CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability.

BACKGROUND: The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvß6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability. METHODS: ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion. RESULTS: In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype. CONCLUSIONS: ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC.

Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells.

CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.

CAR T Cells Targeting Membrane-Bound Hsp70 on Tumor Cells Mimic Hsp70-Primed NK Cells.

Strategies to boost anti-tumor immunity are urgently needed to treat therapy-resistant late-stage cancers, including colorectal cancers (CRCs). Cytokine stimulation and genetic modifications with chimeric antigen receptors (CAR) represent promising strategies to more specifically redirect anti-tumor activities of effector cells like natural killer (NK) and T cells. However, these approaches are critically dependent on tumor-specific antigens while circumventing the suppressive power of the solid tumor microenvironment and avoiding off-tumor toxicities. Previously, we have shown that the stress-inducible heat shock protein 70 (Hsp70) is frequently and specifically expressed on the cell surface of many different, highly aggressive tumors but not normal tissues. We could take advantage of tumors expressing Hsp70 on their membrane ('mHsp70') to attract and engage NK cells after in vitro stimulation with the 14-mer Hsp70 peptide TKDNNLLGRFELSG (TKD) plus low dose interleukin (IL)-2. However, a potential limitation of activated primary NK cells after adoptive transfer is their comparably short life span. T cells are typically long-lived but do not recognize mHsp70 on tumor cells, even after stimulation with TKD/IL-2. To combine the advantages of mHsp70-specificity with longevity, we constructed a CAR having specificity for mHsp70 and retrovirally transduced it into primary T cells. Co-culture of anti-Hsp70 CAR-transduced T cells with mHsp70-positive tumor cells stimulates their functional responsiveness. Herein, we demonstrated that human CRCs with a high mHsp70 expression similarly attract TKD/IL-2 stimulated NK cells and anti-Hsp70 CAR T cells, triggering the release of their lytic effector protein granzyme B (GrB) and the pro-inflammatory cytokine interferon (IFN)-γ, after 4 and 24 hours, respectively. In sum, stimulated NK cells and anti-Hsp70 CAR T cells demonstrated comparable anti-tumor effects, albeit with somewhat differing kinetics. These findings, together with the fact that mHsp70 is expressed on a large variety of different cancer entities, highlight the potential of TKD/IL-2 pre-stimulated NK, as well as anti-Hsp70 CAR T cells to provide a promising direction in the field of targeted, cell-based immunotherapies which can address significant unmet clinical needs in a wide range of cancer settings.

Humanized ß2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

The use of cell-based reporter systems has provided valuable insights into the molecular mechanisms of integrin activation. However, current models have significant drawbacks because their artificially expressed integrins cannot be regulated by either physiological stimuli or endogenous signaling pathways. Here, we report the generation of a Hoxb8 cell line expressing human ß2 integrin that functionally replaced the deleted mouse ortholog. Hoxb8 cells are murine hematopoietic progenitor cells that can be efficiently differentiated into neutrophils and macrophages resembling their primary counterparts. Importantly, these cells can be stimulated by physiological stimuli triggering classical integrin inside-out signaling pathways, ultimately leading to ß2 integrin conformational changes that can be recorded by the conformation-specific antibodies KIM127 and mAb24. Moreover, these cells can be efficiently manipulated via the CRISPR/Cas9 technique or retroviral vector systems. Deletion of the key integrin regulators talin1 and kindlin3 or expression of ß2 integrins with mutations in their binding sites abolished both integrin extension and full activation regardless of whether only one or both activators no longer bind to the integrin. Moreover, humanized ß2 integrin Hoxb8 cells represent a valuable new model for rapidly testing the role of putative integrin regulators in controlling ß2 integrin activity in a physiological context.

Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones.

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

Binding of Rap1 and Riam to Talin1 Fine-Tune ß2 Integrin Activity During Leukocyte Trafficking.

ß2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent ß2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the ß2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that ß2 integrin regulation primarily occurs via these two pathways.

Src family kinase-mediated vesicle trafficking is critical for neutrophil basement membrane penetration.

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.

Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation.

Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talins to regulate integrin activity.

A kindlin-3-leupaxin-paxillin signaling pathway regulates podosome stability.

Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation, requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3-null cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation and downstream signaling.

Direct Rap1/Talin1 interaction regulates platelet and neutrophil integrin activity in mice.

Targeting Talin1 to the plasma membrane is a crucial step in integrin activation, which in leukocytes is mediated by a Rap1/RIAM/Talin1 pathway, whereas in platelets, it is RIAM independent. Recent structural, biochemical, and cell biological studies have suggested direct Rap1/Talin1 interaction as an alternative mechanism to recruit Talin1 to the membrane and induce integrin activation. To test whether this pathway is of relevance in vivo, we generated Rap1 binding-deficient Talin1 knockin (Tln13mut) mice. Although Tln13mut mice showed no obvious abnormalities, their platelets exhibited reduced integrin activation, aggregation, adhesion, and spreading, resulting in prolonged tail-bleeding times and delayed thrombus formation and vessel occlusion in vivo. Surprisingly, neutrophil adhesion to different integrin ligands and ß2 integrin-dependent phagocytosis were also significantly impaired, which caused profound leukocyte adhesion and extravasation defects in Tln13mut mice. In contrast, macrophages exhibited no defect in adhesion or spreading despite reduced integrin activation. Taken together, our findings suggest that direct Rap1/Talin1 interaction is of particular importance in regulating the activity of different integrin classes expressed on platelets and neutrophils, which both depend on fast and dynamic integrin-mediated responses.

Differential requirement of kindlin-3 for T cell progenitor homing to the non-vascularized and vascularized thymus.

The role of integrin-mediated adhesion during T cell progenitor homing to and differentiation within the thymus is ill-defined, mainly due to functional overlap. To circumvent compensation, we disrupted the hematopoietic integrin regulator kindlin-3 in mice and found a progressive thymus atrophy that is primarily caused by an impaired homing capacity of T cell progenitors to the vascularized thymus. Notably, the low shear flow conditions in the vascular system at midgestation allow kindlin-3-deficient fetal liver-derived T cell progenitors to extravasate via pharyngeal vessels and colonize the avascular thymus primordium. Once in the thymus, kindlin-3 promotes intrathymic T cell proliferation by facilitating the integrin-dependent crosstalk with thymic antigen presenting cells, while intrathymic T cell migration, maturation into single positive CD4 and CD8 T cells and release into the circulation proceed without kindlin-3. Thus, kindlin-3 is dispensable for integrin-mediated T cell progenitor adhesion and signalling at low and indispensable at high shear forces.

The Yin and Yang of Tyrosine Kinase Inhibition During Experimental Polymicrobial Sepsis.

Neutrophils are the first cells of our immune system to arrive at the site of inflammation. They release cytokines, e.g., chemokines, to attract further immune cells, but also actively start to phagocytose and kill pathogens. In the case of sepsis, this tightly regulated host defense mechanism can become uncontrolled and hyperactive resulting in severe organ damage. Currently, no effective therapy is available to fight sepsis; therefore, novel treatment targets that could prevent excessive inflammatory responses are warranted. Src Family tyrosine Kinases (SFK), a group of tyrosine kinases, have been shown to play a major role in regulating immune cell recruitment and host defense. Leukocytes with SFK depletion display severe spreading and migration defects along with reduced cytokine production. Thus, we investigated the effects of dasatinib, a tyrosine kinase inhibitor, with a strong inhibitory capacity on SFKs during sterile inflammation and polymicrobial sepsis in mice. We found that dasatinib-treated mice displayed diminished leukocyte adhesion and extravasation in tumor necrosis factor-α-stimulated cremaster muscle venules in vivo. In polymicrobial sepsis, sepsis severity, organ damage, and clinical outcome improved in a dose-dependent fashion pointing toward an optimal therapeutic window for dasatinib dosage during polymicrobial sepsis. Dasatinib treatment may, therefore, provide a balanced immune response by preventing an overshooting inflammatory reaction on the one side and bacterial overgrowth on the other side.

Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation.

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.

Maturation of Platelet Function During Murine Fetal Development In Vivo.

OBJECTIVE: Platelet function has been intensively studied in the adult organism. However, little is known about the function and hemostatic capacity of platelets in the developing fetus as suitable in vivo models are lacking. APPROACH AND RESULTS: To examine fetal platelet function in vivo, we generated a fetal thrombosis model and investigated light/dye-induced thrombus formation by intravital microscopy throughout gestation. We observed that significantly less and unstable thrombi were formed at embryonic day (E) 13.5 compared with E17.5. Flow cytometry revealed significantly lower platelet counts in E13.5 versus E17.5 fetuses versus adult controls. In addition, fetal platelets demonstrated changed activation responses of surface adhesion molecules and reduced P-selectin content and mobilization. Interestingly, we also measured reduced levels of the integrin-activating proteins Kindlin-3, Talin-1, and Rap1 during fetal development. Consistently, fetal platelets demonstrated diminished spreading capacity compared with adults. Transfusion of adult platelets into the fetal circulation led to rapid platelet aggregate formation even in young fetuses. Yet, retrospective data analysis of a neonatal cohort demonstrated no correlation of platelet transfusion with closure of a persistent ductus arteriosus, a process reported to be platelet dependent. CONCLUSIONS: Taken together, we demonstrate an ontogenetic regulation of platelet function in vivo with physiologically low platelet numbers and hyporeactivity early during fetal development shedding new light on hemostatic function during fetal life.

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5ß1, αIIbß3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of ß3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5ß1 with the RGD, but essential to re-enforce the binding of α5ß1/αIIbß3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvß3 integrin levels decrease below a critical level.

Minimal amounts of kindlin-3 suffice for basal platelet and leukocyte functions in mice.

Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1.

Loss of the Rap1 effector RIAM results in leukocyte adhesion deficiency due to impaired ß2 integrin function in mice.

Talin is an integrin adaptor, which controls integrin activity in all hematopoietic cells. How intracellular signals promote talin binding to the integrin tail leading to integrin activation is still poorly understood, especially in leukocytes. In vitro studies identified an integrin activation complex whose formation is initiated by the interaction of active, guanosine triphosphate (GTP)-bound Ras-related protein 1 (Rap1) with the adapter protein Rap1-GTP-interacting adapter molecule (RIAM) followed by the recruitment of talin to the plasma membrane. Unexpectedly, loss-of-function studies in mice have shown that the talin-activating role of RIAM is neither required for development nor for integrin activation in platelets. In this study, we show that leukocyte integrin activation critically depends on RIAM both in vitro and in vivo. RIAM deficiency results in a loss of ß2 integrin activation in multiple leukocyte populations, impaired leukocyte adhesion to inflamed vessels, and accumulation in the circulation. Surprisingly, however, the major leukocyte ß1 integrin family member, α4ß1, was only partially affected by RIAM deficiency in leukocytes. Thus, although talin is an essential, shared regulator of all integrin classes expressed by leukocytes, we report that ß2 and α4 integrins use different RIAM-dependent and -independent pathways to undergo activation by talin.