Induction of stearoyl-CoA desaturase confers cell density-dependent ferroptosis resistance in melanoma.

Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.

Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation.

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

eIF4B enhances ATF4 expression and contributes to cellular adaptation to asparagine limitation in BRAF-mutated A375 melanoma.

ATF4 is a crucial transcription factor in the integrated stress response, a major adaptive signaling pathway activated by tumor microenvironment and therapeutic stresses. BRAF inhibitors, such as vemurafenib, induce ATF4 in BRAF-mutated melanoma cells, but the mechanisms of ATF4 induction are not fully elucidated. Here, we show that ATF4 expression can be upregulated by eukaryotic initiation factor 4B (eIF4B) in BRAF-mutated A375 cells. Indeed, eIF4B knockout (KO) prevented ATF4 induction and activation of the uORF-mediated ATF4 translation mechanism during vemurafenib treatment, which were effectively recovered by the rescue of eIF4B. Transcriptome analysis revealed that eIF4B KO selectively influenced ATF4-target gene expression among the overall gene expression changed by vemurafenib. Interestingly, eIF4B supported cellular proliferation under asparagine-limited conditions, possibly through the eIF4B-ATF4 pathway. Our findings indicate that eIF4B can regulate ATF4 expression, thereby contributing to cellular stress adaptation, which could be targeted as a therapeutic approach against malignancies, including melanoma.

Modulating Macrophage Phenotype by Sustained MicroRNA Delivery Improves Host-Implant Integration.

Biomedical implant failure due to the host's response remains a challenging problem. In particular, the formation of the fibrous capsule is a common barrier for the normal function of implants. Currently, there is mounting evidence indicating that the polarization state of macrophages plays an important role in effecting the foreign body reaction (FBR). This opens up a potential avenue for improving host-implant integration. Here, electrospun poly(caprolactone-co-ethyl ethylene phosphate) nanofiber scaffolds are utilized to deliver microRNAs (miRs) to induce macrophage polarization and modulate FBR. Specifically, C57BL/6 mice that are treated with M2-inducing miRs, Let-7c and miR-124, display relatively thinner fibrous capsule formation around the scaffolds at both Week 2 and 4, as compared to treatment with M1-inducing miR, Anti-Let-7c. Histological analysis shows that the density of blood vessels in the scaffolds are the highest in miR-124 treatment group, followed by Anti-Let-7c and Let-7c treatment groups. Based on immunohistochemical quantifications, these miR-encapsulated nanofiber scaffolds are useful for localized and sustained delivery of functional miRs and are able to modulate macrophage polarization during the first 2 weeks of implantation to result in significant alteration in host-implant integration at longer time points.

Biomimicking Fiber Scaffold as an Effective In Vitro and In Vivo MicroRNA Screening Platform for Directing Tissue Regeneration.

MicroRNAs effectively modulate protein expression and cellular response. Unfortunately, the lack of robust nonviral delivery platforms has limited the therapeutic application of microRNAs. Additionally, there is a shortage of drug-screening platforms that are directly translatable from in vitro to in vivo. Here, a fiber substrate that provides nonviral delivery of microRNAs for in vitro and in vivo microRNA screening is introduced. As a proof of concept, difficult-to-transfect primary neurons are targeted and the efficacy of this system is evaluated in a rat spinal cord injury model. With this platform, enhanced gene-silencing is achieved in neurons as compared to conventional bolus delivery (p < 0.05). Thereafter, four well-recognized microRNAs (miR-21, miR-222, miR-132, and miR-431) and their cocktails are screened systematically. Regardless of age and origin of the neurons, similar trends are observed. Next, this fiber substrate is translated into a 3D system for direct in vivo microRNA screening. Robust nerve ingrowth is observed as early as two weeks after scaffold implantation. Nerve regeneration in response to the microRNA cocktails is similar to in vitro experiments. Altogether, the potential of the fiber platform is demonstrated in providing effective microRNA screening and direct translation into in vivo applications.

Scaffold-Mediated Sustained, Non-viral Delivery of miR-219/miR-338 Promotes CNS Remyelination.

The loss of oligodendrocytes (OLs) and subsequently myelin sheaths following injuries or pathologies in the CNS leads to debilitating functional deficits. Unfortunately, effective methods of remyelination remain limited. Here, we present a scaffolding system that enables sustained non-viral delivery of microRNAs (miRs) to direct OL differentiation, maturation, and myelination. We show that miR-219/miR-338 promoted primary rat OL differentiation and myelination in vitro. Using spinal cord injury as a proof-of-concept, we further demonstrate that miR-219/miR-338 could also be delivered non-virally in vivo using an aligned fiber-hydrogel scaffold to enhance remyelination after a hemi-incision injury at C5 level of Sprague-Dawley rats. Specifically, miR-219/miR-338 mimics were incorporated as complexes with the carrier, TransIT-TKO (TKO), together with neurotrophin-3 (NT-3) within hybrid scaffolds that comprised poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP)-aligned fibers and collagen hydrogel. After 1, 2, and 4 weeks post-treatment, animals that received NT-3 and miR-219/miR-338 treatment preserved a higher number of Olig2+ oligodendroglial lineage cells as compared with those treated with NT-3 and negative scrambled miRs (Neg miRs; p < 0.001). Additionally, miR-219/miR-338 increased the rate and extent of differentiation of OLs. At the host-implant interface, more compact myelin sheaths were observed when animals received miR-219/miR-338. Similarly within the scaffolds, miR-219/miR-338 samples contained significantly more myelin basic protein (MBP) signals (p < 0.01) and higher myelination index (p < 0.05) than Neg miR samples. These findings highlight the potential of this platform to promote remyelination within the CNS.

ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression.

It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. However, it is not clear whether specific ECM components such as collagen or fibronectin differentially regulate such processes, especially in 3D scaffolds. In this study, a series of ECM-functionalized inverted colloidal crystal (ICC) microporous scaffolds were fabricated and their influence on Huh-7.5 cell proliferation, morphology, hepatic-specific functions, and patterns of gene expression were compared. Both collagen and fibronectin promoted albumin production and liver-specific gene expression of Huh-7.5 cells, compared with the bare ICC scaffold. Interestingly, cells in the fibronectin-functionalized scaffold exhibited different aggregation patterns to those in the collagen-functionalized scaffold, a variation that could be related to the distinct mRNA expression levels of cell adhesion-related genes. Based on these results, we can conclude that different ECM proteins, such as fibronectin and collagen, indeed play distinct roles in the phenotypic regulation of cells cultured in a 3D environment.

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Precise Tuning of Facile One-Pot Gelatin Methacryloyl (GelMA) Synthesis.

Gelatin-methacryloyl (GelMA) is one of the most commonly used photopolymerizable biomaterials in bio-applications. However, GelMA synthesis remains suboptimal, as its reaction parameters have not been fully investigated. The goal of this study is to establish an optimal route for effective and controllable GelMA synthesis by systematically examining reaction parameters including carbonate-bicarbonate (CB) buffer molarity, initial pH adjustment, MAA concentration, gelatin concentration, reaction temperature, and reaction time. We employed several analytical techniques in order to determine the degree of substitution (DS) and conducted detailed structural analysis of the synthesized polymer. The results enabled us to optimize GelMA synthesis, showing the optimal conditions to balance the deprotonation of amino groups with minimizing MAA hydrolysis, which led to nearly complete substitution. The optimized conditions (low feed ratio of MAA to gelatin (0.1 mL/g), 0.25 M CB buffer at pH 9, and a gelatin concentration of 10-20%) enable a simplified reaction scheme that produces GelMA with high substitution with just one-step addition of MAA in one pot. Looking forward, these optimal conditions not only enable facile one-pot GelMA synthesis but can also guide researchers to explore the efficient, high methacrylation of other biomacromolecules.

Phenotypic regulation of liver cells in a biofunctionalized three-dimensional hydrogel platform.

Loss of function is a major challenge for hepatocytes that are cultured on two-dimensional (2D) cell culture platforms. Biofunctionalized three-dimensional (3D) scaffolds produced by microfabrication strategies can overcome these limitations by presenting vital environmental cues, strong mechanical properties, and three-dimensional geometry to enable high-fidelity liver tissue engineering. Herein, we report the detailed investigation of hepatocarcinoma (Huh 7.5) cellular behavior in a collagen-functionalized microsphere-templated poly(ethylene glycol) (PEG) hydrogel scaffold which promotes 3D hepatic sheet morphology. Collagen conjugation led to improved liver-specific functions, including albumin production and cytochrome P450 (CYP450) activity. Importantly, the gene expression of numerous cell-adhesion markers was enhanced along with stimulated innate hepatocyte fibronectin production. Taken together, the findings reveal a close connection between hepatic cell morphology and gene expression, offering evidence that surface-coated collagen in the 3D hydrogel platform triggers the upregulation of hepatocyte-specific transcription factors and the secretion of liver metabolic markers.

Natural Sunflower Pollen as a Drug Delivery Vehicle.

In nature, pollen grains play a vital role for encapsulation. Many pollen species exist which are often used as human food supplements. Dynamic image particle analysis, scanning electron microscopy, and confocal microscopy analysis confirmed the size, structural uniformity, and macromolecular encapsulation in sunflower pollen, paving the way to explore natural pollen grains for the encapsulation of therapeutic molecules.

Biofunctionalized Hydrogel Microscaffolds Promote 3D Hepatic Sheet Morphology.

Development of artificial tissues providing the proper geometrical, mechanical, and environmental cues for cells is highly coveted in the field of tissue engineering. Recently, microfabrication strategies in combination with other chemistries have been utilized to capture the architectural complexity of intricate organs, such as the liver, in in vitro platforms. Here it is shown that a biofunctionalized poly (ethylene glycol) (PEG) hydrogel scaffold, fabricated using a sphere-template, facilitates hepatic sheet formation that follows the microscale patterns of the scaffold surface. The design takes advantage of the excellent diffusion properties of porous, uniform 3D hydrogel platforms, and the enhanced-cell-extracellular matrix interaction with the display of conjugated collagen type I, which in turn elicits favorable Huh-7.5 response. Collectively, the experimental findings and corresponding simulations demonstrate the importance of biofunctionalized porous scaffolds and indicate that the microscaffold shows promise in liver tissue engineering applications and provides distinct advantages over current cell sheet and hepatocyte spheroid technologies.

Extracellular Matrix Functionalization and Huh-7.5 Cell Coculture Promote the Hepatic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells in a 3D ICC Hydrogel Scaffold.

In this study, we constructed a microporous hydrogel scaffold with hexagonally packed interconnected cavities and extracellular matrix (ECM)-functionalized interior surface, and systematically investigated the hepatic differentiation of human adipose-derived mesenchymal stem cells (hAD-MSCs) under the influence of three key factors: three-dimensional (3D) geometry, ECM presence, and coculture with hepatocyte-derived cell line. Results confirmed that (i) hepatic differentiation of hAD-MSC is more efficient in a 3D microporous scaffold than in 2D monolayer culture; (ii) the presence of both ECM components (fibronectin and collagen-I) in the scaffold is superior to collagen-I only, highlighting the importance of fibronectin; and (iii) coculture with Huh-7.5 hepatocyte-derived cells promoted liver-specific functions of the hAD-MSC-derived hepatocytes. The optimized differentiation process only took 21 days to complete, a time length that is shorter or at least comparable to previous reports, and more importantly, yielded an albumin production more than 10-fold higher than conventional 2D culture. Our approach of optimizing hAD-MSC hepatic differentiation could provide a potential solution to the challenges such as hepatocyte transplantation or the establishment of human physiologically relevant liver models in vitro.