Subcutaneous Injection In Mice Research Articles

Enhancing the therapeutic window for Spectinamide anti-tuberculosis Agents: Synthesis, Evaluation, and activation of phosphate prodrug 3408

Spectinamides are a novel class of narrow-spectrum antitubercular agents with the potential to treat drug-resistant tuberculosis infections. Spectinamide 1810 has shown a good safety record following subcutaneous injection in mice or infusion in rats but exhibits transient acute toxicity following bolus administration in either species. To improve the therapeutic index of 1810, an injectable prodrug strategy was explored. The injectable phosphate prodrug 3408 has a superior maximum tolerated dose compared to 1810 or Gentamicin. Following intravenous administration in rodents, prodrug 3408 was quickly converted to 1810. The resulting 1810 exposure and pharmacokinetic profile after 3408 administration was identical to equivalent molar amounts of 1810 given directly by intravenous administration. 3408 and the parent 1810 exhibited similar overall efficacy in a BALB/c acute tuberculosis efficacy model. Delivery of 1810 in phosphate prodrug form, therefore, holds the potential to improve further the therapeutic index of an already promising tuberculosis antibiotic.

Enhancing cancer immunotherapy with mannose mimicking glycopolymer nanoparticles induced activation of Dendritic cells

Cancer immunotherapy leverages the immune system’s inherent capacity to combat malignancies. However, effective stimulation of Dendritic cells (DCs) is challenging due to their limited distribution and the immune-suppressive tumor microenvironment. Thus, targeting mannose receptors, which are highly expressed on DCs, represents a promising strategy. This study investigates the development of mannose-based glycopolymer nanoparticles to induce activation of DCs through enhanced antigen presentation. A novel ABA-type triblock bioconjugated glycopolymer (PMn-b-PCL-b-PMn), which mimics mannose was synthesized. This polymer was further modified with Dihexadecyldimethylammonium bromide (DHDAB) to prepare cationic nanoparticles (CMNP) for gene delivery of pCMV-TRP2, an antigenic marker for both melanoma and glioblastoma. The immune response generated by CMNP and the CMNP-TRP2 polyplex was compared to an untreated control following subcutaneous injection in mice. Post-injection cytometric analysis revealed robust DC activation and increased T-cell populations in secondary lymphoid organs, including the spleen and lymph nodes. These findings suggest that CMNP can serve as a potent biomimicking vaccination vehicle against cancer, enhancing the immune response through targeted DCs activation.

Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action.

Recombinant human interferon alpha-2b (rIFN) is widely used in antiviral and anticancer immunotherapy. However, the high efficiency of interferon therapy is accompanied by a number of side effects; this problem requires the design of a new class of interferon molecules with reduced cytotoxicity. In this work, IFN was modified via genetic engineering methods by merging it with the blood plasma protein apolipoprotein A-I in order to reduce acute toxicity and improve the pharmacokinetics of IFN. The chimeric protein was obtained via biosynthesis in the yeast P. pastoris. The yield of ryIFN-ApoA-I protein when cultivated on a shaker in flasks was 30 mg/L; protein purification was carried out using reverse-phase chromatography to a purity of 95-97%. The chimeric protein demonstrated complete preservation of the biological activity of IFN in the model of vesicular stomatitis virus and SARS-CoV-2. In addition, the chimeric form had reduced cytotoxicity towards Vero cells and increased cell viability under viral load conditions compared with commercial IFN-a2b preparations. Analysis of the pharmacokinetic profile of ryIFN-ApoA-I after a single subcutaneous injection in mice showed a 1.8-fold increased half-life of the chimeric protein compared with ryIFN.

Nanocontroller-mediated dissolving hydrogel that can sustainably release cold-mimetic menthol to induce adipocyte browning for treating obesity and its related metabolic disorders

Obesity leads to the development of many metabolic diseases, causing severe health problems. Menthol can induce adipocyte browning and thus has been used to combat obesity. To deliver menthol with a sustained effect, an injectable hydrogel that comprises carboxymethyl chitosan and aldehyde-functionalized alginate that are crosslinked through dynamic Schiff-base linkages is developed to load menthol-cyclodextrin inclusion complexes (IC). To render the as-developed hydrogel soluble after its payload is released, amino acid-loaded liposomes, functioning as nanocontrollers, are covalently grafted onto networks of the hydrogel. Upon subcutaneous injection in mice with diet-induced obesity, the as-developed hydrogel absorbs body fluids and spontaneously swells, expanding and stretching its networks, gradually releasing the loaded IC. Menthol then disassociates from the released IC to induce adipocyte browning, triggering fat consumption and increasing energy expenditure. Meanwhile, the expanded hydrogel networks destabilize the grafted liposomes, which function as built-in nanocontrollers, unleashing their loaded amino acid molecules to disrupt the dynamic Schiff-base linkages, causing hydrogel to dissolve. The thus-developed nanocontroller-mediated dissolving hydrogel realizes the sustained release of menthol for treating obesity and its related metabolic disorders without leaving exogenous hydrogel materials inside the body, and thereby preventing any undesired adverse effects.

Targeting SHP2 reverses BRAF inhibitor tolerance in anaplastic thyroid carcinoma.

To explore the possibility of a combination of dabrafenib and SHP2 inhibitor in the treatment of anaplastic thyroid carcinoma and to provide a new therapeutic strategy for the treatment of anaplastic thyroid cancer. Firstly, a drug resistance model was established, and the expression levels of related RTK were detected by qPCR. Western blot was used to detect the protein expression levels of Akt and MAPK signaling pathways in the control group, single-drug group and two-drug combination group. The gene silencing of SHP2 was achieved by transfection of siRNA and verified by Western blot. CCK8 kit and clone formation assay were used to detect cell proliferation activity. In vivo model of mutant thyroid cancer cells was established by subcutaneous injection of mice and then divided into four groups. Tumor diameter was measured every two days. Immunohistochemistry was used to evaluate the expression of p-ERK, p-AKT and Ki67 in mouse tumors. In this study, dabrafenib-resistant ATC cells were first constructed, and the response of RTKs in drug-resistant cells was upregulated to activate Akt and MER/ERK pathways. The activation of Akt and MEK/ERK pathways in the combination group was significantly inhibited, and the proliferation ability of tumor cells was significantly reduced compared with Dabrafenib, SHP099 group and DMSO group. To verify that SHP099 was not off-target, we also silenced SHP2 expression by transfection with siRNA and obtained the same results. Finally, by building a mouse drug resistance model, we confirmed that dabrafenib and SHP099 can also play a powerful anti-cancer effect in vivo. The SHP2 inhibitor SHP099 can effectively reverse the drug resistance of dabrafenib through inhibiting the reactivated RAS signaling pathway in anaplastic thyroid cancer.The combination of dabrafenib with SHP2 inhibitor has shown significant tumor suppressive effects for dabrafenib-resistant cells and it may be a new therapeutic strategy with longer lasting therapeutic benefits.

Hybrid Polydimethylsiloxane (PDMS) Incorporated Thermogelling System for Effective Liver Cancer Treatment.

For the delivery of anticancer drugs, an injectable in situ hydrogel with thermal responsiveness and prolonged drug release capabilities shows considerable potential. Here, we present a series of thermosensitive in situ hydrogels that serve as drug delivery systems for the treatment of liver cancer. These hydrogels were created by utilizing the polydimethylsiloxane (PDMS) oligomer, polyethylene glycol (PEG) and polypropylene glycol (PPG)'s chemical cross-linking capabilities. Doxorubicin (DOX) was encapsulated in a hydrogel with a hydrophobic core and hydrophilic shell to enhance DOX solubility. Studies into the behavior of in situ produced hydrogels at the microscopic and macroscopic levels revealed that the copolymer solution exhibits a progressive shift from sol to gel as the temperature rises. The hydrogels' chemical composition, thermal properties, rheological characteristics, gelation period, and DOX release behavior were all reported. Subcutaneous injection in mice was used to confirm the injectability. Through the in vitro release of DOX in a PBS solution that mimics the tumor microenvironment, the hydrogel's sustained drug release behavior was confirmed. Additionally, using human hepatocellular hepatoma, the anticancer efficacy of thermogel (DEP-2@DOX) was assessed (HepG2). The carrier polymer material DEP-2 was tested for cytotoxicity using HepG2 cells and its excellent cytocompatibility was confirmed. In conclusion, these thermally responsive injectable hydrogels are prominent potential candidates as drug delivery vehicles for the treatment of hepatocellular carcinoma.

Synthesis of 19F MRI Nanotracers by Dispersion Polymerization-Induced Self-Assembly of N-(2,2,2-Trifluoroethyl)acrylamide in Water.

19F magnetic resonance imaging (MRI) using fluoropolymer tracers has recently emerged as a promising, non-invasive diagnostic tool in modern medicine. However, despite its potential, 19F MRI remains overlooked and underused due to the limited availability or unfavorable properties of fluorinated tracers. Herein, we report a straightforward synthetic route to highly fluorinated 19F MRI nanotracers via aqueous dispersion polymerization-induced self-assembly of a water-soluble fluorinated monomer. A polyethylene glycol-based macromolecular chain-transfer agent was extended by RAFT-mediated N-(2,2,2-trifluoroethyl)acrylamide (TFEAM) polymerization in water, providing fluorine-rich self-assembled nanoparticles in a single step. The resulting nanoparticles had different morphologies and sizes ranging from 60 to 220 nm. After optimizing their structure to maximize the magnetic relaxation of the fluorinated core, we obtained a strong 19F NMR/MRI signal in an aqueous environment. Their non-toxicity was confirmed on primary human dermal fibroblasts. Moreover, we visualized the nanoparticles by 19F MRI, both in vitro (in aqueous phantoms) and in vivo (after subcutaneous injection in mice), thus confirming their biomedical potential.

An injectable hydrogel/staple fiber composite for sustained release of CA4P and doxorubicin for combined chemotherapy of xenografted breast tumor in mice

To prepare an injectable hydrogel/staple fiber composite loaded with combretastain A-4 disodium phosphate (CA4P) and doxorubicin (DOX) and evaluate its antitumor efficacy via intratumoral injection. DOX-loaded PELA staple fibers (FDOX) were prepared using electro-spinning and cryo-cutting, and the drug distribution on the surface of the fibers was observed using a fluorescence microscope, and the encapsulation efficiency and loading capacity of FDOX were determined with a fluorospectro photometer. The fibers were then dispersed in CA4P-loaded PLGA-PEG-PLGA tri-block polymer solution at room temperature to obtain the hydrogel/staple fiber composite (GCA4P/FDOX). The thermo-sensitivity of this composite was determined by a test tube inverting method. An ultraviolet spectrophotometer and a fluorospectrophotometer were used to detect the release profile of CA4P and DOX, respectively. We observed in vivo gel formation of the composite after subcutaneous injection in mice. The in vitro cytotoxicity of GCA4P/FDOX composite in MCF-7 and 4T1 cells was assessed using cell Counting Kit-8 (CCK-8) reagent. In a mouse model bearing breast tumor 4T1 cell xenograft, we evaluated the antitumor efficacy of the composite by monitoring tumor growth within 30 days after intratumoral injection of the composite. HE staining, immunohistochemistry for Ki67 and immunofluorescence (TUNEL) assay were used for pathological examination of the tumor tissues 21 days after the treatments. The average length of FDOX was 4.0±1.3 μm, and its drug loading capacity was (2.69±0.35)% with an encapsulation efficiency of (89.70±0.12)%. DOX was well distributed on the surface of the fibers. When the temperature increased to 37 ℃, the composite rapidly solidified to form a gel in vitro. Drug release behavior test showed that CA4P was completely released from the composite in 5 days and 87% of DOX was released in 30 days. After subcutaneous injection, the composite solidified rapidly without degradation at 24 h after injection. After incubation with GCA4P/FDOX for 72 h, only 30.6% of MCF-7 cells and 28.9% of 4T1 cells were viable. In the tumor-bearing mice, the tumor volume was 771.9±76.9 mm3 in GCA4P/FDOX treatment group at 30 days. Pathological examination revealed obvious necrosis of the tumor tissues and tumor cell apoptosis induced by intratumoral injection of G4A4P/FDOX. As an efficient dual drug delivery system, this hydrogel/staple fiber composite provides a new strategy for local combined chemotherapy of solid tumors.

Irradiation conditioning of adjuvanted, autologous cancer cell membrane nanoparticle vaccines

Cancer vaccines systems based on autologous cancer cell membrane fragments (CMs) have attracted attention due to broad antigen coverage, however, the lack of immunogenicity of conventional cancer CMs bring the limitations of application. Here, the next-generation nanovaccines is constructed using irradiated cancer cell membrane (RM) coated on a polylactic-co-glycolic acid (PLGA) nanoscale delivery system loaded with a TLR-7 agonist (R837) to generate the [email protected] vaccine with prophylactic and therapeutic anti-cancer properties. Irradiation of cell results in enhanced expression of major histocompatibility complex I (MHCI) on the cell membrane. Compared with regular CMs, RMs carry more 8–11 amino-acid MHC-I epitopes and enrich with more damage-associated molecular patterns (DAMPs). When combined with R837, [email protected] promoted maturation of dendritic cells (DCs) and induced DCs secreting cytokines for immune activation. Following subcutaneous injection in mice, [email protected] accumulated in lymph nodes (LNs) and induced antitumor immune responses which further prevented tumor growth. In addition, [email protected] enhanced the therapeutic efficacy of anti-PD-1 treatment. This work provides a new strategy for improving the antitumor immune response and the efficacy of anti-PD-1 treatment. Taken together, our data showed that irradiation of cancer cells and formulation of RMs into adjuvanted nanoparticles would be a promising strategy for generating autologous cancer vaccines.

Gut microbiota-based vaccination engages innate immunity to improve blood glucose control in obese mice

ObjectiveObesity and diabetes increase circulating levels of microbial components derived from the gut microbiota. Individual bacterial factors (i.e., postbiotics) can have opposing effects on blood glucose. MethodsWe tested the net effect of gut bacterial extracts on blood glucose in mice using a microbiota-based vaccination strategy. ResultsMale and female mice had improved glucose and insulin tolerance five weeks after a single subcutaneous injection of a specific dose of a bacterial extract obtained from the luminal contents of the upper small intestine (SI), lower SI, or cecum. Injection of mice with intestinal extracts from germ-free mice revealed that bacteria were required for a microbiota-based vaccination to improve blood glucose control. Vaccination of Nod1−/−, Nod2−/−, and Ripk2−/− mice showed that each of these innate immune proteins was required for bacterial extract injection to improve blood glucose control. A microbiota-based vaccination promoted an immunoglobulin-G (IgG) response directed against bacterial extract antigens, where subcutaneous injection of mice with the luminal contents of the lower SI elicited a bacterial extract-specific IgG response that is compartmentalized to the lower SI of vaccinated mice. A microbiota-based vaccination was associated with an altered microbiota composition in the lower SI and colon of mice. Lean mice only required a single injection of small intestinal-derived bacterial extract, but high fat diet (HFD)-fed, obese mice required prime-boost bacterial extract injections for improvements in blood glucose control. ConclusionsSubversion of the gut barrier by vaccination with a microbiota-based extract engages innate immunity to promote long-lasting improvements in blood glucose control in a dose-dependent manner.

An Injectable Meta-Biomaterial: From Design and Simulation to In Vivo Shaping and Tissue Induction.

A novel type of injectable biomaterial with an elastic softening transition is described. The material enables invivo shaping, followed by induction of 3D stable vascularized tissue. The synthesis of the injectable meta-biomaterial is instructed by extensive numerical simulation as a suspension of irregularly fragmented, highly porous sponge-like microgels. The irregular particle shape dramatically enhances yield strain for invivo stability against deformation. Porosity of the particles, along with friction between internal surfaces, provides the elastic softening transition. This emergent metamaterial property enables the material to reversibly change stiffness during deformation, allowing native tissue properties to be matched over a wide range of deformation amplitudes. After subcutaneous injection in mice, predetermined shapes can be sculpted manually. The 3D shape is maintained during excellent host tissue integration, with induction of vascular connective tissue that persists to the end of one-year follow-up. The geometrical design is compatible with many hydrogel materials, including cell-adhesion motives for cell transplantation. The injectable meta-biomaterial therefore provides new perspectives in soft tissue engineering and regenerative medicine.

Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs.

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

Transformed Canine and Murine Mesenchymal Stem Cells as a Model for Sarcoma with Complex Genomics.

Simple SummarySarcomas are rare cancers of mesenchymal origin, the majority of which are characterized by many copy number alterations, amplifications, or deletions. Because of these complex genomics, it is notoriously difficult to identify driver events of malignant transformation. In this study, we show that murine and canine mesenchymal stem cells (MSCs) can be used to model spontaneous malignant transformation towards sarcomas with complex genomics. We show that these MSCs have an abnormal karyotype, many structural variants, and point mutations at whole genome sequencing analysis, and form sarcomas after injection into mice. Our cross-species analysis reveals that p53 loss is an early event in sarcomagenesis, and it was shown that MSCs with a knock-out in Trp53 transform earlier compared to wild-type MSCs. Our study points to the importance of p53 loss in the transformation process towards sarcomas with complex genomics.Sarcomas are rare mesenchymal tumors with a broad histological spectrum, but they can be divided into two groups based on molecular pathology: sarcomas with simple or complex genomics. Tumors with complex genomics can have aneuploidy and copy number gains and losses, which hampers the detection of early, initiating events in tumorigenesis. Often, no benign precursors are known, which is why good models are essential. The mesenchymal stem cell (MSC) is the presumed cell of origin of sarcoma. In this study, MSCs of murine and canine origin are used as a model to identify driver events for sarcomas with complex genomic alterations as they transform spontaneously after long-term culture. All transformed murine but not canine MSCs formed sarcomas after subcutaneous injection in mice. Using whole genome sequencing, spontaneously transformed murine and canine MSCs displayed a complex karyotype with aneuploidy, point mutations, structural variants, inter-chromosomal translocations, and copy number gains and losses. Cross-species analysis revealed that point mutations in Tp53/Trp53 are common in transformed murine and canine MSCs. Murine MSCs with a cre-recombinase induced deletion of exon 2–10 of Trp53 transformed earlier compared to wild-type murine MSCs, confirming the contribution of loss of p53 to spontaneous transformation. Our comparative approach using transformed murine and canine MSCs points to a crucial role for p53 loss in the formation of sarcomas with complex genomics.

Tumor Growth Analysis of Ewing Sarcoma Cell Lines Using Subcutaneous Xenografts in Mice.

Subcutaneous murine xenograft models are one of the most commonly used in vivo experimental methods in the cancer research field. Due to the lack of appropriate animal models for Ewing sarcoma, subcutaneous murine xenograft models currently offer the simplest way to investigate antineoplastic effects of therapeutics or biological functions of target genes in vivo. In order to properly carry out tumor growth analysis via subcutaneous xenografts of Ewing sarcoma cells many factors should be taken into account beforehand at the planning phase of experiments. Therefore, in this chapter we describe in detail a widely used procedure for subcutaneous injection in mice, focusing on the specific characteristics of Ewing sarcoma cell lines.

Ellagic acid protects dopamine neurons from rotenone-induced neurotoxicity via activation of Nrf2 signalling.

Parkinson's disease (PD) is the second most prevalent central nervous system (CNS) degenerative disease. Oxidative stress is one of key contributors to PD. Nuclear factor erythroid‐2‐related factor 2 (Nrf2) is considered to be a master regulator of many genes involved in anti‐oxidant stress to attenuate cell death. Therefore, activation of Nrf2 signalling provides an effective avenue to treat PD. Ellagic acid (EA), a natural polyphenolic contained in fruits and nuts, possesses amounts of pharmacological activities, such as anti‐oxidant stress and anti‐inflammation. Recent studies have confirmed EA could be used as a neuroprotective agent in neurodegenerative diseases. Here, mice subcutaneous injection of rotenone (ROT)‐induced DA neuronal damage was performed to investigate EA‐mediated neuroprotection. In addition, adult Nrf2 knockout mice and different cell cultures including MN9D‐enciched, MN9D‐BV‐2 and MN9D‐C6 cell co‐cultures were applied to explore the underlying mechanisms. Results demonstrated EA conferred neuroprotection against ROT‐induced DA neurotoxicity. Activation of Nrf2 signalling was involved in EA‐mediated DA neuroprotection, as evidenced by the following observations. First, EA activated Nrf2 signalling in ROT‐induced DA neuronal damage. Second, EA generated neuroprotection with the presence of astroglia and silence of Nrf2 in astroglia abolished EA‐mediated neuroprotection. Third, EA failed to produce DA neuroprotection in Nrf2 knockout mice. In conclusion, this study identified EA protected against DA neuronal loss via an Nrf2‐dependent manner.

Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin ligase PARK2.

Objective: Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed cancer types in China. Recent genomic sequencing analysis indicated the over-activation of Hippo/YAP signaling might play important roles for the carcinogenic process and progression for ESCC patients. However, little is known about the molecular mechanisms that controls Hippo signaling activity in ESCC. Our previous studies indicated that PLCE1-an important risk factor for ESCC-linked to ESCC progression through snail signaling, during this period, we found PARK2 was an important downstream target of PLCE1-snail axis. PARK2 was decreased in ESCC human samples, and correlated with good prognosis in ESCC patients. Further research showed that PARK2 could inhibit YAP, which functions as key downstream effectors of the Hippo pathway. Here, we aim to reveal the molecular mechanisms of PARK2 modulated Hippo pathway in ESCC.Methods: To evaluate the function of PARK2 in ESCC, we used a tissue microarray (TMA) of 223 human ESCC patients and immunohistochemistry to analyze the correlation between PARK2 expression and clinicopathologic variables. Depletion of endogenous PARK2 and YAP from ESCC cells using CRISPR/Cas9 technologies. Flow cytometry and EdU cell proliferation assay were used to detect proliferation of ESCC cells. Nude mice subcutaneous injection and Ki-67 staining were used to evaluate tumor growth in vivo. Migration and invasion assays were performed. In addition, lung metastasis models in mice were used to validate the function of PARK2 in vivo. Identification of PARK2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. The RNA-seq analysis results were validated through quantitative real-time PCR (qRT-PCR) analysis. The protein half-life of YAP was analyzed by Cycloheximide assay, and the TEAD activity was detected by Luciferase reporter assays.Results: Clinical sample of ESCC revealed that low PARK2 expression correlated with late tumor stage (P < 0.001), poor differentiation (P < 0.04), lymph node (P < 0.001) and distant metastasis (P = 0.0087). Multivariate Cox proportional regression analysis further revealed that PARK2 expression (P = 0.032) is an independent prognostic factor for the overall survival of ESCC patients. Besides, the immunohistochemistry results showed that PARK2 negatively correlated with YAP protein level (P < 0.001). PARK2 depletion promotes ESCC progression both through Hippo/YAP axis, while PARK2 overexpression suppresses ESCC tumor progression by Hippo signaling. Co-IP and ubiquitination assays revealed that PARK2 could interact with YAP in the cytosol and promotes YAP K48-linked ubiquitination at K90 sites.Conclusion: Clinical sample analysis and mechanistic study have validated PARK2 as a tumor suppressor for ESCC. Multivariate Cox proportional regression analysis further revealed that PARK2 is an independent prognostic factor for the overall survival of ESCC patients. Cellular and molecular mechanisms in this study showed that PARK2 associated with YAP protein in the cytosol, promoted YAP ubiquitination and proteasome-dependent degradation in ESCC cells. Therefore, as a novel modulator for Hippo signaling, modulation of PARK2 activity or gene expression level could be an appealing strategy to treat esophageal.

A new class of biological materials: Cell membrane-derived hydrogel scaffolds

Biological materials are superior to synthetic biomaterials in biocompatibility and active interactions with cells. Here, a new class of biological materials, cell membrane-derived hydrogel scaffolds are reported for harnessing these advantages. To form macroporous scaffolds, vesicles derived from red blood cell membranes (RBCMs) are chemically crosslinked via cryogelation. The RBCM scaffolds with a pore size of around 70 μm are soft and injectable. Highly biocompatible scaffolds are typically made of superhydrophilic polymers and lack the ability to encapsulate and release hydrophobic drugs in a controlled manner. However, hydrophobic molecules can be efficiently encapsulated inside RBCM scaffolds and be sustainedly released. RBCM scaffolds show low neutrophil infiltration after subcutaneous injection in mice, and a significantly higher number of infiltrated macrophages than methacrylate alginate (MA-alginate) scaffolds. According to gene expression and surface markers, these macrophages have an M2-like phenotype, which is anti-inflammatory and immune suppressive. There are also higher percentages of macrophages presenting immunosuppressive PD-L1 in RBCM-scaffolds than in MA-alginate scaffolds. Interestingly, the concentrations of anti-inflammatory cytokine, IL-10 in both types of scaffolds are higher than those in normal organ tissues. This study sheds light on cell membrane-derived hydrogels, which can actively modulate cells in unique ways unavailable to existing hydrogel scaffolds.

Decreased PKG transcription mediated by PI3K/Akt/FoxO1 pathway is involved in the development of nitroglycerin tolerance

Phosphoinositide 3-kinase (PI3K)/Akt plays a pivotal role in the vascular response. The present study is to determine whether PI3K/Akt pathway in vascular smooth muscle cells is involved in nitroglycerin (NTG) tolerance and the underlying mechanism. Nitrate tolerance of porcine coronary arteries in vitro was induced by incubation of NTG (10−5 M) for 24 h. Nitrate tolerance in vivo was obtained by subcutaneous injection of mice with NTG (20 mg kg−1, tid, 3 days) and the aortas were used. Protein levels of total and phosphorylated Akt, forkhead box protein O1 (FoxO1), and cGMP-dependent protein kinase (PKG) were determined by western blot analysis. Isometric vessel tension was recorded by organ chamber technique. PKG mRNA was determined by real-time PCR. The cellular translocation of FoxO1 was observed by immunofluorescence. Reactive oxygen species (ROS) level was measured by DHE staining. The vascular relaxation to NTG was significantly inhibited in in vivo and in vitro NTG tolerant arteries. Meanwhile, the protein level of phosphorylated Akt at Ser473 was increased in the tolerant arteries. The attenuated relaxation and the augmented Akt-p were ameliorated by LY294002, a specific inhibitor of PI3K. The protein and mRNA expression of PKG were significantly down-regulated in NTG tolerant arteries, which were reversed by LY294002. The level of phosphorylated FoxO1 at Ser256 and its translocation from the nucleus to the cytosol were both increased in NTG tolerance and were also inhibited by LY294002. ROS production was significantly increased in NTG tolerant arteries, which was not be affected by LY294002 but inhibited by N-acetyl-L-cysteine. In conclusion, the present study suggests that PI3K/Akt in vascular smooth muscle is involved in the development of NTG tolerance via inhibiting PKG transcription and the effect is mediated by FoxO1.

Abstract 3118: Oncogenesis modeling in malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is causally associated with exposure to asbestos and accounts for roughly 75% of mesothelioma cancers. It is an aggressive tumor with a median survival time of approximately 12 months. Treatment options are limited, largely palliative, and still based on cisplatin and pemetrexed, a regimen that remains unchanged since 2003. Despite widespread study, the complexities of MPM biology remain poorly described, especially at the point of tumor initiation where development of novel clinical interventional strategies would be expected to yield the most effect. The extended latency period in humans consisting of decades (over 20-30 years) remains a prohibitive barrier to gain accurate, in vivo insights about MPM oncogenic processes. As such, all in vitro MPM models, which are designed to accelerate tumor development as a practical endpoint of study, to date have not yielded clinically translatable molecular targets. Here, we present a novel in vitro model system for exploring the earliest molecular mechanisms contributing to the development of MPM using normal, human mesothelial cell lines (MeT-5a and LP9-hTERT) engineered to harbor constitutively enhanced NFkB signaling activity. Both 3D-sphere and soft agar assays were used to assess cellular transformation status in these induced cells compared to their wild-type counterparts. Heterotopic xenograft implantation studies were performed in NOD scid-gamma (NSG) immunocompromised mice (sub-cutaneous dorsal flank injection) for tumorigenic potential. In all cases, these induced cells (despite the complete absence of asbestos exposure) exhibited a significant malignant-like phenotype. Tumor growth in mouse xenograft models occurred very rapidly, and was marked by extensive vascular recruitment with invasion into surrounding tissues. In addition, these transformed cells expressed typical mesothelioma markers such as Wilms tumor protein (WT1 gene product) as revealed by immunohistofluorescence. This new MPM model system could represent a powerful construct to explore the early molecular events involved in transformation of normal mesothelial cells leading to malignancy. Citation Format: Nathanael D. Pruett, Anand Singh, Nisan Bhattacharyya, Chuong D. Hoang. Oncogenesis modeling in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3118.

Glycosaminoglycan Conjugation for Improving the Duration of Therapeutic Action of Glucagon-Like Peptide-1.

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that plays a crucial role in lowering blood glucose levels and holds promise for treating type II diabetes. In this study, we synthesized GLP-1 derivatives that were conjugated with glycosaminoglycans (GAGs), i.e., chondroitin (CH) or heparosan (HPN), to address the major limitation in their clinical use of GLP-1, which is its short half-life in the body. After exploring a variety of CHs with different molecular sizes and heterobifunctional linkers having different alkyl chains, we obtained CH-conjugated GLP-1 derivatives that stayed in blood circulation much longer (T1/2 elim > 25 h) than unconjugated GLP-1 and showed blood glucose-lowering efficacy up to 120 h after subcutaneous injection in mice. By using the same optimized linker design, we eventually obtained a HPN-conjugated GLP-1 derivative with efficacy lasting 144 h. These results demonstrate that conjugation with GAG is a promising strategy for improving the duration of peptide drugs.