Supplementary materials and methods, supplementary tables, and supplementary figures to support the main manuscript

List of differentially expressed genes from RNA-seq

Glioma is the most common primary central nervous system tumor. The World Health Organization (WHO) 2021 classification of gliomas extended from WHO 2016 in the integration of molecular and histological classifications. WHO 2021 aims to establish a specific diagnosis that could reflect the biological and clinical features of the tumors, and further transform into more specific clinical studies or trials and therapeutics. Major updates of WHO 2021 classification focusing on glioma were reviewed in this article.

The brain is the center of the nervous system controlling other organs. The brain is characterized by extreme complexity of its structure and delicate texture, which makes it difficult to investigate by invasive methods. The advances in semiconductor manufacturing technology led to the development of implantable microelectromechanical system (MEMS)-based devices that can be implanted into the brain with reduced risk of damage to the brain tissue due to their small size and low stiffness. Such devices are now widely applied in the clinic and brain research for understanding brain functions, diagnosing brain-related diseases, and pursuing innovative therapies. Inevitably, these invasive devices cause damage to the brain tissue, such as wounds, inflammation, and scars, eventually leading to dysfunction of the device. Therefore, it is vital to find an optimal design solution for such devices taking into consideration such parameters as size, stiffness and biocompatibility. In this review, we focus on three main brain applications of such devices: detection of electrical signals, intracranial pressure (ICP), and optogenetics. We summarize the current application scope of such MEMS-based devices in brain research and clinical application, and compare them based on their mechanical and biological properties. The understanding of the device design, methods of application, and current development can support neuroscientists and neurologists to make revolutionary discoveries in the brain research field.

Abstract Lung cancers are the leading cause of cancer-related mortality worldwide, and the majority of lung cancers are non–small cell lung carcinoma (NSCLC). Overexpressed or activated EGFR has been associated with a poor prognosis in NSCLC. We previously identified a circular noncoding RNA, hsa_circ_0000190 (C190), as a negative prognostic biomarker of lung cancer. Here, we attempted to dissect the mechanistic function of C190 and test the potential of C190 as a therapeutic target in NSCLC. C190 was upregulated in both NSCLC clinical samples and cell lines. Activation of the EGFR pathway increased C190 expression through a MAPK/ERK-dependent mechanism. Transient and stable overexpression of C190 induced ERK1/2 phosphorylation, proliferation, and migration in vitro and xenograft tumor growth in vivo. RNA sequencing and Expression2Kinases (X2K) analysis indicated that kinases associated with cell-cycle and global translation are involved in C190-activated networks, including CDKs and p70S6K, which were further validated by immunoblotting. CRISPR/Cas13a-mediated knockdown of C190 decreased proliferation and migration of NSCLC cells in vitro and suppressed tumor growth in vivo. TargetScan and CircInteractome databases predicted that C190 targets CDKs by sponging miR-142-5p. Analysis of clinical lung cancer samples showed that C190, CDK1, and CDK6 expressions were significantly higher in advanced-stage lung cancer than in early-stage lung cancer. In summary, C190 is directly involved in EGFR–MAPK–ERK signaling and may serve as a potential therapeutic target for the treatment of NSCLC. Significance: The circRNA C190 is identified as a mediator of multiple pro-oncogenic signaling pathways in lung cancer and can be targeted to suppress tumor progression.

Peptide drugs that target protein–protein interactions have attracted mounting research efforts towards clinical developments over the past decades. Increasing reports have indicated that expression of Musashi 1 (MSI1) is tightly correlated to high grade of cancers as well as enrichment of cancer stem cells. Treatment failure in malignant tumors glioblastoma multiform (GBM) had also been correlated to CSC-regulating properties of MSI1. It is thus imperative to develop new therapeutics that could effectively improve current regimens used in clinics. MSI1 and AGO2 are two emerging oncogenic molecules that both contribute to GBM tumorigenesis through mRNA regulation of targets involved in apoptosis and cell cycle. In this study, we designed peptide arrays covering the C-terminus of MSI1 and identified two peptides (Pep#11 and Pep#26) that could specifically interfere with the binding with AGO2. Our Biacore analyses ascertained binding between the identified peptides and AGO2. Recombinant reporter system Gaussian luciferase and fluorescent bioconjugate techniques were employed to determine biological functions and pharmacokinetic characteristics of these two peptides. Our data suggested that Pep#11 and Pep#26 could function as decoy peptides by mimicking the interaction function of MSI1 with its binding partner AGO2 in vitro and in vivo. Further experiments using GMB animal models corroborated the ability of Pep#11 and Pep#26 in disrupting MSI1/AGO2 interaction and consequently anti-tumorigenicity and prolonged survival rates. These striking therapeutic efficacies orchestrated by the synthetic peptides were attributed to the decoy function to C-terminal MSI1, especially in malignant brain tumors and glioblastoma.

Glioblastoma (GBM) is the most malignant brain tumor which is characterized by high proliferation and migration capacity. The poor survival rate has been attributed to limitations of the current standard therapies. The search for novel biological targets that can effectively hamper tumor progression remains extremely challenging. Previous studies indicated that tumor-associated macrophages (TAMs) are the abundant elements in the tumor microenvironment that are closely implicated in glioma progression and tumor pathogenesis. M2 type TAMs are immunosuppressive and promote GBM proliferation. RNA-binding protein Musashi-1 (MSI1) has recently been identified as a marker of neural stem/progenitor cells, and its high expression has been shown to correlate with the growth of GBM. Nevertheless, the relationship between MSI1 and TAMs in GBM is still unknown. Thus, in our present study, we aimed to investigate the molecular interplay between MSI1 and TAMs in contributing to GBM tumorigenesis. Our data revealed that the secretion of macrophage inhibitory factor 1 (MIF1) is significantly upregulated by MSI1 overexpression in vitro. Importantly, M2 surface markers of THP-1-derived macrophages were induced by recombinant MIF1 and reduced by using MIF1 inhibitor (S,R)-3-(4-hHydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1). Furthermore, GBM tumor model data suggested that the tumor growth, MIF1 expression and M2 macrophage population were significantly downregulated when MSI1 expression was silenced in vivo. Collectively, our findings identified a novel role of MSI1 in the secretion of MIF1 and the consequent polarization of macrophages into the M2 phenotype in promoting GBM tumor progression.

Lung cancers (LC) are the leading cause of cancer-related mortality worldwide, and the majority of LC are non-small cell lung carcinoma (NSCLC). Overexpressed or activated EGFR has been associated with a poor prognosis in NSCLC. We previously identified a circular non-coding RNA, hsa_circ_0000190 (C190), as a negative prognostic biomarker of LC. Here we attempted to dissect the mechanistic function of C190 and test the potential of C190 as a therapeutic target in NSCLC. C190 was upregulated in both NSCLC clinical samples and cell lines. Activation of the EGFR pathway increased C190 expression through a MAPK/ERK-dependent mechanism. Transient and stable overexpression of C190 induced ERK1/2 phosphorylation, proliferation, and migration in vitro and xenograft tumor growth in vivo. RNA sequencing and Expression2Kinases (X2K) analysis indicated that kinases associated with cell cycle and global translation are involved in C190-activated networks, including CDKs and p70S6K, which were further validated by immunoblotting. CRISPR/Cas13a-mediated knockdown of C190 decreased proliferation and migration of NSCLC cells in vitro and suppressed tumor growth in vivo. TargetScan and CircInteractome databases predicted that C190 targets CDKs by sponging miR-142-5p. Analysis of clinical LC samples showed that C190, CDK1, and CDK6 expression were significantly higher in advanced-stage LC than in early-stage LC. In summary, C190 is directly involved in EGFR-MAPK-ERK signaling and may serve as a potential therapeutic target for the treatment of NSCLC.

Glioblastoma (GBM) is one of the most devastating cancers, with an approximate median survival of only 16 months. Although some new insights into the fantastic heterogeneity of this kind of brain tumor have been revealed in recent studies, all subclasses of GBM still demonstrate highly aggressive invasion properties to the surrounding parenchyma. This behavior has become the main obstruction to current curative therapies as invasive GBM cells migrate away from these foci after surgical therapies. Therefore, this review aimed to provide a relatively comprehensive study of GBM invasion mechanisms, which contains an intricate network of interactions and signaling pathways with the extracellular matrix (ECM). Among these related molecules, TGF-β, the ECM, Akt, and microRNAs are most significant in terms of cellular procedures related to GBM motility and invasion. Moreover, we also review data indicating that Musashi-1 (MSI1), a neural RNA-binding protein (RBP), regulates GBM motility and invasion, maintains stem cell populations in GBM, and promotes drug-resistant GBM phenotypes by stimulating necessary oncogenic signaling pathways through binding and regulating mRNA stability. Importantly, these necessary oncogenic signaling pathways have a close connection with TGF-β, ECM, and Akt. Thus, it appears promising to find MSI-specific inhibitors or RNA interference-based treatments to prevent the actions of these molecules despite using RBPs, which are known as hard therapeutic targets. In summary, this review aims to provide a better understanding of these signaling pathways to help in developing novel therapeutic approaches with better outcomes in preclinical studies.

Lung cancer (LC) causes the majority of cancer-related deaths. Circular RNAs (circRNAs) were reported to play roles in cancers by targeting pro- and anti-oncogenic miRNAs. However, the mechanisms of circRNAs in LC progression and their prognostic value of treatment response remain unclear. By using next generation sequencing (NGS) of LC cell lines' transcriptomes, we identified highly overexpressed hsa_circ_0000190 and hsa_circ_000164 as potential biomarkers. By using the highly sensitive RT-ddPCR method, these circRNAs were shown to be secreted by cell lines and were detected in human blood. Clinical validation by RT-ddPCR was carried out on 272 (231 LC patients and 41 controls) blood samples. Higher hsa_circ_0000190 levels were associated with larger tumor size (p < 0.0001), worse histological type of adenocarcinoma (p = 0.0028), later stage (p < 0.0001), more distant metastatic organs (p = 0.0039), extrathoracic metastasis (p = 0.0004), and poor survival (p = 0.047) and prognosis. Using liquid biopsy-based RT-ddPCR, we discovered the correlation between increased hsa_circ_0000190 plasma level (p < 0.0001) and higher programmed death-ligand 1 (PD-L1) level in tumor (p = 0.0283). Notably, long-term follow-up of the immunotherapy treated cases showed that upregulated plasma hsa_circ_0000190 level correlated with poor response to systemic therapy and immunotherapy (p = 0.0002, 0.0058, respectively). Secretory circRNAs are detectable in blood by LB-based RT-ddPCR and may serve as blood-based biomarkers to monitor disease progression and treatment efficacy.

Carcinomatous progression and recurrence are the main therapeutic challenges frequently faced by patients with refractory tumors. However, the underlined molecular mechanism remains obscure. Methods: We found Musashi-1 (MSI1) transported into cytosol under stress condition by confocal microscopy and cell fractionation. Argonaute 2 (AGO2) was then identified as a cytosolic binding partner of MSI1 by Mass Spectrametry, immunoprecipitation, and recombinant protein pull-down assay. We used RNA-IP to determine the MSI1/AGO2 associated regions on downstream target mRNAs. Finally, we overexpressed C-terminus of MSI1 to disrupt endogenous MSI1/AGO2 interaction and confirm it effects on tmor progression. Results: Malignant tumors exhibit elevated level of cytosolic Musashi-1 (MSI1), which translocates into cytosol in response to stress and promote tumor progression. Cytosolic MSI1 forms a complex with AGO2 and stabilize or destabilize its target mRNAs by respectively binding to their 3´ untranslated region or coding domain sequence. Both MSI1 translocation and MSI1/AGO2 binding are essential for promoting tumor progression. Blocking MSI1 shuttling by either chemical inhibition or point mutation attenuates the growth of GBM-xenografts in mice. Importantly, overexpression of the C-terminus of MSI1 disrupts endogenous MSI1/AGO2 interaction and effectively reduces stress-induced tumor progression. Conclusion: Our findings highlight novel molecular functions of MSI1 during stress-induced carcinomatous recurrence, and suggest a new therapeutic strategy for refractory malignancies by targeting MSI1 translocation and its interaction with AGOs.

Atypical teratoid/rhabdoid tumor (ATRT) is a rare pediatric brain tumor with extremely high aggressiveness and poor prognosis. The tumor microenvironment is regulated by a complex interaction among distinct cell types, yet the crosstalk between tumor-associated mesenchymal stem cells (tMSCs) and naïve ATRT cells are unclear. In this study, we sought to identify the secretory factor(s) that is responsible for the tMSC-mediated regulation of ATRT migration. Comparing with ATRT cell alone, co-culture of tMSCs or addition of its conditioned medium (tMSC-CM) promoted the migration of ATRT, and this effect could be abrogated by exosome release inhibitor GW4869. The exosomes in tMSC-CM were detected by transmission electron microscope and flow cytometry. ATRT naïve cell-derived conditioned media (ATRT-CM) also enhanced the exosome secretion from tMSCs, indicating the interplay between ATRT cells and tMSCs. Microarray analysis revealed that, compared with that in bone marrow-derived MSCs, microRNA155 is the most upregulated microRNA in the tMSC-CM. Tracing the PK67-labeled exosomes secreted from tMSCs confirmed their incorporation into naïve ATRT cells. After entering ATRT cells, miR155 promoted ATRT cell migration by directly targeting SMARCA4. Knockdown of SMARCA4 mimicked the miR155-driven ATRT cell migration, whereas SMARCA4 overexpression or the delivery of exosomes with miR155 knockdown suppressed the migration. Furthermore, abrogation of exosome release with GW4869 reduced the tumorigenesis of the xenograft containing naïve ATRT cells and tMSCs in immunocompromised recipients. In conclusion, our data have demonstrated that tMSCs secreted miR155-enriched exosomes, and the exosome incorporation and miR155 delivery further promoted migration in ATRT cells via a SMARCA4-dependent mechanism.

Background: Glioblastoma multiforme (GBM) is the most malignant brain tumor, and there is no effective treatment strategy. Patients with GBM have a median overall survival of only 14.6 months. Current treatment consists of safe and maximal surgical excision, followed by concurrent chemoradiotherapy and maintenance chemotherapy. There are several obstacles that hinder the effectiveness of this aggressive treatment. Temozolomide (TMZ) is an oral alkylating drug that acts through alkylating the O-6 position of guanine in DNA that leads to cell death. However, the expression and enzymatic activity of the DNA repair protein MGMT limits the therapeutic benefit from treatment with TMZ. MGMT reduces the efficacy of alkylating drugs by removing the methyl or alkyl group from damaged O-6-methylguanine. Expression levels of MGMT play an important role in the outcome of GBM patients. miRNAs are a group of small regulatory RNAs that control target gene expression by binding to mRNAs. miR-142-3p has been found to be an important factor in the development and maintenance of the oncogenic state. Results: In this study, we sought to investigate whether miR-142-3p can regulate MGMT gene expression in GBM cells. Here, we show that miR-142-3p downregulates MGMT expression through binding to the 3'-UTR of MGMT mRNA, thus affecting protein translation. Responsiveness to TMZ was significantly enhanced after transfection with miR-142-3p. Overexpression of miR-142-3p also sensitized GBM cells to alkylating drugs. Conclusion: Above all, our findings demonstrate that miR-142-3p plays a critical role in regulating MGMT expression, has great potential for future clinical applications, and acts as a new diagnostic marker for this intractable disease.

Musashi-1 (MSI1), one of the RNA-binding proteins, is abundantly found not only in neural stem cells but also in several cancer tissues and has been reported to act as a positive regulator of cancer progression. Growing evidence indicates that PKR and eIF2α play pivotal roles in the stimulation of stress granule formation as well as in the subsequent translation modulation in response to stressful conditions; however, little is known about whether MSI1 is involved in this PKR/eIF2α cancer stem cell-enhancing machinery. In this study, we demonstrated that MSI1 promotes human glioblastoma multiforme (GBM) stem cells and enhances chemoresistance when exposed to sublethal stress. The overexpression of MSI1 leads to a protective effect in mitigating drug-induced cell death, thus facilitating the formation of chemoresistant stress granules (SGs) in response to arsenic trioxide (ATO) treatment. SG components, such as PKR and eIF2α, were dominantly activated and assembled, while ATO was engaged. The activated PKR and eIF2α contribute to the downstream enhancement of stem cell genes, thereby promoting the progression of GBM. The silencing of MSI1 or PKR both obviously withdrew the phenomena. Taken together, our findings indicate that MSI1 plays a leading role in stress granule formation that grants cancer stem cell properties and chemoresistant stress granules in GBM, in response to stressful conditions via the PKR/eIF2α signalling cascade.

Glioblastoma Multiforme (GBM) is a lethal primary brain tumor with poor survival lifespan and dismal outcome. Surgical resection of GBM is greatly limited due to the biological significance of brain, giving rise to tumor relapse in GBM patients. Transactive response DNA binding protein-43 (TDP-43) is a DNA/RNA-binding protein known for causing neurodegenerative diseases through post-translational modification; but little is known about its involvement in cancer development. In this study, we found that nutrient deprivation in GBM cell lines elevated TDP-43 expression by a mechanism of evasion from ubiquitin-dependent proteolytic pathway, and subsequently activated the autophagy process. Exogenous overexpression of TDP-43 consistently activated autophagy and suppressed stress-induced apoptosis. The inhibition of autophagy in TDP-43-overexpressing cells effectively increased the apoptotic population under nutrition shortage. Furthermore, we demonstrated that HDAC6 was involved in the activation of autophagy in TDP-43-overexpressing GBM cell lines. The treatment with SAHA, a universal HDAC inhibitor, significantly reduced TDP-43-mediated anti-apoptotic effect. Additionally, the results of immunohistochemistry showed that TDP-43 and HDAC6 collaborated in GBM-tumor lesions and negatively correlated with the relapse-free survival of GBM patients. Taken together, our results suggest that the TDP-43-HDAC6 signaling axis functions as a stress responsive pathway in GBM tumorigenesis and combats nutrient deprivation stress via activating autophagy, while inhibition of HDAC6 overpowers the pathway and provides a novel therapeutic strategy against GBM.

Background: Glioblastoma multiforme (GBM) is the most lethal type of adult brain cancer and performs outrageous growth and resistance regardless of adjuvant chemotherapies, eventually contributing to tumor recurrence and poor outcomes. Considering the common heterogeneity of cancer cells, the imbalanced regulatory mechanism could be switched on/off and contribute to drug resistance. Moreover, the subpopulation of GBM cells was recently discovered to share similar phenotypes with neural stem cells. These cancer stem cells (CSCs) promote the potency of tumor initiation. As a result, targeting of glioma stem cells has become the dominant way of improving the therapeutic outcome against GBM and extending the life span of patients. Among the biomarkers of CSCs, CD-133 (prominin-1) has been known to effectively isolate CSCs from cancer population, including GBM; however, the underlying mechanism of how stemness genes manipulate CSC-associated phenotypes, such as tumor initiation and relapse, is still unclear.Methods: Tumorigenicity, drug resistance and embryonic stem cell markers were examined in primary CD133-positive (CD133(+)) GBM cells and CD133(+) subpopulation. Sternness signature of CD133(+) GBM cells was identified using microarray analysis. Stem cell potency, tumorigenicity and drug resistance were also tested in differential expression of SOX2 in GBM cells.Results: In this study, high tumorigenic and drug resistance was noticed in primary CD-133(+) GBM cells; meanwhile, plenty of embryonic stem cell markers were also elevated in the CD-133(+) subpopulation. Using microarray analysis, we identified SOX2 as the most enriched gene among the stemness signature in CD133(+) GBM cells. Overexpression of SOX2 consistently enhanced the stem cell potency in the GBM cell lines, whereas knockdown of SOX2 dramatically withdrew CD133 expression in CD133(+) GBM cells. Additionally, we silenced SOX2 expression using RNAi system, which abrogated the ability of tumor initiation as well as drug resistance of CD133(+) GBM cells, suggesting that SOX2 plays a crucial role in regulating tumorigenicity in CD133(+) GBM cells.Conclusion: SOX2 plays a crucial role in regulating tumorigenicity in CD133(+) GBM cells. Our results not only revealed the genetic plasticity contributing to drug resistance and sternness but also demonstrated the dominant role of SOX2 in maintenance of GBM CSCs, which may provide a novel therapeutic target to overcome the conundrum of poor survival of brain cancers. Copyright (C) 2016, the Chinese Medical Association. Published by Elsevier Taiwan LLC.

BACKGROUND:Atypical and anaplastic meningiomas have much higher recurrence rates after surgical resection compared with benign meningiomas, but the role of adjuvant radiosurgery remains unclear. This study was undertaken to evaluate the outcomes of gamma knife radiosurgery for patients with atypical and anaplastic meningiomas.METHODS:In this retrospective analysis of a prospectively maintained database, 46 patients with histologically proven atypical or anaplastic meningiomas by current World Health Organization (WHO) criteria underwent postoperative Gamma Knife radiosurgery between 1993 and 2013. The median follow-up period was 32.6 months. The median tumor volume and margin dose were 11.7 mL (range, 2-53 mL) and 13.1 Gy (range, 12.0-16.5 Gy), respectively.RESULTS:Local control at 3 and 5 years was 50.6% and 32.1%, respectively. Gender (P = 0.013) and marginal dose less than or equal to 13 Gy (P = 0.049) were associated with the local control. The 3- and 5-year overall survival for patients with WHO grade II was 97.1% and 88.3%, respectively, compared with 66.7% and 66.7% for patients with WHO grade III meningiomas. Radiation therapy before Gamma Knife radiosurgery (GKRS; P = 0.018) and tumor grade (P = 0.019) were the factors associated with a worse overall survival rate. Fourteen patients (30.4%) developed adverse radiation effects after GKRS treatment, and all were Radiation Therapy Oncology Group grade I.CONCLUSIONS:Postoperative GKRS treatment for patients with atypical and anaplastic meningioma is challenging. More aggressive treatment, including of safely maximizing the extent of surgical resection and using a higher margin dose (>13Gy), should be applied to achieve better local control.

Glioblastoma multiform (GBM) is one of the most lethal human malignant brain tumors with high risks of recurrence and poor treatment outcomes. The RNA-binding protein Musashi-1 (MSI1) is a marker of neural stem/progenitor cells. Recent study showed that high expression level of MSI1 positively correlates with advanced grade of GBM, where MSI1 increases the growth of GBM. Herein, we explore the roles of MSI1 as well as the underlying mechanisms in the regulation of drug resistance and tumorigenesis of GBM cells. Our results demonstrated that overexpression of MSI1 effectively protected GBM cells from drug-induced apoptosis through down-regulating pro-apoptotic genes; whereas inhibition of AKT withdrew the MSI1-induced anti-apoptosis and cell survival. We further showed that MSI1 robustly promoted the secretion of the pro-inflammatory cytokine IL-6, which was governed by AKT activity. Autonomously, the secreted IL-6 enhanced AKT activity in an autocrine/paracrine manner, forming a positive feedback regulatory loop with the MSI1-AKT pathway. Our results conclusively demonstrated a novel drug resistance mechanism in GBM cells that MSI1 inhibits drug-induced apoptosis through AKT/IL6 regulatory circuit. MSI1 regulates both cellular signaling and tumor-microenvironmental cytokine secretion to create an intra- and intercellular niche for GBM to survive from chemo-drug attack.

Intracranial germ-cell tumors (GCTs) represent 10-15% of all pediatric brain tumors in East Asia. There is a wide histopathological spectrum of intracranial GCTs. Germinomas and nongerminomatous GCTs are the two major classifications. It is difficult to distinguish different subtypes of intracranial GCTs based solely on imaging studies, however, some tumor markers, such as α-fetoprotein or β-human chorionic gonadotropin, are helpful for diagnosis. In this study we present the case of a 13-year-old girl with an intracranial mixed GCT containing a hepatocellular carcinoma and germinoma without a primary liver tumor. Based on this unique pathological diagnosis, a series of treatments were applied, including surgery for gross tumor removal, adjuvant radiotherapy, and chemotherapy. Long-term follow up indicates fair disease control.