Short-Stranded Zein Fibers for Muscle Tissue Engineering in Alginate-Based Composite Hydrogels.

Cultivated meat is a nascent technology that aims to create an environmentally and animal-friendly alternative to conventional meat. Producing skeletal muscle tissue in an animal-free system allowing for high levels of myofusion and maturation is important for the nutritional and sensorial value of cultivated meat. Alginate is an attractive biomaterial to support muscle formation as it is food-safe, sustainable and cheap and can be crosslinked using non-toxic methods. Although alginate can be functionalized to promote cell attachment, limitations in its mechanical properties, including form, viscosity, and stress relaxation, hinder the cellular capacity for myogenic differentiation and maturation in alginate-based hydrogels. Here, we show that the addition of electrospun short-stranded zein fibers increased hydrogel degradation, resulting in faster compaction, improved cell-gel interaction, and enhanced alignment of bovine muscle precursor cells. We conclude that fiber-hydrogel composites are a promising approach to support optimal formation of 3D constructs, by improving tissue stability and thus prolonging culture duration. Together, this improves muscle-related protein content by facilitating myogenic differentiation and priming muscle organoids for maturation.

Tumor-Targeting Immune System Engagers (ISErs) Activate Human Neutrophils after Binding to Cancer Cells.

Immune system engagers (ISErs) make up a new class of immunotherapeutics against cancer. They comprise two or more tumor-targeting peptides and an immune-stimulating effector peptide connected by inert polymer linkers. They are produced by solid phase peptide synthesis and share the specific targeting activities of antibodies (IgGs) but are much smaller in size and exploit a different immune-stimulating mechanism. Two ISErs (Y-9 and Y-59) that bind to the cancer cell markers integrin α3 and EphA2, respectively, are analyzed here with respect to their immune cell stimulation. We have previously shown that they activate formyl peptide receptors on myeloid immune cells and induce respiratory burst in neutrophils and myeloid chemotaxis in solution. It remained, however, unclear whether these molecules can stimulate immune cells while bound to tumor cells, an essential step in the hypothesized mode of action. Here, we demonstrate that ISEr Y-9 induced respiratory burst and caused a change in the shape of neutrophils when bound to the surface of protein A beads as a model of tumor cells. More importantly, tumor cell lines carrying receptor-bound Y-9 or Y-59 also activated neutrophils, evidenced by a significant change in shape. Interestingly, similar activation was induced by the supernatants of the cells incubated with ISEr, indicating that ISErs released from tumor cells, intact or degraded into fragments, significantly contributed to immune stimulation. These findings provide new evidence for the mode of action of ISErs, namely by targeting cancer cells and subsequently provoking an innate immune response against them.

Synthetic Cancer-Targeting Innate Immune Stimulators Give Insights into Avidity Effects.

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid-phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two "binder" peptides that target ephrin A2 and integrin α3 receptors and an "effector" peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.

Synthetic integrin-binding immune stimulators target cancer cells and prevent tumor formation.

Immuno-oncology approaches mainly utilize monoclonal antibodies or protein-based scaffolds that bind with high affinity to cancer cells and can generate an immune response. Peptides can also bind with high affinity to cancer cells and are intermediate in size between antibodies and small molecules. They are also synthetically accessible and therefore easily modified to optimize their stability, binding affinity and selectivity. Here we describe the design of immune system engagers (ISErs), a novel class of synthetic peptide-based compounds that bind specifically to cancer cells and stimulate the immune system. A prototype, Y9, targets integrin α3, which is overexpressed on several cancer cells, and activates the immune system via a formyl methionine-containing effector peptide. Injection of Y9 leads to immune cell infiltration into tissue and prevents tumor formation in a guinea pig model. The anti-tumor activity and synthetic accessibility of Y9 illustrate that ISErs could be applied to a wide variety of targets and diseases.

Amyloid-beta42 signals tau hyperphosphorylation and compromises neuronal viability by disrupting alkylacylglycerophosphocholine metabolism.

Perturbation of lipid second messenger networks is associated with the impairment of synaptic function in Alzheimer disease. Underlying molecular mechanisms are unclear. Here, we used an unbiased lipidomic approach to profile alkylacylglycerophosphocholine second messengers in diseased tissue. We found that specific isoforms defined by a palmitic acid (16:0) at the sn-1 position, namely 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and 1-O-hexadecyl-sn-glycero-3-phosphocholine (C16:0 lyso-PAF), were elevated in the temporal cortex of Alzheimer disease patients, transgenic mice expressing human familial disease-mutant amyloid precursor protein, and human neurons directly exposed to amyloid-beta(42) oligomers. Acute intraneuronal accumulation of C16:0 PAF but not C16:0 lyso-PAF initiated cyclin-dependent kinase 5-mediated hyperphosphorylation of tau on Alzheimer disease-specific epitopes. Chronic elevation caused a caspase 2 and 3/7-dependent cascade resulting in neuronal death. Pharmacological inhibition of C16:0 PAF signaling, or molecular strategies increasing hydrolysis of C16:0 PAF to C16:0 lyso-PAF, protected human neurons from amyloid-beta(42) toxicity. Together, these data provide mechanistic insight into how disruptions in lipid metabolism can determine neuronal response to accumulating oligomeric amyloid-beta(42).

Androgens inhibit the proliferation of a variant of the human prostate cancer cell line LNCaP.

UNLABELLED: The paradoxical androgen response of R2, a subline of the human prostate cancer cell line LNCaP, is described here. Two androgens (DHT and R1881) decreased, in a dose-dependent manner, R2 cell proliferation and [3H]thymidine incorporation. These ligand and cell specific effects were accompanied by an increase in the metabolism of the vital dye MTT and in cell protein content. Both androgens increased the doubling time and the percentage of G0-G1 cells. No evidence of androgen-induced apoptosis was found. Cloning allowed the selection of two cell populations on the basis of the response to 10 nM of R1881. Long term culture of uncloned R2 cells with R1881 modified reversibly the pattern of androgen response. R2 was compared to the androgen-stimulated LNCaP-FGC subline to investigate the causes of their different androgen responsiveness. The androgen receptor (number, affinity for hormones and antihormones, sedimentation constant and molecular weight) and androgen receptor genes (exon size and exon 8 sequence) were found to be identical in the two sublines. EGF stimulated LNCaP-FGC but not R2. Both cells were slightly stimulated by basic FGF but were insensitive to IGF-I and TGF beta 1. IN CONCLUSION: (1) androgens inhibit the proliferation of R2 cells possibly by introducing a G0-G1 block; (2) this inhibition is incomplete because, at least in part, the R2 cell population is heterogeneous; (3) chronic androgen treatment induces reversible cell adaptation; and (4) there is no evidence that the loss of the classical stimulatory effect of androgen on cell proliferation and the gain of inhibitory effect are due to androgen receptor alteration or to a specific action of one of the four growth factors tested.