting for more than 25% of all newly reported cancer cases and remains the second leading cause of cancer-related deaths in men in USA in 2023. The success of early treatments has resulted in a 98% 5-year survival rate for early-stage PC. However, resistance to androgen ablation can lead to metastatic castration-resistant prostate cancer (mCRPC), with a significant drop in the survival rate to ~30%. The metastatic cascade involves changes in adhesive, metabolic and signaling properties of PC cells, allowing them to survive, proliferate and thrive in their new microenvironment. Kindlin-2 (K2), a cytoskeletal adapter protein, is highly expressed in cancer, including PC cells and tumors, contributing to their tumorigenic properties. The present study focuses on three K2 functions relevant to PC metastasis: 1)Reduction in K2 levels, achieved through CRISPR/Cas9, profoundly affected the tumorigenic properties of PC cell lines LNCaP (androgen dependent), DU145, and PC3 (both androgen independent), influencing colony formation, invasion, and adhesion to integrin ligands. While reduction in K2 was observed, K1 and K3 levels in these cell lines was not altered by K2KO. 2)The E3 ubiquitin ligase, Parkin, known for its role in Parkinson’s disease, has emerged as a tumor suppressor inhibiting various cancer hallmarks. Our recently published studies showed that Parkin induces ubiquitination of specific lysines in K2, leading to the hypothesis that Parkin acts as a tumor suppressor by targeting K2 for degradation. Stable over-expression of Parkin inhibited adhesion to fibrinogen (Fg), while loss of function Parkin mutants failed to do so, supporting a role of Parkin in K2 turnover and its impact on mitochondrial dynamics. We are currently investigating the effect of K2-lysines mutants as well as loss of function Parkin mutants on the progression and metastasis of PC tumors in vivo.3)K2's established function is the regulation of integrin activation, affecting adhesion receptors' transition from low to high affinity/avidity states. The K2-integrin axis is pivotal for understanding oncogenic responses, and its role in the prostate microenvironment in PC progression and metastasis is under exploration using our newly established K2-floxed mouse. The K2-floxed mice are currently being crossed with mice harboring prostate-epithelial specific cre under control of the probasin (Pbsn) promoter. These animal studies will shed light on K2's contribution to PC development and progression in vivo, providing insights into its role in the prostate microenvironment. To sum up, this study emphasizes the crucial role of K2 in the tumorigenic properties of PC cells and tumors. By incorporating CRISPR/Cas9 and innovative mouse models, the study seeks to shed the lights on the mechanisms through which K2 contributes to PC development and progression. Citation Format: Lamyae El Khalki, Wei Wang, Neelum A. Yousaf Zai, Justin Szpendyk, Elzbieta Pluskota, Katarzyna Bialkowska, Edward F. Plow, Lucia R. Languino, Dario C. Altieri, Khalid Sossey-Alaoui. Role of kindlin-2 in prostate cancer progression and metastasis [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 4380.

PH-Responsive, Self-Assembled Ruthenium Nanodrug: Dual Impact on Lysosomes and DNA for Synergistic Chemotherapy and Immunogenic Cell Death.

Several DNA-damaging antitumor agents, including ruthenium complexes, induce immunogenic cell death (ICD). In this study, an arginyl-glycyl-aspartic acid (RGD) peptide-modified carboline ruthenium complex (KS-Ru) is synthesized as a chemotherapeutic nanodrug and an ICD inducer. The RGD peptide, an integrin ligand, provides tumor-specific targeting and promotes self-assembly of the KS-Ru complex. The pH-responsive self-assembly is assessed through transmission and scanning electron microscopy. Additionally, in vitro cytotoxic activity and anti-metastasis ability are evaluated using MTT and Transwell assays, respectively, along with cellular immunofluorescence staining and imaging flow cytometry. The ability of the complex to inhibit primary tumor formation and lung metastasis in vivo is evaluated using Lewis lung cancer and A549 xenograft models. Furthermore, the tumor immune microenvironment is evaluated using single-cell flow mass cytometry. KS-Ru translocates to the nucleus, causing DNA damage and inducing ICD. Within the lysosomes, KS-Ru self-assembled into nanoflowers, leading to lysosomal swelling and apoptosis. Notably, the as-synthesized pH-dependent ruthenium nanomedicine achieves dual functionality-chemotherapy and immunotherapy. Moreover, the pH-responsive self-assembly of KS-Ru enables simultaneous mechanisms in the lysosome and nucleus, thereby lowering the likelihood of drug resistance. This study provides valuable insight for the design of novel ruthenium-based nanoantitumor drugs.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the β5 integrin

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvβ5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin β5 complexes with the insulin receptor beta (IRβ) in skeletal muscle, resulting in dephosphorylation of IRβ and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between β5 and IRβ impacts IRβ phosphorylation status. We show in invitro and invivo in skeletal muscle in mice that antibody-mediated blockade of the β5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRβ resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The β5-PTP1B complex is recruited by MFGE8 to IRβ leading to termination of canonical insulin signaling. β5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and β5 in regulating insulin signaling.

Abstract C053: Multi-omics guided design of lymphoid organoids to study cooperative cell signaling in lymphomas

Abstract Our collaborative research (Cell1) used 1001 patients of diffuse large B-cell lymphoma (DLBCL) to show that genetic alterations interact with clinical factors to impact overall survival (OS) in DLBCL. B cell receptor (BCR) signaling-driven activated B cell-like (ABC) DLBCL subtypes is the most chemo-resistant subtype to the frontline therapy R-CHOP, with 40% of patients experiencing no response or relapse. We have systematically examined disparities in lymphoma outcomes and identified African-American (AA) patients as a poor-risk population in DLBCL, with a diagnosis age 10 years younger than other racial groups. We found that AA-DLBCL patients experienced inferior 5-year OS compared to non-Hispanic whites (38% vs 46%)2,3. We performed the first-ever characterization of genetic alterations among AA-DLBCL patients and demonstrated distinct mutation patterns across DLBCL arising in AA ancestry groups4. While some of the most frequent genetic mutations in AA-DLBCL are related to chromatin and epigenetics, few studies have investigated racial disparities in patients with aggressive ABC DLBCLs. The mechanisms that determine this racial inequality are unknown but may lie in the particular spectrum of mutations that act in concert with intricate survival signals imparted by the lymph node tumor microenvironment (Ly-TME). The pathogenesis of ABC DLBCLs has been linked to constitutive activation of and cooperation between the BCR and Toll-like receptor (TLR) pathways. These pathways are emerging as novel therapeutic targets, with a particular interest in MALT1 (mucosa-associated lymphoid tissue translocation protein 1). However, the genetic heterogeneity of ABC DLBCLs and the cooperative signaling mediated by the Ly-TME have led to discrepancies in drug responses. As a first step towards understanding Ly-TME in DLBCLs (Nature Materials5), we used DLBCL patient samples from multiple cohorts, covering >1100 samples and imaging analysis to determine immune and ECM cues in the lymphoid TME. These samples were not classified into racial categories. We designed a biomaterials-based modular lymphoma organoid that presents specific integrin ligands at controlled densities to DLBCLs. Human ABC-DLBCLs responded to MALT1 inhibition in an integrin-ligand-dependent manner. TLR activation with TLR agonists rescued cells from MALT1 inhibitors, possibly due to increased MALT1 expression. The inclusion of T-cell signals increased MALT1 expression and rescued cells from MALT1 inhibitor-mediated killing in cell lines and human PDX organoids. Changes in RNA-seq and phosphorylation in the TLR & BCR pathways were used to inform effective combination therapy approaches with MALT1 inhibitors in vitro and in vivo5. Ongoing work is determining the racial disparities in AA lymphomas through complex interactions between Ly-TME and DLBCLs.

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Cerebral arterioles express the laminin subunits α4 and α5 in conjunction with α6β4 integrin, but strongly downregulate laminin α4 during hypoxia-induced arteriogenic remodeling

Previous studies have shown that expression of the endothelial laminin receptor α6β4 integrin in the brain is uniquely restricted to arterioles. As exposure to chronic mild hypoxia (CMH, 8 % O2) stimulates robust angiogenic and arteriogenic remodeling responses in the brain, the goal of this study was to determine how CMH influences cerebrovascular expression of the β4 integrin as well as its potential ligands, laminin 411 and 511, containing the α4 and α5 laminin subunits respectively, and then define how aging impacts this expression. We observed the following: (i) CMH launched a robust arteriogenic remodeling response both in the young (10 weeks) and aged (20 months) brain, correlating with an increased number of β4 integrin+ vessels, (ii) while the laminin α4 subunit is expressed evenly across all cerebral blood vessels, laminin α5 was highly expressed preferentially on β4 integrin+ arterioles, (iii) CMH-induced arteriolar remodeling was associated with strong downregulation of the laminin α4 subunit but no change in the laminin α5 subunit, (iv) in addition to its expression on arterioles, β4 integrin was also expressed at lower levels on capillaries specifically in white matter (WM) tracts but not in the grey matter (GM), and (v), these observations were consistent in both the brain and spinal cord, and age had no obvious impact. Taken together, our findings suggest that laminin 511 may be a specific ligand for α6β4 integrin and that dynamic switching of the laminin subunits α4 and α5 might play an instructive role in arteriogenic remodeling. Furthermore, β4 integrin expression differentiates WM from GM capillaries, highlighting a novel and important difference.

Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia

We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate Insulin Like Growth Factor 1 Receptor (IGF1R) signaling in leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL in vitro, implicating additional myeloid-mediated signals in leukemia progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion activates downstream focal adhesion kinase (FAK)/ proline-rich tyrosine kinase 2 (PYK2), which are required for myeloid-mediated T-ALL support, partly through activation of IGF1R. Blocking integrin ligands or inhibiting FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated adhesion or FAK/PYK2 also reduces survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate with higher FAK signaling and myeloid gene signatures and are associated with an inferior prognosis in pediatric T-ALL patients. Together, these findings demonstrate that integrin activation and downstream FAK/PYK2 signaling are important mechanisms underlying myeloid-mediated support of T-ALL progression.

Effect of Dimeric Disintegrins Isolated from Vipera lebetina obtusa Venom on Glioblastoma Cellular Responses.

Integrins play a fundamental role in the migration and invasiveness of glioblastoma (GBM) cells, making them suitable targets for innovative cancer therapy. The aim of this study was to evaluate the effect of the RGD homodimeric disintegrin VLO4, isolated from Vipera lebetina obtusa venom, on the adhesion, spreading, migration, and survival of LBC3, LN18, and LN229 cell lines. This disintegrin, as a potent antagonist for α5β1 integrin, showed pro-adhesive properties for these cell lines, the highest for LN229 and the lowest for LBC3. Glioblastoma cells displayed significant differences in the spreading on the immobilized VLO4 and the natural α5β1 integrin ligand, fibronectin. Solubilized VLO4 showed different cytotoxicity and pro-apoptotic properties among tested cell lines, with the highest against LN18 and none against LN229. Moreover, VLO4 revealed an inhibitory effect on the migration of LBC3 and LN18 cell lines, in contrast to LN229 cells, which were not sensitive to this disintegrin. However, LN229 migration was impaired by VLO5, a disintegrin antagonistic to integrin α9β1, used in combination with VLO4. A possible mechanism of action of VLO4 may be related to the downregulation of α5β1 integrin subunit expression, as revealed by Western blot. VLO4 also inhibited cell proliferation and induced caspase-dependent apoptosis in LBC3 and LN18 cell lines. These results indicate that targeting α5β1 integrin by related VLO4 compounds may be useful in the development of integrin-targeted therapy for glioblastoma.

Virtual Screening of Protein Data Bank via Docking Simulation Identified the Role of Integrins in Growth Factor Signaling, the Allosteric Activation of Integrins, and P-Selectin as a New Integrin Ligand.

Integrins were originally identified as receptors for extracellular matrix (ECM) and cell-surface molecules (e.g., VCAM-1 and ICAM-1). Later, we discovered that many soluble growth factors/cytokines bind to integrins and play a critical role in growth factor/cytokine signaling (growth factor-integrin crosstalk). We performed a virtual screening of protein data bank (PDB) using docking simulations with the integrin headpiece as a target. We showed that several growth factors (e.g., FGF1 and IGF1) induce a integrin-growth factor-cognate receptor ternary complex on the surface. Growth factor/cytokine mutants defective in integrin binding were defective in signaling functions and act as antagonists of growth factor signaling. Unexpectedly, several growth factor/cytokines activated integrins by binding to the allosteric site (site 2) in the integrin headpiece, which is distinct from the classical ligand (RGD)-binding site (site 1). Since 25-hydroxycholesterol, a major inflammatory mediator, binds to site 2, activates integrins, and induces inflammatory signaling (e.g., IL-6 and TNFα secretion), it has been proposed that site 2 is involved in inflammatory signaling. We showed that several inflammatory factors (CX3CL1, CXCL12, CCL5, sPLA2-IIA, and P-selectin) bind to site 2 and activate integrins. We propose that site 2 is involved in the pro-inflammatory action of these proteins and a potential therapeutic target. It has been well-established that platelet integrin αIIbβ3 is activated by signals from the inside of platelets induced by platelet agonists (inside-out signaling). In addition to the canonical inside-out signaling, we showed that αIIbβ3 can be allosterically activated by inflammatory cytokines/chemokines that are stored in platelet granules (e.g., CCL5, CXCL12) in the absence of inside-out signaling (e.g., soluble integrins in cell-free conditions). Thus, the allosteric activation may be involved in αIIbβ3 activation, platelet aggregation, and thrombosis. Inhibitory chemokine PF4 (CXCL4) binds to site 2 but did not activate integrins, Unexpectedly, we found that PF4/anti-PF4 complex was able to activate integrins, indicating that the anti-PF4 antibody changed the phenotype of PF4 from inhibitory to inflammatory. Since autoantibodies to PF4 are detected in vaccine-induced thrombocytopenic thrombosis (VIPP) and autoimmune diseases (e.g., SLE, and rheumatoid arthritis), we propose that this phenomenon is related to the pathogenesis of these diseases. P-selectin is known to bind exclusively to glycans (e.g., sLex) and involved in cell-cell interaction by binding to PSGL-1 (CD62P glycoprotein ligand-1). Unexpectedly, through docking simulation, we discovered that the P-selectin C-type lectin domain functions as an integrin ligand. It is interesting that no one has studied whether P-selectin binds to integrins in the last few decades. The integrin-binding site and glycan-binding site were close but distinct. Also, P-selectin lectin domain bound to site 2 and allosterically activated integrins.

A simple and robust nanosystem for photoacoustic imaging of bladder cancer based on α5β1-targeted gold nanorods

BackgroundEarly detection and removal of bladder cancer in patients is crucial to prevent tumor recurrence and progression. Because current imaging techniques may fail to detect small lesions of in situ carcinomas, patients with bladder cancer often relapse after initial diagnosis, thereby requiring frequent follow-up and treatments.ResultsIn an attempt to obtain a sensitive and high-resolution imaging modality for bladder cancer, we have developed a photoacoustic imaging approach based on the use of PEGylated gold nanorods (GNRs) as a contrast agent, functionalized with the peptide cyclic [CphgisoDGRG] (Iso4), a selective ligand of α5β1 integrin expressed by bladder cancer cells. This product (called GNRs@PEG-Iso4) was produced by a simple two-step procedure based on GNRs activation with lipoic acid-polyethyleneglycol(PEG-5KDa)-maleimide and functionalization with peptide Iso4. Biochemical and biological studies showed that GNRs@PEG-Iso4 can efficiently recognize purified integrin α5β1 and α5β1-positive bladder cancer cells. GNRs@PEG-Iso4 was stable and did not aggregate in urine or in 5% sodium chloride, or after freeze/thaw cycles or prolonged exposure to 55 °C, and, even more importantly, do not settle after instillation into the bladder. Intravesical instillation of GNRs@PEG-Iso4 into mice bearing orthotopic MB49-Luc bladder tumors, followed by photoacoustic imaging, efficiently detected small cancer lesions. The binding to tumor lesions was competed by a neutralizing anti-α5β1 integrin antibody; furthermore, no binding was observed to healthy bladders (α5β1-negative), pointing to a specific targeting mechanism.ConclusionGNRs@PEG-Iso4 represents a simple and robust contrast agent for photoacoustic imaging and diagnosis of small bladder cancer lesions.Graphical

Optimizing the enzymatic release of MMAE from isoDGR-based small molecule drug conjugate by incorporation of a GPLG-PABC enzymatically cleavable linker.

Antibody-Drug Conjugates (ADCs) and Small Molecule-Drug Conjugates (SMDCs) represent successful examples of targeted drug-delivery technologies for overcoming unwanted side effects of conventional chemotherapy in cancer treatment. In both strategies, a cytotoxic payload is connected to the tumor homing moiety through a linker that releases the drug inside or in proximity of the tumor cell, and that represents a key component for the final therapeutic effect of the conjugate. Here, we show that the replacement of the Val-Ala-p-aminobenzyloxycarbamate linker with the Gly-Pro-Leu-Gly-p-aminobenzyloxycarbamate (GPLG-PABC) sequence as enzymatically cleavable linker in the SMDC bearing the cyclo[DKP-isoDGR] αVβ3 integrin ligand as tumor homing moiety and the monomethyl auristatin E (MMAE) as cytotoxic payload led to a 4-fold more potent anti-tumoral effect of the final conjugate on different cancer cell lines. In addition, the synthesized conjugate resulted to be significantly more potent than the free MMAE when tested following the "kiss-and-run" protocol, and the relative potency were clearly consistent with the expression of the αVβ3 integrin receptor in the considered cancer cell lines. In vitro enzymatic cleavage tests showed that the GPLG-PABC linker is cleaved by lysosomal enzymes, and that the released drug is observable already after 15min of incubation. Although additional data are needed to fully characterize the releasing capacity of GPLG-PABC linker, our findings are of therapeutic significance since we are introducing an alternative to other well-established enzymatically sensitive peptide sequences that might be used in the future for generating more efficient and less toxic drug delivery systems.

Co-treatment of Naringenin and Ketoprofen-RGD Suppresses Cell Proliferation via Calmodulin/PDE/cAMP/PKA Axis Pathway in Leukemia and Ovarian Cancer Cells.

Naringenin (Nar) has anti-inflammatory and anticarcinogenic properties. Arginine-glycine- aspartate (RGD) is a tripeptidic sequence used as an integrin ligand and targeting system for delivering chemotherapeutic agents to cancer cells. In this study, the inhibitory effects of Nar and ketoprofen-RGD on leukemia and ovarian cancer cells (K562 and SKOV3) were explored for the first time, focusing on their proliferation activity and their anti-inflammatory capacity. Analyses were conducted on the calmodulin (CaM)-dependent phosphodiesterase 1 (PDE1) activation by ketoprofen-RGD, Nar, and their combination. These drugs' effects on protein kinase A (PKA) activation, intracellular cyclic adenosine monophosphate (cAMP) level, and PDE1 inhibition were identified. Later, it was also evaluated if ketoprofen-RGD alone or in combination with Nar had anti-inflammatory effects. Nar improved the antagonizing consequences of ketoprofen-RGD on the CaM protein, which hinders PDE1, improving PKA activity and cAMP levels. A mixture of ketoprofen-RGD and Nar and ketoprofen-RGD alone diminished K562 and SKOV3 cell viability through the cAMP/PKA pathway by inhibiting PDE1 and CaM. These two compounds showed anti-inflammatory effects on both cell lines. This study indicated for the first time that combining ketoprofen-RGD and Nar can be a promising anti-inflammatory therapeutic regimen for treating leukemia and ovarian cancer.

Thyroid hormone membrane receptor binding and transcriptional regulation in the sea urchin Strongylocentrotus purpuratus.

Thyroid hormones (THs) are small amino acid derived signaling molecules with broad physiological and developmental functions in animals. Specifically, their function in metamorphic development, ion regulation, angiogenesis and many others have been studied in detail in mammals and some other vertebrates. Despite extensive reports showing pharmacological responses of invertebrate species to THs, little is known about TH signaling mechanisms outside of vertebrates. Previous work in sea urchins suggests that non-genomic mechanisms are activated by TH ligands. Here we show that several THs bind to sea urchin (Strongylocentrotus purpuratus) cell membrane extracts and are displaced by ligands of RGD-binding integrins. A transcriptional analysis across sea urchin developmental stages shows activation of genomic and non-genomic pathways in response to TH exposure, suggesting that both pathways are activated by THs in sea urchin embryos and larvae. We also provide evidence associating TH regulation of gene expression with TH response elements in the genome. In ontogeny, we found more differentially expressed genes in older larvae compared to gastrula stages. In contrast to gastrula stages, the acceleration of skeletogenesis by thyroxine in older larvae is not fully inhibited by competitive ligands or inhibitors of the integrin membrane receptor pathway, suggesting that THs likely activate multiple pathways. Our data confirms a signaling function of THs in sea urchin development and suggests that both genomic and non-genomic mechanisms play a role, with genomic signaling being more prominent during later stages of larval development.