Stress-Induced Tinnitus in a Rat Model: Transcriptomics of the Prefrontal Cortex and Hippocampus.

OBJECTIVES: The molecular mechanisms by which stress leads to the development of tinnitus are not yet well understood. This study aimed to identify brain changes in a stress-induced tinnitus (ST) animal model through transcriptome analysis of the prefrontal lobe and hippocampus. METHODS: Twenty Sprague-Dawley rats were subjected to restraint stress for 2 h. Following the gap prepulse inhibition of the acoustic startle (GPIAS) reflex test to assess tinnitus development, the prefrontal lobes and hippocampi of the brains were harvested from 15 rats: five with evident tinnitus (ST), five with noticeable non-tinnitus (stress-induced non-tinnitus; SNT), and five without stress (control group). Comparative RNA-seq analysis was conducted to examine gene expression profiles. RESULTS: In comparison to the control group, the ST group exhibited 971 and 463 differentially expressed genes (DEGs) in the prefrontal lobe and hippocampus, respectively (FDR < 0.05). The SNT group showed a largely similar gene expression to the control group. Enrichment analysis of the prefrontal lobe revealed the downregulation of gene sets associated with neurotransmitter and synapse-related functions and the upregulation of cell cycle-related gene sets in the ST group. In the hippocampus, there were significantly downregulated gene sets associated with steroid production and upregulated gene sets related to the extracellular matrix in the ST group. Immune-related gene sets were upregulated in both the prefrontal lobe and hippocampus. CONCLUSION: Our research presents evidence that differences in genetic expression in the prefrontal lobe and hippocampus after exposure to stress play a significant role in the development of tinnitus. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Evaluating the Dermatological Benefits of Snowberry (Symphoricarpos albus): A Comparative Analysis of Extracts and Fermented Products from Different Plant Parts.

Skin ageing is influenced by both intrinsic and extrinsic factors, with excessive ultraviolet (UV) exposure being a significant contributor. Such exposure can lead to moisture loss, sagging, increased wrinkling, and decreased skin elasticity. Prolonged UV exposure negatively impacts the extracellular matrix by reducing collagen, hyaluronic acid, and aquaporin 3 (AQP-3) levels. Fermentation, which involves microorganisms, can produce and transform beneficial substances for human health. Natural product fermentation using lactic acid bacteria have demonstrated antioxidant, anti-inflammatory, antibacterial, whitening, and anti-wrinkle properties. Snowberry, traditionally used as an antiemetic, purgative, and anti-inflammatory agent, is now also used as an immune stimulant and for treating digestive disorders and colds. However, research on the skin benefits of Fermented Snowberry Extracts remains limited. Thus, we aimed to evaluate the skin benefits of snowberry by investigating its moisturising and anti-wrinkle effects, comparing extracts from different parts of the snowberry plant with those subjected to fermentation using Lactobacillus plantarum. Chlorophyll-free extracts were prepared from various parts of the snowberry plant, and ferments were created using Lactobacillus plantarum. The extracts and ferments were analysed using high-performance liquid chromatography (HPLC) to determine and compare their chemical compositions. Moisturising and anti-ageing tests were conducted to assess the efficacy of the extracts and ferments on the skin. The gallic acid content remained unchanged across all parts of the snowberry before and after fermentation. However, Fermented Snowberry Leaf Extracts exhibited a slight decrease in chlorogenic acid content but a significant increase in ferulic acid content. The Fermented Snowberry Fruit Extract demonstrated increased chlorogenic acid and a notable rise in ferulic acid compared to its non-fermented counterpart. Skin efficacy tests revealed that Fermented Snowberry Leaf and Fruit Extracts enhanced the expression of AQP-3, HAS-3, and COL1A1. These extracts exhibited distinct phenolic component profiles, indicating potential skin benefits such as improved moisture retention and protection against ageing. These findings suggest that Fermented Snowberry Extracts could be developed into effective skincare products, providing a natural alternative for enhancing skin hydration and reducing signs of ageing.

Intravesical instillation-based mTOR-STAT3 dual targeting for bladder cancer treatment.

BACKGROUND: Recent intravesical administration of adenoviral vectors, either as a single injection or in combination with immune checkpoint inhibitors, exemplified by cretostimogene grenadenorepvec and nadofaragene firadenovec, has demonstrated remarkable efficacy in clinical trials for non-muscle invasive bladder cancer. Despite their ability to induce an enhanced immune reaction within the lesion, the intracellular survival signaling of cancer cells has not been thoroughly addressed. METHODS: An analysis of the prognostic data revealed a high probability of therapeutic efficacy with simultaneous inhibition of mTOR and STAT3. Considering the challenges of limited pharmaco-accessibility to the bladder due to its pathophysiological structure and the partially undruggable nature of target molecules, we designed a dual siRNA system targeting both mRNAs. Subsequently, this dual siRNA system was encoded into the adenovirus 5/3 (Ad 5/3) to enhance in vivo delivery efficiency. RESULTS: Gene-targeting efficacy was assessed using cells isolated from xenografted tumors using a single-cell analysis system. Our strategy demonstrated a balanced downregulation of mTOR and STAT3 at the single-cell resolution, both in vitro and in vivo. This approach reduced tumor growth in bladder cancer xenograft and orthotopic animal experiments. In addition, increased infiltration of CD8+ T cells was observed in a humanized mouse model. We provided helpful and safe tissue distribution data for intravesical therapy of siRNAs coding adenoviruses. CONCLUSIONS: The bi-specific siRNA strategy, encapsulated in an adenovirus, could be a promising tool to augment cancer treatment efficacy and overcome conventional therapy limitations associated with "undruggability." Hence, we propose that dual targeting of mTOR and STAT3 is an advantageous strategy for intravesical therapy using adenoviruses.

Comparison of Glioblastoma Cell Culture Platforms Based on Transcriptional Similarity with Paired Tissue.

No standardized in vitro cell culture models for glioblastoma (GBM) have yet been established, excluding the traditional two-dimensional culture. GBM tumorspheres (TSs) have been highlighted as a good model platform for testing drug effects and characterizing specific features of GBM, but a detailed evaluation of their suitability and comparative performance is lacking. Here, we isolated GBM TSs and extracellular matrices (ECM) from tissues obtained from newly diagnosed IDH1 wild-type GBM patients and cultured GBM TSs on five different culture platforms: (1) ordinary TS culture liquid media (LM), (2) collagen-based three-dimensional (3D) matrix, (3) patient typical ECM-based 3D matrix, (4) patient tumor ECM-based 3D matrix, and (5) mouse brain. For evaluation, we obtained transcriptome data from all cultured GBM TSs using microarrays. The LM platform exhibited the most similar transcriptional program to paired tissues based on GBM genes, stemness- and invasiveness-related genes, transcription factor activity, and canonical signaling pathways. GBM TSs can be cultured via an easy-to-handle and cost- and time-efficient LM platform while preserving the transcriptional program of the originating tissues without supplementing the ECM or embedding it into the mouse brain. In addition to applications in basic cancer research, GBM TSs cultured in LM may also serve as patient avatars in drug screening and pre-clinical evaluation of targeted therapy and as standardized and clinically relevant models for precision medicine.

Intratumoral IL-12 delivery via mesenchymal stem cells combined with PD-1 blockade leads to long-term antitumor immunity in a mouse glioblastoma model.

BACKGROUND: Although PD-1 blockade is effective for treating several types of cancer, the efficacy of this agent in glioblastoma is largely limited. To overcome non-responders and the immunosuppressive tumor microenvironment, combinational immunotherapeutic strategies with anti-PD-1 need to be considered. Here, we developed IL-12-secreting mesenchymal stem cells (MSC_IL-12) with glioblastoma tropism and evaluated the therapeutic effects of anti-PD-1, MSC_IL-12, and their combination against glioblastoma. METHODS: Therapeutic responses were evaluated using an immunocompetent mouse orthotopic model. Tumor-infiltrating lymphocytes (TILs) were analyzed using immunofluorescent imaging. Single-cell transcriptome was obtained from mouse brains after treatments. RESULTS: Anti-PD-1 and MSC_IL-12 showed complete tumor remission in 25.0% (4/16) and 23.1% (3/13) of glioblastoma-implanted mice, respectively, and their combination yielded synergistic antitumor efficacy indicated by 50.0% (6/12) of complete tumor remission. Analyses of TILs revealed that anti-PD-1 increased CD8+ T cells, while MSC_IL-12 led to infiltration of CD4+ T cells and NK cells. Both therapies reduced frequencies of Tregs. All these aspects observed in each monotherapy group were superimposed in the combination group. Notably, no tumor growth was observed upon rechallenge in cured mice, indicating long-term immunity against glioblastoma provoked by the therapies. Single-cell RNA-seq data confirmed these results and revealed that the combined treatment led to immune-favorable tumor microenvironment-CD4+, CD8+ T cells, effector memory T cells, and activated microglia were increased, whereas exhausted T cells, Tregs, and M2 polarized microglia were reduced. CONCLUSION: Anti-PD-1 and MSC_IL-12 monotherapies show long-term therapeutic responses, and their combination further enhances antitumor efficacy against glioblastoma via inducing immune-favorable tumor microenvironment.

Establishment of tumor microenvironment-preserving organoid model from patients with intracranial meningioma.

BACKGROUND: Although meningioma is the most common primary brain tumor, treatments rely on surgery and radiotherapy, and recurrent meningiomas have no standard therapeutic options due to a lack of clinically relevant research models. Current meningioma cell lines or organoids cannot reflect biological features of patient tumors since they undergo transformation along culture and consist of only tumor cells without microenvironment. We aim to establish patient-derived meningioma organoids (MNOs) preserving diverse cell types representative of the tumor microenvironment. METHODS: The biological features of MNOs were evaluated using WST, LDH, and collagen-based 3D invasion assays. Cellular identities in MNOs were confirmed by immunohistochemistry (IHC). Genetic alteration profiles of MNOs and their corresponding parental tumors were obtained by whole-exome sequencing. RESULTS: MNOs were established from four patients with meningioma (two grade 1 and two grade 2) at a 100% succession rate. Exclusion of enzymatic dissociation-reaggregation steps endowed MNOs with original histology and tumor microenvironment. In addition, we used a liquid media culture system instead of embedding samples into Matrigel, resulting in an easy-to-handle, cost-efficient, and time-saving system. MNOs maintained their functionality and morphology after long-term culture (> 9 wk) and repeated cryopreserving-recovery cycles. The similarities between MNOs and their corresponding parental tumors were confirmed by both IHC and whole-exome sequencing. As a representative application, we utilized MNOs in drug screening, and mifepristone, an antagonist of progesterone receptor, showed prominent antitumor efficacy with respect to viability, invasiveness, and protein expression. CONCLUSION: Taken together, our MNO model overcame limitations of previous meningioma models and showed superior resemblance to parental tumors. Thus, our model could facilitate translational research identifying and selecting drugs for meningioma in the era of precision medicine.

Anti-Inflammatory Activity of Vacuum Distillate from Panax ginseng Root on LPS-Induced RAW264.7 Cells.

Panax ginseng has been widely applied as an important herb in traditional medicine to treat numerous human disorders. However, the inflammatory regulation effect of P. ginseng distillate (GSD) has not yet been fully assessed. To determine whether GSD can ameliorate inflammatory processes, a GSD was prepared using the vacuum distillation process for the first time, and the regulation effect on lipopolysaccharide-induced macrophages was assessed. The results showed that GSD effectively inhibited nitric oxide (NO) formation and activation of inducible nitric oxide synthase (iNOS) mRNA in murine macrophage cell, but not cyclooxygenase-2 production. The mRNA expression pattern of tumor necrosis factor alpha and IL-6 were also reduced by GSD. Furthermore, we confirmed that GSD exerted its anti-inflammatory effects by downregulating c-Jun NH2-terminal kinase (JNK) phosphorylation, the extracellular signal-regulated kinase phosphorylation, and signaling pathway of nuclear factor kappa B (NF-κB). Our findings revealed that the inflammatory regulation activity of GSD could be induced by iNOS and NO formation inhibition mediated by regulation of nuclear factor kappa B and p38/JNK MAPK pathways.

Classification of IDH wild-type glioblastoma tumorspheres into low- and high-invasion groups based on their transcriptional program.

BACKGROUND: Glioblastoma (GBM), one of the most lethal tumors, exhibits a highly infiltrative phenotype. Here, we identified transcription factors (TFs) that collectively modulate invasion-related genes in GBM. METHODS: The invasiveness of tumorspheres (TSs) were quantified using collagen-based 3D invasion assays. TF activities were quantified by enrichment analysis using GBM transcriptome, and confirmed by cell-magnified analysis of proteome imaging. Invasion-associated TFs were knocked down using siRNA or shRNA, and TSs were orthotopically implanted into mice. RESULTS: After classifying 23 patient-derived GBM TSs into low- and high-invasion groups, we identified active TFs in each group-PCBP1 for low invasion, and STAT3 and SRF for high invasion. Knockdown of these TFs reversed the phenotype and invasion-associated-marker expression of GBM TSs. Notably, MRI revealed consistent patterns of invasiveness between TSs and the originating tumors, with an association between high invasiveness and poor prognosis. Compared to controls, mice implanted with STAT3- or SRF-downregulated GBM TSs showed reduced normal tissue infiltration and tumor growth, and prolonged survival, indicating a therapeutic response. CONCLUSIONS: Our integrative transcriptome analysis revealed three invasion-associated TFs in GBM. Based on the relationship among the transcriptional program, invasive phenotype, and prognosis, we suggest these TFs as potential targets for GBM therapy.

Single-cell RNA sequencing of a poorly metastatic melanoma cell line and its subclones with high lung and brain metastasis potential reveals gene expression signature of metastasis with prognostic implication.

The molecular mechanisms underlying melanoma metastasis remain poorly understood. In this study, we aimed to delineate the mechanisms underlying gene expression alterations during metastatic potential acquisition and characterize the metastatic subclones within primary cell lines. We performed single-cell RNA sequencing of a poorly metastatic melanoma cell line (WM239A) and its subclones with high metastatic potential to the lung (113/6-4L) and the brain (131/4-5B1 and 131/4-5B2). Unsupervised clustering of 8173 melanoma cells identified three distinct clusters according to cell type ('Primary', 'Lung' and 'Brain' clusters) with differential expression of MITF and AXL pathways and putative cancer and cell cycle drivers, with the lung cluster expressing intermediate but distinct gene profiles between primary and brain clusters. Principal component (PC) analysis revealed that PC2 (the second PC), which was positively associated with MITF expression and negatively with AXL pathways, primarily segregated cell types, in addition to PC1 of the cell cycle pathway. Pseudotime trajectory and RNA velocity analyses suggested the existence of cellular subsets with metastatic potential in the Primary cluster and an association between PC2 signature alteration and metastasis potential acquisition. Analysis of The Cancer Genome Atlas melanoma samples by clustering into PC2-high and -low clusters by quartiles of PC2 signature expression revealed that the PC2-high cluster was an independent significant factor for poor prognosis (p-value = 0.003) with distinct genomic and transcriptomic characteristics, compared to the PC2-low cluster. In conclusion, we identified signatures of melanoma metastasis with prognostic significance and putative pro-metastatic subclones within a primary cell line.

Suppression of Metastatic Ovarian Cancer Cells by Bepridil, a Calcium Channel Blocker.

Although surgery followed by platinum-based therapy is effective as a standard treatment in the early stages of ovarian cancer, the majority of cases are diagnosed at advanced stages, leading to poor prognosis. Thus, the identification of novel therapeutic drugs is needed. In this study, we assessed the effectiveness of bepridil-a calcium channel blocker-in ovarian cancer cells using two cell lines: SKOV-3, and SKOV-3-13 (a highly metastatic clone of SKOV-3). Treatment of these cell lines with bepridil significantly reduced cell viability, migration, and invasion. Notably, SKOV-3-13 was more sensitive to bepridil than SKOV-3. The TGF-ß1-induced epithelial-mesenchymal transition (EMT)-like phenotype was reversed by treatment with bepridil in both cell lines. Consistently, expression levels of EMT-related markers, including vimentin, ß-catenin, and Snail, were also substantially decreased by the treatment with bepridil. An in vivo mouse xenograft model was used to confirm these findings. Tumor growth was significantly reduced by bepridil treatment in SKOV-3-13-inoculated mice, and immunohistochemistry showed consistently decreased expression of EMT-related markers. Our findings are the first to report anticancer effects of bepridil in ovarian cancer, and they suggest that bepridil holds significant promise as an effective therapeutic agent for targeting metastatic ovarian cancer.

Single-cell RNA sequencing reveals a pro-metastatic subpopulation and the driver transcription factor NFE2L1 in ovarian cancer cells.

BACKGROUND: Although cytoreductive surgery followed by adjuvant chemotherapy is effective as a standard treatment for early-stage ovarian cancer, the majority of ovarian cancer cases are diagnosed at the advanced stages with dissemination to the peritoneal cavity, leading to a poor prognosis. Therefore, it is crucial to understand the cellular and molecular mechanisms underlying metastasis and identify novel therapeutic targets. OBJECTIVE: In this study, we aimed to elucidate the mechanisms underlying gene expression alterations during the acquisition of metastatic potential and characterize the metastatic subpopulations within ovarian cancer cells. METHODS: We conducted single-cell RNA sequencing of two human ovarian cancer cell lines: SKOV-3 and SKOV-3-13, a highly metastatic subclone of SKOV-3. Suppression of NFE2L1 expression was performed through siRNA-mediated knockdown and CRISPR-Cas9-mediated knockout. RESULTS: Clustering and pseudotime trajectory analysis revealed pro-metastatic subpopulation within these cells. Furthermore, gene set enrichment analysis and prognosis analysis indicated that NFE2L1 could be a key transcription factor in the acquisition of metastasis potential. Inhibition of NFE2L1 significantly reduced migration and viability of both cells. In addition, NFE2L1 knockout cells exhibited significantly reduced tumor growth in a mouse xenograft model, recapitulating in silico and in vitro results. CONCLUSION: The results presented in this study deepen our understanding of the molecular pathogenesis of ovarian cancer metastasis with the ultimate goal of developing treatments targeting pro-metastatic subclones prior to metastasis.

Mesenchymal Stem-Like Cells Derived from the Ventricle More Effectively Enhance Invasiveness of Glioblastoma Than Those Derived from the Tumor.

PURPOSE: Glioblastoma (GBM) is one of the most lethal human tumors with a highly infiltrative phenotype. Our previous studies showed that GBM originates in the subventricular zone, and that tumor-derived mesenchymal stem-like cells (tMSLCs) promote the invasiveness of GBM tumorspheres (TSs). Here, we extend these studies in terms of ventricles using several types of GBM patient-derived cells. MATERIALS AND METHODS: The invasiveness of GBM TSs and ventricle spheres (VSs) were quantified via collagen-based 3D invasion assays. Gene expression profiles were obtained from microarray data. A mouse orthotopic xenograft model was used for in vivo experiments. RESULTS: After molecular and functional characterization of ventricle-derived mesenchymal stem-like cells (vMSLCs), we investigated the effects of these cells on the invasiveness of GBM TSs. We found that vMSLC-conditioned media (CM) significantly accelerated the invasiveness of GBM TSs and VSs, compared to the control and even tMSLC-CM. Transcriptome analyses revealed that vMSLC secreted significantly higher levels of several invasiveness-associated cytokines. Moreover, differentially expressed genes between vMSLCs and tMSLCs were enriched for migration, adhesion, and chemotaxis-related gene sets, providing a mechanistic basis for vMSLC-induced invasion of GBM TSs. In vivo experiments using a mouse orthotopic xenograft model confirmed vMSLC-induced increases in the invasiveness of GBM TSs. CONCLUSION: Although vMSLCs are non-tumorigenic, this study adds to our understanding of how GBM cells acquire infiltrative features by vMSLCs, which are present in the region where GBM genesis originates.

C5α secreted by tumor mesenchymal stem-like cells mediates resistance to 5-aminolevulinic acid-based photodynamic therapy against glioblastoma tumorspheres.

PURPOSE: Advancements in photodynamic diagnosis (PDD) and photodynamic therapy (PDT) as a standard care in cancer therapy have been limited. This study is aimed to investigate the clinical availability of 5-aminolevulinic acid (5-ALA)-based PDD and PDT in glioblastoma (GBM) patient-derived tumorspheres (TSs) and mouse orthotopic xenograft model. METHODS: PDT was performed using a 635 nm light-emitting diode (LED). Transcriptome profiles were obtained from microarray data. For knockdown of C5α, siRNA was transfected into tumor mesenchymal stem-like cells (tMSLCs). The invasiveness of TSs was quantified using collagen-based 3D invasion assays. RESULTS: Treatment with 1 mM 5 ALA induced distinct protoporphyrin IX (PpIX) fluorescence in GBM TSs, but not in non-tumor cells or tissues, including tMSLCs. These observations were negatively correlated with the expression levels of FECH, which catalyzes the conversion of accumulated PpIX to heme. Furthermore, the 5-ALA-treated GBM TSs were sensitive to PDT, thereby significantly decreasing cell viability and invasiveness. Notably, the effects of PDT were abolished by culturing TSs with tMSLC-conditioned media. Transcriptome analysis revealed diverse tMSLC-secreted chemokines, including C5α, and their correlations with the expression of stemness- or mesenchymal transition-associated genes. By adding or inhibiting C5α, we confirmed that acquired resistance to PDT was induced via tMSLC-secreted C5α. CONCLUSIONS: Our results show substantial therapeutic effects of 5-ALA-based PDT on GBM TSs, suggesting C5α as a key molecule responsible for PDT resistance. These findings could trigger PDT as a standard clinical modality for the treatment of GBM.

Assessing Spatial Distribution of Multicellular Self-Assembly Enables the Prediction of Phenotypic Heterogeneity in Glioblastoma.

Phenotypic heterogeneity of glioblastomas is a leading determinant of therapeutic resistance and treatment failure. However, functional assessment of the heterogeneity of glioblastomas is lacking. We developed a self-assembly-based assessment system that predicts inter/intracellular heterogeneity and phenotype associations, such as cell proliferation, invasiveness, drug responses, and gene expression profiles. Under physical constraints for cellular interactions, mixed populations of glioblastoma cells are sorted to form a segregated architecture, depending on their preference for binding to cells of the same phenotype. Cells distributed at the periphery exhibit a reduced temozolomide (TMZ) response and are associated with poor patient survival, whereas cells in the core of the aggregates exhibit a significant response to TMZ. Our results suggest that the multicellular self-assembly pattern is indicative of the intertumoral and intra-patient heterogeneity of glioblastomas, and is predictive of the therapeutic response.

Human allogenic γδ T cells kill patient-derived glioblastoma cells expressing high levels of DNAM-1 ligands.

Adoptive transfer of γδ T cells is a novel immunotherapeutic approach to glioblastoma. Few recent studies have shown the efficacy of γδ T cells against glioblastoma, but no previous studies have identified the ligand-receptor interactions between γδ T cells and glioblastoma cells. Here, we identify those ligand-receptor interactions and provide a basis for using γδ T cells to treat glioblastoma. Vγ9Vδ2 T cells were generated from peripheral blood mononuclear cells of healthy donors using artificial antigen presenting cells. MICA, ULBP, PVR and Nectin-2 expression in 10 patient-derived glioblastoma (PDG) cells were analyzed. The in vitro cytokine secretion from the γδ T cells and their cytotoxicity toward the PDG cells were also analyzed. The in vivo anti-tumor effects were evaluated using a U87 orthotopic xenograft glioblastoma model. Expression of ligands and cytotoxicity of the γδ T cells varied among the PDG cells. IFN-γ and Granzyme B secretion levels were significantly higher when γδ Tcells were co-cultured with high-susceptible PDG cells than when they were co-cultured with low-susceptible PDG cells. Cytotoxicity correlated significantly with the expression levels of DNAM-1 ligands of the PDG cells. Blocking DNAM-1 resulted in a decrease in γδ T cell-mediated cytotoxicity and cytokine secretion. Intratumoral injection of γδ T cells showed anti-tumor effects in an orthotopic mouse model. Allogenic γδ T cells showed potent anti-tumor effects on glioblastoma in a DNAM-1 axis dependent manner. Our findings will facilitate the development of clinical strategies using γδ T cells for glioblastoma treatment.

Dual inhibition of CPT1A and G6PD suppresses glioblastoma tumorspheres.

PURPOSE: Limited treatment options are currently available for glioblastoma (GBM), an extremely lethal type of brain cancer. For a variety of tumor types, bioenergetic deprivation through inhibition of cancer-specific metabolic pathways has proven to be an effective therapeutic strategy. Here, we evaluated the therapeutic effects and underlying mechanisms of dual inhibition of carnitine palmitoyltransferase 1A (CPT1A) and glucose-6-phosphate dehydrogenase (G6PD) critical for fatty acid oxidation (FAO) and the pentose phosphate pathway (PPP), respectively, against GBM tumorspheres (TSs). METHODS: Therapeutic efficacy against GBM TSs was determined by assessing cell viability, neurosphere formation, and 3D invasion. Liquid chromatography-mass spectrometry (LC-MS) and RNA sequencing were employed for metabolite and gene expression profiling, respectively. Anticancer efficacy in vivo was examined using an orthotopic xenograft model. RESULTS: CPT1A and G6PD were highly expressed in GBM tumor tissues. Notably, siRNA-mediated knockdown of both genes led to reduced viability, ATP levels, and expression of genes associated with stemness and invasiveness. Similar results were obtained upon combined treatment with etomoxir and dehydroepiandrosterone (DHEA). Transcriptome analyses further confirmed these results. Data from LC-MS analysis showed that this treatment regimen induced a considerable reduction in the levels of metabolites associated with the TCA cycle and PPP. Additionally, the combination of etomoxir and DHEA inhibited tumor growth and extended survival in orthotopic xenograft model mice. CONCLUSION: Our collective findings support the utility of dual suppression of CPT1A and G6PD with selective inhibitors, etomoxir and DHEA, as an efficacious therapeutic approach for GBM.

Estradiol-17 ß levels as a tool for sex determination in Farmed Anguilla japonica.

In this study, the levels of plasma estradiol-17ß (E2) in farmed Anguilla japonica were measured to determine their sex. The analyses were performed for two different size groups (large group, Total length (TL): 61-69 cm; small group, TL: 53-60 cm). The anatomical and histological observations showed that the large group consisted of 29% males and 71% females; the small group, 54% males and 45% females. The gonad histology showed that in the large group, 88% of the eels had immature gonads with ongoing sexual differentiation, 12% were mature with completed sexual differentiation. In the small group, 87% of the eels had immature gonads. The plasma E2 hormone levels were higher in the females of both sizes. In the large group, the average plasma E2 in females was 415 pg/ml, which was significantly higher than the average of 109 pg/ml in males (P < 0.05). In the small group, the average plasma E2 hormone level was 618 pg/ml, which was much higher than the average of 108 pg/ml in males. Quantitative real-time PCR showed that zygote arrest 1 (zar 1) and zona pellucida glycoprotein 3 (zp3) were more highly expressed in females than male. In the H-E staining, an eel in the oil droplet containing ovary stage had a high level of plasma E2 (1500 pg/ml), while an eel with testis in the spermatocyte stage had a low (60 pg/ml). E2 is a potentially useful tool and could play an important role in sex determination in broodstocks.

Etomoxir, a carnitine palmitoyltransferase 1 inhibitor, combined with temozolomide reduces stemness and invasiveness in patient-derived glioblastoma tumorspheres.

INTRODUCTION: The importance of fatty acid oxidation (FAO) in the bioenergetics of glioblastoma (GBM) is being realized. Etomoxir (ETO), a carnitine palmitoyltransferase 1 (CPT1) inhibitor exerts cytotoxic effects in GBM, which involve interrupting the FAO pathway. We hypothesized that FAO inhibition could affect the outcomes of current standard temozolomide (TMZ) chemotherapy against GBM. METHODS: The FAO-related gene expression was compared between GBM and the tumor-free cortex. Using four different GBM tumorspheres (TSs), the effects of ETO and/or TMZ was analyzed on cell viability, tricarboxylate (TCA) cycle intermediates and adenosine triphosphate (ATP) production to assess metabolic changes. Alterations in tumor stemness, invasiveness, and associated transcriptional changes were also measured. Mouse orthotopic xenograft model was used to elucidate the combinatory effect of TMZ and ETO. RESULTS: GBM tissues exhibited overexpression of FAO-related genes, especially CPT1A, compared to the tumor-free cortex. The combined use of ETO and TMZ further inhibited TCA cycle and ATP production than single uses. This combination treatment showed superior suppression effects compared to treatment with individual agents on the viability, stemness, and invasiveness of GBM TSs, as well as better downregulation of FAO-related gene expression. The results of in vivo study showed prolonged survival outcomes in the combination treatment group. CONCLUSION: ETO, an FAO inhibitor, causes a lethal energy reduction in the GBM TSs. When used in combination with TMZ, ETO effectively reduces GBM cell stemness and invasiveness and further improves survival. These results suggest a potential novel treatment option for GBM.

Tissue Niche Miniature of Glioblastoma Patient Treated with Nano-Awakeners to Induce Suicide of Cancer Stem Cells.

Patient-specific cancer therapies can evolve by vitalizing the mother tissue-like cancer niche, cellular profile, genetic signature, and drug responsiveness. This evolution has enabled the elucidation of a key mechanism along with development of the mechanism-driven therapy. After surgical treatment, glioblastoma (GBM) patients require prompt therapy within 14 days in a patient-specific manner. Hence, this study approaches direct culture of GBM patient tissue (1 mm diameter) in a microchannel network chip. Cancer vasculature-mimetic perfusion can support the preservation of the mother tissue-like characteristic signatures and microenvironment. When temozolomide and radiation are administered within 1 day, the responsiveness of the tissue in the chip reflected the clinical outcomes, thereby overcoming the time-consuming process of cell and organoid culture. When the tissue chip culture is continued, the intact GBM signature gets lost, and the outward migration of stem cells from the tissue origin increases, indicating a leaving-home effect on the family dismantle. Nanovesicle production using GBM stem cells enables self-chasing of the cells that escape the temozolomide effect owing to quiescence. The anti-PTPRZ1 peptide display and temozolomide loading to nanovesicles awakes cancer stem cells from the quiescent stage to death. This study suggests a GBM clinic-driven avatar platform and mechanism-learned nanotherapy for translation.

A lignan from Alnus japonica inhibits glioblastoma tumorspheres by suppression of FOXM1.

Forkhead Box M1 (FOXM1) is known to regulate cell proliferation, apoptosis and tumorigenesis. The lignan, (-)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS), from Alnus japonica has shown anti-cancer effects against colon cancer cells by suppressing FOXM1. The present study hypothesized that DFS can have anti-cancer effects against glioblastoma (GBM) tumorspheres (TSs). Immunoprecipitation and luciferase reporter assays were performed to evaluate the ability of DFS to suppress nuclear translocation of ß-catenin through ß-catenin/FOXM1 binding. DFS-pretreated GBM TSs were evaluated to assess the ability of DFS to inhibit GBM TSs and their transcriptional profiles. The in vivo efficacy was examined in orthotopic xenograft models of GBM. Expression of FOXM1 was higher in GBM than in normal tissues. DFS-induced FOXM1 protein degradation blocked ß-catenin translocation into the nucleus and consequently suppressed downstream target genes of FOXM1 pathways. DFS inhibited cell viability and ATP levels, while increasing apoptosis, and it reduced tumorsphere formation and the invasiveness of GBM TSs. And DFS reduced the activities of transcription factors related to tumorigenesis, stemness, and invasiveness. DFS significantly inhibited tumor growth and prolonged the survival rate of mice in orthotopic xenograft models of GBM. It suggests that DFS inhibits the proliferation of GBM TSs by suppressing FOXM1. DFS may be a potential therapeutic agent to treat GBM.