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.

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis. Tumor mesenchymal stem-like cells (tMSLCs) in GBM tissues secrete complement component 5a (C5a), which promotes tumor malignancy. This study aimed to investigate the impact of C5a on GBM tumorspheres (GBM-TS) and to evaluate the therapeutic potential of C5a inhibition using the C5a antagonist, W54011. METHODS RNA sequencing (RNAseq) was performed on tumor tissues to identify differentiaaly expressing genes (DEG) and perform gene set enrichment analysia (GSEA) to correlate with patient survival based on C5aR1 expression. GBM-TS and tMSLCs were cultured to create a conditioned medium (CM) containing C5a, which was used to stimulate GBM-TS. The effect of C5a on GBM-TS proliferation, invasion, and stemness was determined using WST/ATP assay, 3D invasion assay, and neurosphere formation assay. Western blot and RNAseq analysis were performed to validate the results. In vivo studies were conducted using orthotopic xenograft mouse models injected with GBM-TS alone or with CM and treated with W54011. RESULTS Patients with high C5aR1 expression had a poor prognosis. Treatment with CM increased proliferation, invasion, and stemness of GBM-TS, but W54011 reversed these effects. Treatment with CM induced epithelial-messenchymal transition (EMT) in GBM TSs, whereas W54011 restored the spherical morphology and induced apoptosis. Our results were supported by transcriptome analysis and marker expression in Western blots. In the orthotopic xenograft mouse model, co-injection of GBM-TS and CM resulted in larger tumors and poorer survival, while treatment with W54011 reduced tumor size. CONCLUSIONS C5a promotes the growth, invasion, and stemness of GBM-TS, and inhibition of C5a using W54011 may have therapeutic potential for patients with high C5a expression. These results provide insight into the underlying mechanisms of GBM progression and establish a knowledge base for the clinical efficacy of W54011 in GBM treatment.

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 β-catenin through β-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 β-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.

BACKGROUND: Cancer cells depended on cytosolic nicotinamide adenine dinucleotide (NADH) transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. KN612 (N-phenylmaleimide), a MAS inhibitor, is known to interfere with cancer growth by reducing ATP production, supported by several studies except on glioblastoma (GBM). Therefore, this study was designed to elucidate whether MAS could be an aimable target in GBM. METHODS: We compared expression levels of MAS conforming enzymes between normal and GBM samples. Gene expression profiles were analyzed using RNA-sequencing. Mitochondrial activity was measured by oxygen consumption (OCR), tetramethylrhodamine-ethylester (TMRE) staining, and liquid chromatograph-tandem mass spectrometer (LC-MS/MS). Also, biological functions were measured by cell viability, ATP levels, NADH levels, stemness, and invasiveness. In vivo efficacies were confirmed using a mouse orthotopic xenograft model. RESULTS: An analysis of the microarray database revealed that expression levels of several MAS enzymes including OGC (SLC25A11) were elevated in GBM. Through RNA sequencing, it was confirmed that KN612 accurately targeted SLC25A11 and decreased its expression. KN612-treated cells showed decreased viability, ATP production, and NADH levels compared with control cells. Under the same conditions, a significant decrease in stemness, invasion, and MMP was confirmed. In addition, KN612 confirmed remarkable therapeutic responses in a mouse orthotopic xenograft model. CONCLUSION: Our results show that KN612 effectively inhibits cancer cells both at the cellular level and at the in vivo level. This shows that targeting MAS could be a potential treatment option in addition to the currently limited standard GBM therapy.

Human glioblastoma (GBM), originating from the subventricular zone (SVZ), occurs due to molecular disruptions in chromosomes. Most GBM tissues exhibit definitive chromosomal patterns: copy-number-variations (CNV) in chromosome 7 (gain) and 10 (loss), known as the earliest molecular events. Herein, we hypothesised that the origin-cells in SVZ of GBM patients can provide clues regarding these chromosomal alterations. We compared bulk RNA sequencing (RNAseq) data of GBM tumor tissue (n=126), tumour free GBM SVZ (n=40), and tumor-free control SVZ of non-glial tumor (n=9). Paired single-cell-level RNAseq samples of tumor free GBM SVZ (n=7) and GBM tumor tissue (n=10), were done to see cell specific CNVs. Using human SVZ and GBM samples as a background, we generated origin-cell and origin-cell-derived tumour cell using CRISPR/Cas9. In this work, we identified two GBM-origin-cell types with stem-cell signatures during single-cell level analyses of 60 SVZ tissue samples obtained from tumor-free regions of GBM patients. Furthermore, single-cell level analysis revealed that two origin-cell types in SVZ harbor ongoing patterns of CNV alterations. Among the origin-cells found in the SVZ of GBM patients, NO-like cells showed neural progenitor plus oligodendrocyte progenitor (NO) signature, while AN-like cells showed astrocyte plus neural stem cell (AN) signature. For the interconnectedness, we subjected single-cell-level RNA-seq data to ligand-receptor connection analysis. In the stem cell mode, NO-like cells was connected to AN-like cells in SVZ samples and while in the tumor samples, cycling cell was connected to AN-like cells. NO-like cells was common in TERT promoter wildtype GBM and AN-like cells was more common in TERT promoter mutant GBM. CRISPR/Cas9 models revealed accumulation copy-number alterations from non-tumorigenic origin-cells to tumor cells. These two origin-cells (NO-like cells and AN-like cells) derived from the SVZ of the adult human brain will facilitate the understanding of GBM genesis and development of potential novel therapeutic targets.

Abstract BACKGROUND: Cancer cells depended on cytosolic nicotinamide adenine dinucleotide (NADH) transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. KN612 (N-phenylmaleimide), a MAS inhibitor, is known to interfere with cancer growth by reducing ATP production, supported by several studies except on glioblastoma (GBM). Therefore, this study was designed to elucidate whether MAS could be an aimable target in GBM. METHODS: We compared expression levels of MAS conforming enzymes between normal and GBM samples. Gene expression profiles were analyzed using RNA-sequencing. Mitochondrial activity was measured by oxygen consumption (OCR), tetramethylrhodamine-ethylester (TMRE) staining, and liquid chromatograph-tandem mass spectrometer (LC-MS/MS). Also, biological functions were measured by cell viability, ATP levels, NADH levels, stemness, and invasiveness. In vivo efficacies were confirmed using a mouse orthotopic xenograft model. RESULTS: An analysis of the microarray database revealed that expression levels of several MAS enzymes including OGC (SLC25A11) were elevated in GBM. Through RNA sequencing, it was confirmed that KN612 accurately targeted SLC25A11 and decreased its expression. KN612-treated cells showed decreased viability, ATP production, and NADH levels compared with control cells. Under the same conditions, a significant decrease in stemness, invasion, and MMP was confirmed. In addition, KN612 confirmed remarkable therapeutic responses in a mouse orthotopic xenograft model. CONCLUSION: Our results show that KN612 effectively inhibits cancer cells both at the cellular level and at the in vivo level. This shows that targeting MAS could be a potential treatment option in addition to the currently limited standard GBM therapy.

Abstract Background: Although glioblastoma (GBM) is the most common primary brain tumor, the best available treatment options are still associated with poor prognosis. Recently, the Hippo/YAP signaling pathway has emerged as an important driver of GBM. Nevertheless, extensive studies have not yet been focused on the importance of phosphorylation event in regulating endogenous YAP activity in GBM. Here, we sought to elucidate that the modulation and stabilization of YAP/TAZ in Hippo pathway promote GBM progression. Methods: A core gene expression signature reflecting Silence of Hippo pathway (SOH) was developed in GBM patient samples to determine whether SOH enhances the tumor progression. In GBM tumorsphere (TS), mRNA levels of YAP1 were determined by microarray, and nuclear YAP1 expression level and its correlation with tumor aggressiveness were assessed through nucleus cytosolic fractionation, quantitative confocal microscopy, Western blot, and TEAD4 reporter assay. Cell proliferation, stemness, and invasive properties were also measured after treatment of YAP siRNA or its inhibitor verteporfin (VP). Results: GBM patients with SOH signature associated with poor prognosis and shorter survival. GBM TSs exhibited various phosphorylation states of YAP and could be distinguished by its phosphorylation status. GBM TSs with reduced YAP phosphorylation, including TS15-88, and other GBM TSs with higher YAP phosphorylation, which includes TS13-64, showed aggressive or less-aggressive cancer phenotypes, respectively. The siRNA-mediated knockdown of YAP1 significantly suppressed proliferation of TSs, and the results were confirmed using VP. Knockdown of YAP1 not only attenuated the invasiveness and stemness of TSs. Conclusion: We showed that reduced phosphorylation of endogenous YAP contributes to GBM progression. Moreover, we found that GBM TSs can be classified into two groups based on their YAP phosphorylation. These associations emphasize the YAP signaling network as new therapeutic opportunities in diagnosing and treating GBM. Citation Format: Yoojung Oh, Eui Hyun Kim, Dongkyu Lee, Junseong Park, Ju Hyung Moon, Hyun Woo Park, Jong Hee Chang, Seok-Gu Kang. Classification of glioblastoma tumorsphere depending on the regulatory mechanisms of the Hippo pathway [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A26.

Following the publication of the above article and a Corrigendum published in July 2018, the authors have noted an additional error, associated with the presentation of Fig. 1C. Fig 1C showed that β‑N‑methylamino‑L‑alanine induces neuronal apoptotic cell death; however, an error was made in the compilation of this figure and an incorrect band image was selected for α‑actinin, the loading control panel for Fig. 1C. A corrected version of Fig. 1 is shown opposite, incorporating the correct α‑actinin protein bands in Fig. 1C. This change affects neither the interpretation of the data nor conclusions of this work. We regret that this further error went unnoticed at the time, and thank the Editor for allowing us the opportunity to publish this additional Corrigendum. [the original article was published in the Molecular Medicine Reports 14: 4873‑4880, 2016; DOI: 10.3892/mmr.2016.5802].

β-N-methylamino-L-alanine (BMAA) is a neurotoxin that is closely associated with the incidence of amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease. In cultured neuronal cells, BMAA notably induces the upregulation of endoplasmic reticulum (ER) chaperons and activates the unfolded protein response (UPR) receptor pathways of protein kinase RNA‑like endoplasmic reticulum kinase, inositol‑requiring kinase 1 and transcription factor 6. The ER stress‑specific protein CCAAT/‑enhancer‑binding protein homologous protein (CHOP) affords pro‑apoptotic responses that cause mitochondrial damage and caspase activation. BMAA also induces the activation of mitogen‑activated protein kinase member c‑JUN N‑terminal kinase, p38 and extracellular signal‑regulated kinase, which have been suggested to be involved in the signaling pathway of UPR‑mediated apoptosis. Inhibition of ER stress using ER stress antagonist, salubrinal, attenuated the expression of CHOP and alleviated neuronal death. Overexpression of heat shock protein 70 suppressed the activation of UPR receptors and UPR‑evoked apoptotic signaling. The present findings demonstrated that ER stress induced by BMAA is the important mediator of neuronal injury and apoptotic death, and suggests development in novel therapeutic strategies for treatment.

Abstract Previously, the authors have identified that the acquired drug resistance to BRAF inhibitor, PLX4032 in BRAF (V600E) mutant anaplastic thyroid cancer promotes not only tumor progression and proliferation, but also migration and invasion of cancer through upregulated epithelial-to-mesenchymal transition (EMT). The underlying mechanism to the acquired resistance to BRAF inhibition involves c-Met-mediated reactivation of PI3K/AKT pathway. Therefore combinatorial dual targeted therapy of BRAF and c-Met inhibition has shown to reverse EMT and show maximal antitumor effect. Previously, the authors have developed a novel in vivo imaging strategy using CD44-targetable near-infrared (NIR)-sensitive supramolecular hydrogels (NIRSHs) for the recognition of CD44-expressing cancer cells. In the present study, we applied this NIR-sensitive molecular imaging probe in detecting the upregulated EMT changes in PLX4032-treated 8505C cells. The CD44-targetable NIRSHs were fabricated by polyplexing Cy5.5-conjugated polyethyleimine and hyaluronic acid in an aqueous medium. Ectopic xenograft mouse models were prepared by injecting 8505C cells at the flank of male athymic nude BALB/c mice, aged 6 weeks. After confirming tumor formation at 3 weeks post-injection, the mice were randomly divided into four groups and were each treated under different conditions; DMSO, PLX4032, PHA665752, PLX4032 and PHA665752. After 3 weeks, the pre-established NIRSH probes were injected and confirmed by IVIS imaging. The injected NIRSH probes showed highest uptake in the PLX4032 single treatment group and lowest uptake in the PLX4032 and PHA665752 combination group. Sizes of tumor were verified by MRI which showed correlations with the NIRSH fluorescence imagings. The results suggest that CD44-targetable NIRSHs imaging shows potential as a non-invasive in vivo imaging tool in detecting the increased invasion potential of cancer cells and monitoring appropriate therapeutic effects. Citation Format: Hyung Kwon Byeon, Minhee Ku, Yeon Ju Yang, Min Hee Cho, Yoojung Oh, Jae Wook Kim, Myung Jin Ban, Ji-Hoon Kim, Da Hee Kim, Joo Hyun Kim, Jaemoon Yang, Yoon Woo Koh. CD44-specific supramolecular hydrogels for fluorescence molecular imaging of EMT induced BRAF mutant thyroid cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 881. doi:10.1158/1538-7445.AM2017-881