Adhesive Ligands Research Articles

Glycoside Residues on Platelet's Surface Regulate Platelet Function, Apoptosis and Binding of Coagulation Complexes in Patients with Immune Thrombocytopaenia

Introduction: Platelet surface glycoproteins (GPs) are highly glycosylated and are key elements for platelet function since most of them constitute receptors for adhesion ligands. However, exact role of their glycan composition is not clear. Under normal conditions, platelets contain sialic acid in the carbohydrate side chains of their GPs, and it has been described that alterations in the degree of their sialinization can affect the clearance of platelets. This mechanism has been proposed as involved in etiopathogenesis of immune thrombocytopaenia (ITP), mainly in those patients who do not respond to treatments. Thus, after the loss of sialic acid, there would be a greater exposure of galactose and of N-acetyl-glucosamine residues on the surface of circulating platelets to hepatic Ashwell-Morell receptors, which could induce their phagocytosis and platelet clearance. On the other hand, procoagulant platelets, defined as the platelet subpopulation that binds functional prothrombinase, exposed on their surface increased levels of P-selectin and GPIb, two glycan rich GPs. So, it is tempting to speculate that changes in glycan residues on platelet surface may induce changes in their function. Aim: We aimed to assess in ITP patients whether changes in platelet glycosylation, mainly the loss of sialic acid, may condition platelet function, apoptosis and binding of prothrombinase complex. Methods: This is an observational, prospective and transversal study approved by Ethics Committee from La Paz University Hospital. One hundred and eight patients with chronic primary ITP (68 with a platelet count ≥30x103 platelets/µL and 40 with a platelet count <30x103 platelets/µL) and 132 healthy controls were included after signing the informed consent. Platelet activation markers were determined in platelet rich plasma; whereas platelet glycosylation, binding of prothrombinase, annexin V and caspase's activities were assayed in washed platelets. Samples were analyzed by flow cytometry. Table 1 shows lectins tested and their sugar-binding specificity. Data were analyzed with GraphPad Prism 6.0 software. Results: Platelets from ITP patients with a platelet count <30x103/µL exposed less sialic acid in correspondence to an enhanced binding of lectins to non-sialylated residues. Moreover, levels of α1,6-Fucose, a glycan residue which could directly regulate antibody-dependent cellular cytotoxicity, and of α-Mannose, which could be recognized by the mannose binding lectin and activate complement pathway, were increased in platelets from these ITP patients. In accordance, sialic acid loss and consequent platelet surface exposure of other glycoside residues were inversely related to platelet count and ability to be activated (Table 1). These differences in glycosylation observed in ITP patients with a platelet count <30x103/µL were accompanied by a less ability of platelets to be activated (Figure 1), an increased exposure of phosphatidylserine and higher caspase activites (Figure 2). Moreover, increased exposure of phosphatidylserine and of N-acetyl-glucosamine residues (measured through the binding of WGA) enhanced binding of prothrombinase complex (Figure 3). Conclusion: Changes in glycoside composition of GPs on platelet's surface impaired their functional capacity, increases their apoptosis and modifies conditions for the binding of coagulation proteins. These modifications in platelet's glycoside residues seem to be related to severity of ITP. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772) and and Platelet Disorder Support Association. EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Butta: Grifols: Research Funding; Novartis: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; SOBI: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Speakers Bureau. Alvarez Román:Grifols: Research Funding; Bayer: Consultancy; Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau. Martín:SOBI: Research Funding; Pfizer: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau. Rivas Pollmar:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Justo Sanz:Takeda: Current Employment. García Barcenilla:NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Pfizer,: Speakers Bureau; Roche: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Speakers Bureau. Canales:Celgene: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria; Roche: Honoraria; Gilead: Honoraria; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Sandoz: Honoraria; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding.

Matrix Stiffness-Regulated Growth of Breast Tumor Spheroids and Their Response to Chemotherapy.

Interactions between tumor cells and the extracellular matrix (ECM) are an important factor contributing to therapy failure in cancer patients. Current in vitro breast cancer spheroid models examining the role of mechanical properties on spheroid response to chemotherapy are limited by the use of two-dimensional cell culture, as well as simultaneous variation in hydrogel matrix stiffness and other properties, e.g., hydrogel composition, pore size, and cell adhesion ligand density. In addition, currently used hydrogel matrices do not replicate the filamentous ECM architecture in a breast tumor microenvironment. Here, we report a collagen-alginate hydrogel with a filamentous architecture and a 20-fold variation in stiffness, achieved independently of other properties, used for the evaluation of estrogen receptor-positive breast cancer spheroid response to doxorubicin. The variation in hydrogel mechanical properties was achieved by altering the degree of cross-linking of alginate molecules. We show that soft hydrogels promote the growth of larger MCF-7 tumor spheroids with a lower fraction of proliferating cells and enhance spheroid resistance to doxorubicin. Notably, the stiffness-dependent chemotherapeutic response of the spheroids was temporally mediated: it became apparent at sufficiently long cell culture times, when the matrix stiffness has influenced the spheroid growth. These findings highlight the significance of decoupling matrix stiffness from other characteristics in studies of chemotherapeutic resistance of tumor spheroids and in development of drug screening platforms.

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

Chemically Orthogonal Protein Ligation Domains for Independent Control of Hydrogel Modification with Adhesive Ligands and Growth Factors.

The twin, chemically orthogonal protein ligation domains, SpyCatcher and SnoopCatcher, were used to link two engineered proteins into poly(ethylene glycol) (PEG) hydrogels in order to control both endothelial cell adhesion and material-mediated pro-mitotic stimulation. SpyCatcher was appended with an N-terminal adhesion ligand RGDS to form RGDS-SC, and SnoopCatcher was appended with the vascular endothelial growth factor (VEGF)-mimetic peptide QK to form QK-SnpC. QK-SnpC formed a spontaneous covalent bond with SnoopTag peptide with 40% reaction efficiency, both in solution, in a PEG gel containing SnoopTag peptide, and in a PEG gel with both SnoopTag and SpyTag sites. QK-SnpC added to cell culture media enhanced endothelial cell proliferation compared to a negative control, and was statistically indistinguishable from the positive control of 130 pM VEGF165. Endothelial cells seeded onto PEG gels presenting both RGDS-SC and QK-SnpC showed ∼50% of cells actively proliferating (defined as Ki67+), compared to ∼31% of cells seeded on gels presenting RGDS-SC alone. These results show that complementary nondiffusing biochemical signals can be linked into PEG-DA hydrogels simultaneously using 'Catcher-based ligation strategies, thereby inducing more nuanced cell-material interactions.

Cell Migration Driven by Self-Generated Integrin Ligand Gradient on Ligand-Labile Surfaces

Integrin-ligand interaction mediates the adhesion and migration of many metazoan cells. Here, we report a unique mode of cell migration elicited by the lability of integrin ligands. We found that stationary cells spontaneously turn migratory on substrates where integrin ligands are subject to depletion by cellular force. Using TGT, a rupturable molecular linker, we quantitatively tuned the rate of ligand rupture by cellular force and tested platelets (anucleate cells), CHO-K1 cells (nucleated cells), and other cell types on TGT surfaces. These originally stationary cells readily turn motile on the uniform TGT surface, and their motility is correlated with the ligand depletion rate caused by cells. We named this new migration mode ligand-depleting (LD) migration. Through both experiments and simulations, we revealed the biophysical mechanism of LD migration. We found that the cells create and maintain a gradient of ligand surface density underneath the cell body by constantly rupturing local ligands, and the gradient in turn drives and guides cell migration. This is reminiscent of the phenomenon that some liquid droplets or solid beads can spontaneously move on homogeneous surfaces by chemically forming and maintaining a local gradient of surface energy. Here, we showed that cells, as living systems, can harness a similar mechanism to migrate. LD migration is beneficial for cells to maintain adhesion on ligand-labile surfaces, and might also play a role in the migration of cancer cells, immune cells, and platelets that deplete adhesive ligands of the matrix.

Abstract 314: Micropatterning supported method for cancer spheroid assays under flow and high throughput CAR-T cell potency assay with single cell resolution

Abstract Tumor spheroids, organoids, and cell culture models demand defined cell-cell and cell-matrix interactions, ideally with specific cell attachment on adhesion sites for multicellular spheroids or single cells. Our bioinert based micropatterning technology allows for the creation of adhesion spots while the surrounding passivated area is biologically inert, not interfering with the assay.We use this micro-patterning of adhesion ligands on highly passivated surfaces to generate homogenously distributed spheroids within a microfluidic channel system which can be perfused. Spheroids can either be directly generated by a simple injection of a large number of cells and a self-sorting process or by the addition of pre-formed spheroids, which will bind to the patterned adhesion sites. Perfusion of the spheroid chip enables the homogenous supply of all spheroids with nutrients and oxygen, and also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. Applying a flow increases the overall spheroid growth, but condenses the cell aggregate, as well as influences the drug triggered protein expression response towards a more in vivo-like reaction.We also use this technique to generate arrays of homogenously distributed single cancer cells to evaluate cytotoxic T cell activity on a single cell level. By optical analysis combined with machine-learning based image processing of cell / spheroid arrays we obtained efficient label-free analysis of each cancer cell - T cell interaction over time. Citation Format: Elias Horn, Miriam Balles, Angelika J. Fischbeck, Jan Schwarz, Dane A. Thyssen, David W. Drell, Elfriede Noessner, Roman Zantl. Micropatterning supported method for cancer spheroid assays under flow and high throughput CAR-T cell potency assay with single cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 314.

PVR and ICAM-1 on Blast Crisis CML Stem and Progenitor Cells with TKI Resistance Confer Susceptibility to NK Cells.

The BCR-ABL1 fusion gene generating an oncogenic tyrosine kinase is a hallmark of chronic myeloid leukemia (CML), which can be successfully targeted by BCR-ABL1 tyrosine kinase inhibitors (TKIs). However, treatment-free remission has been achieved in a minority of patients due to evolving TKI resistance and intolerance. Primary or acquired resistance to the approved TKIs and progression to blast crisis (BC), thus, remain a major clinical challenge that requires alternative therapeutic strategies. Here, we first demonstrate that donor natural killer (NK) cells prepared using a protocol adopted in clinical trials can efficiently eliminate CML-BC blasts, with TKI resistance regardless of BCR-ABL1 mutations, and preferentially target CD34+CD38− leukemic stem cells (LSC), a potential source of disease relapse. Mechanistically, the predominant expression of PVR, a ligand for the NK cell-activating DNAM-1 receptor, in concert with ICAM-1, a ligand for NK cell adhesion, confer this susceptibility to NK cells, despite the lack of ligands for NKG2D, a principal NK cell activating receptor, as an immune evasion mechanism. With these mechanistic insights, our findings provide a proof-of-concept that donor NK cell-based therapy is a viable strategy for overcoming TKI resistance in CML, particularly the advanced, multi-TKI-resistant CML with dismal outcome.

A modular polymer microbead angiogenesis scaffold to characterize the effects of adhesion ligand density on angiogenic sprouting

Three-dimensional micro-physiological in vitro representations of human tissues and organs are emerging as important models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. An enduring need is to create microvascular networks within such 3D models, particularly for tissues with high metabolic demand such as the liver, pancreas, and the central nervous system. Here we report a facile approach to drive angiogenesis in nascent 3D culture models by embedding degradable hydrogel microbeads coated with induced pluripotent stem cell-derived endothelial cells (MB-iPSC-ECs) in a dense epithelial tissue. Specifically, we describe an approach to optimize microbead scaffold cues, independent of the external environment, by evaluating the iPSC-EC to microbead adhesion properties and how they influence the propensity of cells to both coat microbeads uniformly and undergo sprouting angiogenesis. We encapsulated MB-iPSC-ECs in PEG hydrogels, systematically varied the relative concentration of integrin-targeting peptide motifs in the microbead and surrounding environment, and found that an optimal microbead scaffold ligand regime of 0.1–0.25 mM promotes iPSC-EC monolayer formation and subsequent invasion into the synthetic matrix. We used these results to predict the regime of adhesion ligand required to promote angiogenesis of MB-iPSC-ECs in a co-culture hepatocarcinoma (HEPG2) microtissue model. This modular degradable microbead platform has the potential to promote angiogenic sprouting, which may ultimately support vascularization of a variety of cell-dense tissues.

Hydrogel Properties May Influence Mesenchymal Stem Cell Lineage Progression Through Modulating GAPDH Activity

Cell traction forces, biochemical signals, and cell metabolism are each known to regulate mesenchymal stem cell (MSC) differentiation. Biomaterials have therefore been designed to manipulate cell traction force (via their viscoelasticity and adhesion ligands) to guide MSC fate decisions. Similarly, the type and density of biochemical signals presented by a material have been tailored to influence MSC differentiation. However, the potential impact of biomaterial properties on regulating MSC differentiation through modulating cell metabolism is relatively unstudied. Here, we present data indicating that hydrogel elastic modulus and mesh size regulate MSC differentiation in part through modulating the activity of the metabolic enzyme glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Toward this end, we first confirm that the differentiation profile of MSCs cultured in 2D on highly elastic, covalently crosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels differs substantially from that of MSCs encapsulated within the same hydrogel formulations, indicating a dependence in 3D on a variable(s) beyond elastic modulus. Further results indicate that the GAPDH activity of MSCs in 3D hydrogels is a function of elastic modulus and mesh size, suggesting that GAPDH activity may be one of these variables. Studies in 2D supported a positive correlation between hydrogel elastic modulus and GAPDH activity. Additionally, inhibition of GAPDH activity on 2D surfaces induced alterations in the profiles of key differentiation markers, indicating that GAPDH activity can impact lineage progression. Cumulatively, these findings suggest that the potential impact of hydrogel properties on cell metabolism should be considered when evaluating biomaterial-driven MSC differentiation. Cell traction forces, biochemical signals, and cell metabolism are each known to regulate mesenchymal stem cell (MSC) differentiation. Biomaterials have therefore been designed to manipulate cell traction force (via their viscoelasticity and adhesion ligands) to guide MSC fate decisions. Similarly, the type and density of biochemical signals presented by a material have been tailored to influence MSC differentiation. However, the impact of biomaterial properties on regulating MSC differentiation through modulating cell metabolism is relatively unstudied. Here, we present data indicating that hydrogel elastic modulus and mesh size regulate MSC differentiation in part through modulating the activity of the metabolic enzyme glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

Ligand Diffusion Enables Force-Independent Cell Adhesion via Activating α5β1 Integrin and Initiating Rac and RhoA Signaling.

Cells reside in a dynamic microenvironment in which adhesive ligand availability, density, and diffusivity are key factors regulating cellular behavior. Here, the cellular response to integrin-binding ligand dynamics by directly controlling ligand diffusivity via tunable ligand-surface interactions is investigated. Interestingly, cell spread on the surfaces with fast ligand diffusion is independent of myosin-based force generation. Fast ligand diffusion enhances α5β1 but not αvβ3 integrin activation and initiates Rac and RhoA but not ROCK signaling, resulting in lamellipodium-based fast cell spreading. Meanwhile, on surfaces with immobile ligands, αvβ3 and α5β1 integrins synergistically initiate intracellular-force-based canonical mechanotransduction pathways to enhance cell adhesion and osteogenic differentiation of stem cells. These results indicate the presence of heretofore-unrecognized pathways, distinct from canonical actomyosin-driven mechanisms, that are capable of promoting cell adhesion.

Actin Alpha 2 (ACTA2) Downregulation Inhibits Neural Stem Cell Migration through Rho GTPase Activation.

Although neural stem cells (NSCs) could migrate towards lesions after central nervous system (CNS) injury, the migration ability always is restricted due to the disturbed composition and density of the adhesion ligands and extracellular matrix (ECM) gradient after CNS injury. To date, various methods have been developed to enhance NSC migration and a number of factors, which are affecting NSC migration potential, have been identified. Here, primary NSCs were cultured and the expression of actin alpha 2 (ACTA2) in NSCs was determined using reverse transcription polymerase chain reaction (RT-PCR) and immunostaining. Next, the role of ACTA2 in regulating NSC migration and the potential mechanism was explored. Our results demonstrated that ACTA2 expressed in NSCs. Meanwhile, downregulated ACTA2 using siRNA inhibited NSC migration through hindering actin filament polymerization via increasing RhoA expression and decreasing Rac1 expression. The present study might enrich the basic knowledge of ACTA2 in NSC migration and open an avenue for enhancing NSC migration potential, subsequently providing an intervention target for functional recovery after CNS injury.

Extracellular matrix mechanics regulate transfection and SOX9-directed differentiation of mesenchymal stem cells

Gene delivery within hydrogel matrices can potentially direct mesenchymal stem cells (MSCs) towards a chondrogenic fate to promote regeneration of cartilage. Here, we investigated whether the mechanical properties of the hydrogel containing the gene delivery systems could enhance transfection and chondrogenic programming of primary human bone marrow-derived MSCs. We developed collagen-I-alginate interpenetrating polymer network hydrogels with tunable stiffness and adhesion properties. The hydrogels were activated with nanocomplexed SOX9 polynucleotides to direct chondrogenic differentiation of MSCs. MSCs transfected within the hydrogels showed higher expression of chondrogenic markers compared to MSCs transfected in 2D prior to encapsulation. The nanocomplex uptake and resulting expression of transfected SOX9 were jointly enhanced by increased stiffness and cell-adhesion ligand density in the hydrogels. Further, transfection of SOX9 effectively induced MSCs chondrogenesis and reduced markers of hypertrophy compared to control matrices. These findings highlight the importance of matrix stiffness and adhesion as design parameters in gene-activated matrices for regenerative medicine. Statement of SignificanceGene-activated matrices (GAMs) are biodegradable polymer networks integrating gene therapies, and they are promising technologies for supporting tissue regeneration. Despite this interest, there is still limited information on how to rationally design these systems. Here, we provide a systematic study of the effect of matrix stiffness and cell adhesion ligands on gene transfer efficiency. We show that high stiffness and the presence of cell-binding sites promote transfection efficiency and that this result is related to more efficient internalization and trafficking of the gene therapies. GAMs with optimized mechanical properties can induce cartilage formation and result in tissues with better characteristics for articular cartilage tissue engineering as compared to previously described standard methods.

Cell-Cell Adhesion-Driven Contact Guidance and Its Effect on Human Mesenchymal Stem Cell Differentiation.

Contact guidance has been extensively explored using patterned adhesion functionalities that predominantly mimic cell-matrix interactions. Whether contact guidance can also be driven by other types of interactions, such as cell-cell adhesion, still remains a question. Herein, this query is addressed by engineering a set of microstrip patterns of (i) cell-cell adhesion ligands and (ii) segregated cell-cell and cell-matrix ligands as a simple yet versatile set of platforms for the guidance of spreading, adhesion, and differentiation of mesenchymal stem cells. It was unprecedently found that micropatterns of cell-cell adhesion ligands can induce contact guidance. Surprisingly, it was found that patterns of alternating cell-matrix and cell-cell strips also induce contact guidance despite providing a spatial continuum for cell adhesion. This guidance is believed to be due to the difference between the potencies of the two adhesions. Furthermore, patterns that combine the two segregated adhesion functionalities were shown to induce more human mesenchymal stem cell osteogenic differentiation than monofunctional patterns. This work provides new insight into the functional crosstalk between cell-cell and cell-matrix adhesions and, overall, further highlights the ubiquitous impact of the biochemical anisotropy of the extracellular environment on cell function.

Control of adhesive ligand density for modulation of nucleus pulposus cell phenotype

Cells of the nucleus pulposus have been observed to undergo a shift from their notochordal-like juvenile phenotype to a more fibroblast-like state with age and maturation. It has been demonstrated that culture of degenerative adult human nucleus pulposus cells upon soft (<1 kPa) full length laminin-containing hydrogel substrates promotes increased levels of a panel of markers associated with the juvenile nucleus pulposus cell phenotype. In the current work, we observed an ability to use soft polymeric substrates functionalized with short laminin-mimetic peptide sequences to recapitulate the behaviors elicited by soft, full-length laminin containing materials. Furthermore, our work suggests an ability to mimic features of soft systems through control of peptide density upon stiffer substrates. Specifically, results suggest that stiffer polymer-peptide hydrogel substrates can be used to promote the expression of a more juvenile-like phenotype for cells of the nucleus pulposus by reducing adhesive ligand presentation. Here we show how polymer stiffness combined with adhesive ligand presentation can be controlled to be supportive of nucleus pulposus cell phenotype and biosynthesis.

Radiation induced regulation of endothelial adhesion receptors

The synergy of modern immunotherapeutics and radiation has proven to be a promising strategy to combat cancer. However, fundamental questions regarding the underlying mechanisms of these therapies are unanswered. While clinical studies have demonstrated that radiation can potentially boost immune cell infiltration and tumor immunogenicity, the clinicians’ aim to use radiation to improve immune cell infiltration and turn so called ‘cold’ tumors with little infiltration into ‘hot’ tumors lacks mechanistic research data until today. We hypothesize that alterations of the tumor immune cell composition result from modified endothelial adhesion receptor expression mediating immune cell recruitment from the blood. Thus, in order to investigate the tumor vascular endothelium, we quantified infiltrating immune cells and examined the regulation of endothelial adhesion ligands following different fractionations of radiation. We applied two different radiation doses: 1x10Gy (a dose thought to improve immunogenicity), and 1x2Gy (conventional/normofractionation), analyzed immune cell infiltration via flow cytometry, and investigated the expression of endothelial molecules like ICAM‐1, ICAM‐2, VCAM‐1, E‐selectin, and P‐selectin using real time PCR and histology‐based approaches in the tumor and healthy vasculature. We analyzed transplantable lung tumors of Kras;Trp53 mutant mice 1h, 4h, 8h, 24h, 3d, 5d, and 8d after radiation. Importantly, we observed a generalized decrease of tissue immune cells within the first 24h with recovering populations at day 3 after radiation in all groups. Furthermore, we found that the expression of some endothelial adhesion receptors changes in a dose‐dependent manner, but others are not affected. For instance, ICAM‐2 remains unchanged in both groups, but tumor vasculature shows a rapid upregulation of E‐selectin within the first hour after radiation, followed by a downregulation and a second increase after 2–3 days. The lower fractions (2Gy) elicit an augmented upregulation of VCAM‐1 after 24h peaking at day 3. This is of particular interest as VCAM‐1 is vital to T cell recruitment and currently most research indicates that high doses are needed to improve tumor immunogenicity. However, we observed a more robust repopulation of both CD4 and CD8 T cells following 10Gy – a finding that might be due to a marked downregulation of ICAM‐1 following 2Gy doses that was not observed following 10Gy. An improved understanding of the endothelial adhesion receptor regulation depending on radiation fractionation is of therapeutic relevance: novel immune checkpoint inhibition strategies require efficient T cell recruitment to the tumor to be effective and so do, for instance, isolated and modified T cell (so called CAR‐T cell) therapies: Our results are the first to indicate that endothelial ligand regulation differs depending on radiation dose.Support or Funding InformationDFG (HE 6810/3‐1 to J.M.H., HE 6897/2‐1 to G.H.S.) and the CMMC (CAP‐13 to J.M.H. and CAP‐16 to G.H.S. as well as B02 to G.H.S. and J.M.H.)

Tunable fibrin-alginate interpenetrating network hydrogels to support cell spreading and network formation

Hydrogels are effective platforms for use as artificial extracellular matrices, cell carriers, and to present bioactive cues. Two common natural polymers, fibrin and alginate, are broadly used to form hydrogels and have numerous advantages over synthetic materials. Fibrin is a provisional matrix containing native adhesion motifs for cell engagement, yet the interplay between mechanical properties, degradation, and gelation rate is difficult to decouple. Conversely, alginate is highly tunable yet bioinert and requires modification to present necessary adhesion ligands. To address these challenges, we developed a fibrin-alginate interpenetrating network (IPN) hydrogel to combine the desirable adhesion and stimulatory characteristics of fibrin with the tunable mechanical properties of alginate. We tested its efficacy by examining capillary network formation with entrapped co-cultures of mesenchymal stromal cells (MSCs) and endothelial cells (ECs). We manipulated thrombin concentration and alginate crosslinking density independently to modulate the fibrin structure, mesh size, degradation, and biomechanical properties of these constructs. In IPNs of lower stiffness, we observed a significant increase in total cell area (1.7 × 105 ± 7.9 × 104 µm2) and decrease in circularity (0.56 ± 0.03) compared to cells encapsulated in stiffer IPNs (4.0 × 104 ± 1.5 × 104 µm2 and 0.77 ± 0.09, respectively). Fibrinogen content did not influence capillary network formation. However, higher fibrinogen content led to greater retention of these networks confirmed via increased spreading and presence of F-actin at 7 days. This is an elegant platform to decouple cell adhesion and hydrogel bulk stiffness that will be broadly useful for cell instruction and delivery. Statement of significanceHydrogels are widely used as drug and cell delivery vehicles and as artificial extracellular matrices to study cellular responses. However, there are limited opportunities to simultaneously control mechanical properties and degradation while mimicking the complex native adhesion motifs and ligands known to encourage cell engagement with the hydrogel. In this study, we describe a fibrin-alginate interpenetrating network (IPN) hydrogel designed to balance the compliance and provisional qualities of fibrin with the mechanical stability and tunability of alginate to interrogate these contributions on cell response. We used clinically relevant cell sources, a co-culture of endothelial cells and mesenchymal stromal cells, to test its efficacy in supporting capillary formation in vitro. These data demonstrate the promise of this IPN for use in tissue engineering.

Engineering PEG-based hydrogels to foster efficient endothelial network formation in free-swelling and confined microenvironments

In vitro tissue engineered models are poised to have significant impact on disease modeling and preclinical drug development. Reliable methods to induce microvascular networks in such microphysiological systems are needed to improve the size and physiological function of these models. By systematically engineering several physical and biomolecular properties of the cellular microenvironment (including crosslinking density, polymer density, adhesion ligand concentration, and degradability), we establish design principles that describe how synthetic matrix properties influence vascular morphogenesis in modular and tunable hydrogels based on commercial 8-arm poly (ethylene glycol) (PEG8a) macromers. We apply these design principles to generate endothelial networks that exhibit consistent morphology throughout depths of hydrogel greater than 1 mm. These PEG8a-based hydrogels have relatively high volumetric swelling ratios (>1.5), which limits their utility in confined environments such as microfluidic devices. To overcome this limitation, we mitigate swelling by incorporating a highly functional PEG-grafted alpha-helical poly (propargyl-l-glutamate) (PPLGgPEG) macromer along with the canonical 8-arm PEG8a macromer in gel formation. This hydrogel platform supports enhanced endothelial morphogenesis in neutral-swelling environments. Finally, we incorporate PEG8a-PPLGgPEG gels into microfluidic devices and demonstrate improved diffusion kinetics and microvascular network formation in situ compared to PEG8a-based gels.

Micropatterned Adhesion Sites for Spheroid Cultivation Under Flow

The focus of cell based assays in recent years switched from 2D to 3D applications using spheroids and organoid cell assemblies due to the higher validity of the experimental outcome concerning in vivo relevance. However, spheroid generation, continues media exchange for good nutrients supply and high quality imaging over a long period of time is often very laborious. We use a perfusable channel system with micropatterned adhesion ligands on a highly passivated coverslip, which enables high resolution fluorescence microscopy, to generate numerous homogenously distributed spheroids in parallel within one channel. In this system spheroids can either be directly generated by simple injection of a large number of cells and a self-sorting process or by addition of preformed spheroids, which will bind to the patterned adhesion sites. Due to the immobilization of spheroids to the adhesion spots, long-term observation of the same spheroids can be performed with many spheroids in parallel. Perfusion of the spheroid chip at low flow rates not only enables the homogenous supply of all spheroids with nutrients, but also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. With our micropatterned channel system we were able to show that by applying a flow the overall spheroid growth increases over time and spheroid size is very homogenous. By applying very high flow rates spheroids can be harvested from the surfaces, collected and further analyzed. The flow also influences the drug response of spheroids towards a more in vivo like reaction, showing that 3D spheroid cultivation under flow is the method of choice for generating relevant experimental results.

Gradient Hydrogels.

Gradient hydrogels represent a pivotal and expanding direction of three-dimensional cell culture. Since various types of gradients play an important role in physiological and pathological processes in vivo, recreation of these gradients in vitro allows a better understanding of cellular behavior, intercellular and cell-matrix interactions. Moreover, gradient hydrogels can advance the creation of functionally improved and physiologically relevant tissue engineered constructs. Another application of gradient hydrogels is the optimization of the 3D in vitro microenvironment (e.g., in terms of hydrogel stiffness or concentration of adhesion ligands). Tunable hydrogels provide researchers with a versatile toolbox to manufacture such gradients in vitro. In this chapter different types of in vivo and in vitro gradients in hydrogels will be presented. Equipment and methods for various gradient fabrications will be discussed. Furthermore, methods of gradient characterizations in hydrogels will be reported. As one of the most recent developments, the influence of low oxygen concentration on cells, as well as the creation and characterization of oxygen gradients in hydrogels will be described. In the last part, achievements in the creation of multiple combinatorial gradients will be presented. The aim of this chapter is to give the reader an overview on existing techniques and biological importance of gradient hydrogels in basic science as well as in applied research.

Mechanisms of eosinophilic inflammation.

Eosinophils play roles in the pathogenesis of various diseases. In order to accumulate within sites of inflammation, eosinophils must adhere to, and migrate across the microvasculature. These processes are largely controlled by type 2-immune responses; interleukin (IL)-4 and IL-13 induce the expression of endothelial adhesion molecule vascular cell adhesion molecule-1 (VCAM-1), a representative adhesive ligand for eosinophils, while also stimulating generations of CC chemokines from structural cells, including epithelial cells. VCAM-1 and CC chemokines synergistically induce transmigration of eosinophils to the tissue inflammation site. Another type 2 cytokine, IL-5, prolongs survival, and enhances the effector functions of eosinophils. Recently, accumulating evidence has established that corticosteroid-resistant group 2 innate lymphoid cells are cellular sources for IL-5. Another immunological mechanism that may be contributing to eosinophilic inflammation involves type 1 immune system-associated molecules such as interferons and IP-10. In addition to these immune pathways, lipid mediators, such as cysteinyl leukotrienes, directly provoke the infiltration and activation of eosinophils. Extracellular matrix proteins including periostin also induce the adhesion and activation of eosinophils. Finally, activated neutrophils can also induce eosinophil transmigration. In summary, various mechanisms are involved within eosinophilic inflammation, and effective therapeutic strategies targeting these pathways should be established.