Identification of Metabolomic Markers in Frozen or Formalin-Fixed and Paraffin-Embedded Samples of Diffuse Glioma from Adults.

The aim of this study was to identify metabolomic signatures associated with the gliomagenesis pathway (IDH-mutant or IDH-wt) and tumor grade of diffuse gliomas (DGs) according to the 2021 WHO classification on frozen samples and to evaluate the diagnostic performances of these signatures in tumor samples that are formalin-fixed and paraffin-embedded (FFPE). An untargeted metabolomic study was performed using liquid chromatography/mass spectrometry on a cohort of 213 DG samples. Logistic regression with LASSO penalization was used on the frozen samples to build classification models in order to identify IDH-mutant vs. IDH-wildtype DG and high-grade vs low-grade DG samples. 2-Hydroxyglutarate (2HG) was a metabolite of interest to predict IDH mutational status and aminoadipic acid (AAA) and guanidinoacetic acid (GAA) were significantly associated with grade. The diagnostic performances of the models were 82.6% AUC, 70.6% sensitivity and 80.4% specificity for 2HG to predict IDH status and 84.7% AUC, 78.1% sensitivity and 73.4% specificity for AAA and GAA to predict grade from FFPE samples. Thus, this study showed that AAA and GAA are two novel metabolites of interest in DG and that metabolomic data can be useful in the classification of DG, both in frozen and FFPE samples.

Trends in Light and Temperature Sensitivity Recommendations among Licensed Biotechnology Drug Products.

PURPOSE: Inherent structural and functional properties of biotechnology-derived therapeutic biologics make them susceptible to light- and temperature-induced degradation and consequently can influence their quality. Photosensitivity of therapeutic proteins continues to be examined, but the commonalities and trends of storage conditions and information about light and temperature sensitivity among currently licensed therapeutic proteins has not been previously surveyed. METHODS: Using a comprehensive and relational database approach, we conducted a scientific survey of all licensed biotechnology-derived drug products with the goal of providing evidence-based information about recommended storage conditions of formulations sorted by light- and temperature-related attributes as described for each product at licensure. RESULTS: We report the prevalence of indications for light and temperature sensitivity in formulations categorized by their presentation type, number of doses, container type, dosage form and active molecule type. We also report the storage temperature range across formulations and diluents for reconstitution and dilution. Formulations with excipients that potentially facilitate light-induced and thermal degradation were also noted. CONCLUSIONS: The result of our analysis indicates that light and temperature sensitivity are prevalent across therapeutic protein formulations. However, when a formulation is reconstituted or diluted, both light and temperature sensitivity are less clear. In addition, light and temperature sensitivity are more well defined in liquid formulations than lyophilized powder formulations, and more well defined in products manufactured in autoinjectors, prefilled-syringes, and pens than products in vials. Overall, our report provides a data-driven summary of storage conditions among therapeutic protein formulations to support the development of future biologic drug products.

Niclosamide Revitalizes Sorafenib through Insulin-like Growth Factor 1 Receptor (IGF-1R)/Stemness and Metabolic Changes in Hepatocellular Carcinoma.

Sorafenib is the first approved systemic targeting agent for advanced HCC; however, when used alone, drug resistance can result in considerably reduced efficacy. Here, we demonstrate that niclosamide, an antihelminthic agent approved by the US Food and Drug Administration, can be repurposed to increase sorafenib sensitivity in sorafenib-resistant HCC cells. We generated sorafenib-resistant HCC cell lines (HepG2215_R and Hep3B_R) with elevated IGF-1R levels and strong properties in terms of stemness and epithelial-mesenchymal transition. Niclosamide was found to increase sorafenib sensitivity effectively in both cell lines and their organoids. The underlying mechanism involves the modulation of cancer stemness, IGF-1R/p-IGF1R/OCT4, and metabolic changes. The combination of sorafenib and niclosamide, but not linsitinib, effectively suppressed the IGF-1R/OCT4 expressions, yielded a synergistic combination index (CI), and attenuated stemness-related properties such as secondary tumor sphere formation and cell migration in sorafenib-resistant HCC cells. Notably, niclosamide significantly suppressed the sorafenib-induced IGF-1R phosphorylation prompted by IGF-1 treatment. Niclosamide effectively downregulated the sorafenib-induced gene expression associated with glycolysis (GLUT1, HK2, LDHA, and PEPCK), stemness (OCT4), and drug resistance (ABCG2) and enhanced the ability of sorafenib to reduce the mitochondrial membrane potential in vitro. The synergistic effect of a combination of niclosamide and sorafenib in vivo was further demonstrated by the decreased tumor size and tumor volume resulting from apoptosis regulation. Our results suggest that niclosamide can enhance sorafenib sensitivity in sorafenib-resistant HCC cells through IGF-1R/stemness regulation and metabolic changes. Our findings highlight a practical clinical strategy for enhancing sorafenib sensitivity in HCC.

Perivascular Stromal Cells Instruct Glioblastoma Invasion, Proliferation, and Therapeutic Response within an Engineered Brain Perivascular Niche Model.

Glioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. A 3D in vitro model of the brain perivascular niche developed by encapsulating brain-derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel is described. It is shown that brain perivascular stromal cells, namely pericytes and astrocytes, contribute to vascular architecture and maturation. Cocultures of patient-derived GBM tumor cells with brain microvascular cells are used to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provide unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, it is shown that engineered perivascular models provide an important resource to evaluate mechanisms by which intercellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient-specific disease phenotypes.

Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines.

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines.

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

Sibling bullying among Vietnamese children: the relation with peer bullying and subjective well-being.

BACKGROUND: Siblings play an important role in a child's life. However, many children often experience sibling bullying. This study investigates differences in sibling victimization by sex, age, a parent's absence from the home due to employment, or a child's privacy and the relationship between sibling victimization, peer victimization, and the child's well-being. PARTICIPANTS AND PROCEDURE: Participants were Vietnamese children participating in the third wave of the International Survey of Children's Well-Being. The study included 1537 children (811 boys and 726 girls) attending public schools, age 10-14 years (M = 11.29, SD = 1.15). RESULTS: The results show that over half of children with siblings in this study reported being victimized by a sibling. Younger children were bullied more often than older children. Children whose father worked away from home reported an increase in bullying behavior from their siblings. Children sharing a room with siblings reported being bullied more by siblings. CONCLUSIONS: The results indicated a positive correlation between sibling victimization and peer victimization and a negative relationship between being bullied and a child's subjective well-being.

A Yes-Associated Protein (YAP) and Insulin-Like Growth Factor 1 Receptor (IGF-1R) Signaling Loop Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma.

The role of a YAP-IGF-1R signaling loop in HCC resistance to sorafenib remains unknown. METHOD: Sorafenib-resistant cells were generated by treating naïve cells (HepG2215 and Hep3B) with sorafenib. Different cancer cell lines from databases were analyzed through the ONCOMINE web server. BIOSTORM-LIHC patient tissues (46 nonresponders and 21 responders to sorafenib) were used to compare YAP mRNA levels. The HepG2215_R-derived xenograft in SCID mice was used as an in vivo model. HCC tissues from a patient with sorafenib failure were used to examine differences in YAP and IGF-R signaling. RESULTS: Positive associations exist among the levels of YAP, IGF-1R, and EMT markers in HCC tissues and the levels of these proteins increased with sorafenib failure, with a trend of tumor-margin distribution in vivo. Blocking YAP downregulated IGF-1R signaling-related proteins, while IGF-1/2 treatment enhanced the nuclear translocation of YAP in HCC cells through PI3K-mTOR regulation. The combination of YAP-specific inhibitor verteporfin (VP) and sorafenib effectively decreased cell viability in a synergistic manner, evidenced by the combination index (CI). CONCLUSION: A YAP-IGF-1R signaling loop may play a role in HCC sorafenib resistance and could provide novel potential targets for combination therapy with sorafenib to overcome drug resistance in HCC.

Glycosaminoglycan content of a mineralized collagen scaffold promotes mesenchymal stem cell secretion of factors to modulate angiogenesis and monocyte differentiation.

Effective design of biomaterials to aid regenerative repair of craniomaxillofacial (CMF) bone defects requires approaches that modulate the complex interplay between exogenously added progenitor cells and cells in the wound microenvironment, such as osteoblasts, osteoclasts, endothelial cells, and immune cells. We are exploring the role of the glycosaminoglycan (GAG) content in a class of mineralized collagen scaffolds recently shown to promote osteogenesis and healing of craniofacial bone defects. We previously showed that incorporating chondroitin-6-sulfate or heparin improved mineral deposition by seeded human mesenchymal stem cells (hMSCs). Here, we examine the effect of varying scaffold GAG content on hMSC behavior, and their ability to modulate osteoclastogenesis, vasculogenesis, and the immune response. We report the role of hMSC-conditioned media produced in scaffolds containing chondroitin-6-sulfate (CS6), chondroitin-4-sulfate (CS4), or heparin (Heparin) GAGs on endothelial tube formation and monocyte differentiation. Notably, endogenous production by hMSCs within Heparin scaffolds most significantly inhibits osteoclastogenesis via secreted osteoprotegerin (OPG), while the secretome generated by CS6 scaffolds reduced pro-inflammatory immune response and increased endothelial tube formation. All conditioned media down-regulated many pro- and anti-inflammatory cytokines, such as IL6, IL-1ß, and CCL18 and CCL17 respectively. Together, these findings demonstrate that modifying mineralized collagen scaffold GAG content can both directly (hMSC activity) and indirectly (production of secreted factors) influence overall osteogenic potential and mineral biosynthesis as well as angiogenic potential and monocyte differentiation towards osteoclastic and macrophage lineages. Scaffold GAG content is therefore a powerful stimulus to modulate reciprocal signaling between multiple cell populations within the bone healing microenvironment.

Cell and Gene Therapy for Anemia: Hematopoietic Stem Cells and Gene Editing.

Hereditary anemia has various manifestations, such as sickle cell disease (SCD), Fanconi anemia, glucose-6-phosphate dehydrogenase deficiency (G6PDD), and thalassemia. The available management strategies for these disorders are still unsatisfactory and do not eliminate the main causes. As genetic aberrations are the main causes of all forms of hereditary anemia, the optimal approach involves repairing the defective gene, possibly through the transplantation of normal hematopoietic stem cells (HSCs) from a normal matching donor or through gene therapy approaches (either in vivo or ex vivo) to correct the patient's HSCs. To clearly illustrate the importance of cell and gene therapy in hereditary anemia, this paper provides a review of the genetic aberration, epidemiology, clinical features, current management, and cell and gene therapy endeavors related to SCD, thalassemia, Fanconi anemia, and G6PDD. Moreover, we expound the future research direction of HSC derivation from induced pluripotent stem cells (iPSCs), strategies to edit HSCs, gene therapy risk mitigation, and their clinical perspectives. In conclusion, gene-corrected hematopoietic stem cell transplantation has promising outcomes for SCD, Fanconi anemia, and thalassemia, and it may overcome the limitation of the source of allogenic bone marrow transplantation.