Glioblastoma Multiforme Samples Research Articles

CD2AP promotes the progression of glioblastoma multiforme via TRIM5-mediated NF-kB signaling

CD2-associated protein (CD2AP) is a scaffolding/adaptive protein that regulates intercellular adhesion and multiple signaling pathways. Although emerging evidence suggests that CD2AP is associated with several malignant tumors, there is no study investigating the expression and biological significance of CD2AP in glioblastoma multiforme (GBM). Here by studying public datasets, we found that CD2AP expression was significantly elevated in GBM and that glioma patients with increased CD2AP expression had a worse prognosis. We also confirmed the increase of CD2AP expression in clinical GBM samples and GBM cell lines. CD2AP overexpression in GBM cells promoted their proliferation, colony formation, migration, and invasion in vitro and their tumorigenesis in vivo, and reduced cell apoptosis both at basal levels and in response to temozolomide. While CD2AP knockdown had the opposite effects. Mechanistically, we revealed that CD2AP interacted with TRIM5, an NF-κB modulator. CD2AP overexpression and knockdown increased and decreased TRIM5 levels as well as the NF-κB activity, respectively. Moreover, downregulation of TRIM5 reversed elevated NF-κB activity in GBM cells with CD2AP overexpression; and inhibition of the NF-κB activity attenuated malignant features of GBM cells with CD2AP overexpression. Our findings demonstrate that CD2AP promotes GBM progression through activating TRIM5-mediated NF-κB signaling and that downregulation of CD2AP can attenuate GBM malignancy, suggesting that CD2AP may become a biomarker and the CD2AP-TRIM5-NF-κB axis may become a therapeutic target for GBM.

Involvement of oncomiRs miR-23, miR-24, and miR-27 in the regulation of alternative polyadenylation in glioblastoma via CFIm25 cleavage factor.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. The cleavage factor Im 25 (CFIm25), a crucial component of the CFIm complex, plays a key role in regulating the length of the mRNA 3'-UTR and has been implicated in various cancers, including GBM. This study sought to investigate the regulatory influence of specific microRNAs (miRNAs) on CFIm25 expression in GBM, a highly aggressive brain tumor. Bioinformatics analysis identified miRNA candidates targeting CFIm25 mRNA, and gene expression profiles from the NCBI database (GSE90603) were used for further analysis. Expression levels of CFIm25 and selected miRNAs were assessed using qRT-PCR in GBM clinical samples (n = 20) and non-malignant brain tissues (n = 5). Additionally, the MTT assay was performed to examine the effect of miRNA overexpression on U251 cell viability. Lentivectors expressing the identified miRNAs were employed to experimentally validate their regulatory role on CFIm25 in U251 cell lines, and Western blot analysis was conducted to determine CFIm25 protein levels. We observed significantly increased levels of miR-23, miR-24, and miR-27 expression, associated with a marked reduction in CFIm25 expression in GBM samples compared to non-malignant brain tissues. In particular, overexpression of miR-23, miR-24, and miR-27 in U251 cells resulted in CFIm25 downregulation at both the mRNA and protein levels, while their inhibition increased CFIm25 and reduced cell proliferation. These observations strongly implicate miR-23, miR-24, and miR-27 in regulating CFIm25 expression in GBM, emphasizing their potential as promising therapeutic targets for enhancing treatment responses in glioblastoma.

The role of ALDH1A1 in glioblastoma proliferation and invasion

High-grade gliomas, including glioblastoma multiforme (GBM), continue to be a leading aggressive brain tumor in adults, marked by its rapid growth and invasive nature. Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), an enzyme, plays a significant role in tumor progression, yet its function in high-grade gliomas is still poorly investigated. In this study, we evaluated ALDH1A1 levels in clinical samples of GBM. We also assessed the prognostic significance of ALDH1A1 expression in GBM and LGG (low grade glioma) patients using TCGA (The Cancer Genome Atlas) database analysis. The MTT and transwell assays were utilized to examine cell growth and the invasive capability of U87 cells, respectively. We quantitatively examined markers for cell proliferation (Ki-67 and cyclin D1) and invasion (MMP2 and 9). A Western blot test was conducted to determine the downstream signaling of ALDH1A1. We found a notable increase in ALDH1A1 expression in high-grade gliomas compared to their low-grade counterparts. U87 cells that overexpressed ALDH1A1 showed increased cell growth and invasion. We found that ALDH1A1 promotes the phosphorylation of AKT, and inhibiting AKT phosphorylation mitigates the ALDH1A1's effects on tumor growth and migration. In summary, our findings suggest ALDH1A1 as a potential therapeutic target for GBM treatment.

Expression of the apelin receptor, a novel potential therapeutic target, and its endogenous ligands in diverse stem cell populations in human glioblastoma.

Glioblastoma multiforme (GBM) is one of the most common and lethal forms of brain cancer, carrying a very poor prognosis (median survival of ~15 months post-diagnosis). Treatment typically involves invasive surgical resection of the tumour mass, followed by radiotherapy and adjuvant chemotherapy using the alkylating agent temozolomide, but over half of patients do not respond to this drug and considerable resistance is observed. Tumour heterogeneity is the main cause of therapeutic failure, where diverse progenitor glioblastoma stem cell (GSC) lineages in the microenvironment drive tumour recurrence and therapeutic resistance. The apelin receptor is a class A GPCR that binds two endogenous peptide ligands, apelin and ELA, and plays a role in the proliferation and survival of cancer cells. Here, we used quantitative whole slide immunofluorescent imaging of human GBM samples to characterise expression of the apelin receptor and both its ligands in the distinct GSC lineages, namely neural-progenitor-like cells (NPCs), oligodendrocyte-progenitor-like cells (OPCs), and mesenchymal-like cells (MES), as well as reactive astrocytic cells. The data confirm the presence of the apelin receptor as a tractable drug target that is common across the key cell populations driving tumour growth and maintenance, offering a potential novel therapeutic approach for patients with GBM.

Genetic Optimization in Uncovering Biologically Meaningful Gene Biomarkers for Glioblastoma Subtypes

Background: Glioblastoma multiforme (GBM) is a highly aggressive brain cancer known for its challenging survival rates; it is characterized by distinct subtypes, such as the proneural and mesenchymal states. The development of targeted therapies is critically dependent on a thorough understanding of these subtypes. Advances in single-cell RNA-sequencing (scRNA-seq) have opened new avenues for identifying subtype-specific gene biomarkers, which are essential for innovative treatments. Methods: This study introduces a genetic optimization algorithm designed to select a precise set of genes that clearly differentiate between the proneural and mesenchymal GBM subtypes. By integrating differential gene expression analysis with gene variability assessments, our dual-criterion strategy ensures the selection of genes that are not only differentially expressed between subtypes but also exhibit consistent variability patterns. This approach enhances the biological relevance of identified biomarkers. We applied this algorithm to scRNA-seq data from GBM samples, focusing on the discovery of subtype-specific gene biomarkers. Results: The application of our genetic optimization algorithm to scRNA-seq data successfully identified significant genes that are closely associated with the fundamental characteristics of GBM. These genes show a strong potential to distinguish between the proneural and mesenchymal subtypes, offering insights into the molecular underpinnings of GBM heterogeneity. Conclusions: This study introduces a novel approach for biomarker discovery in GBM that is potentially applicable to other complex diseases. By leveraging scRNA-seq data, our method contributes to the development of targeted therapies, highlighting the importance of precise biomarker identification in personalized medicine.

IFITM3 promotes glioblastoma stem cell-mediated angiogenesis via regulating JAK/STAT3/bFGF signaling pathway

Interferon-induced transmembrane protein 3 (IFITM3) has been previously verified to be an endosomal protein that prevents viral infection. Recent findings suggested IFITM3 as a key factor in tumor invasion and progression. To clarify the role and molecular mechanism of IFITM3 in Glioblastoma multiforme (GBM) progression, we investigated the expression of IFITM3 in glioma datasets culled from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA). Primary GBM stem cells (GSCs) were cultured and identified in vitro. Loss-of-function and gain-of-function experiments were established by using shRNAs and lentiviral vectors targeting IFITM3. Co-culture system of GSCs and vascular endothelial cells was constructed in a Transwell chamber. Tube formation and spheroid-based angiogenesis assays were performed to determine the angiogenic capacity of endothelial cells. Results revealed that IFITM3 is elevated in GBM samples and predictive of adverse outcome. Mechanistically, GSCs-derived IFITM3 causes activation of Jak2/STAT3 signaling and leads to robust secretion of bFGF into tumor environment, which eventually results in enhanced angiogenesis. Taken together, these evidence indicated IFITM3 as an essential factor in GBM angiogenesis. Our findings provide a new insight into mechanism by which IFITM3 modulates GBM angiogenesis.

LINC01366 and LINC01433 in Glioblastoma Multiforme: A Potential Role at the Intersection of Inflammation and Angiogenesis.

Glioblastoma multiforme (GBM) is an aggressive cancer with a poor prognosis. Inflammation and angiogenesis are important processes in GBM that are interrelated. In this study, bioinformatic investigations were performed to detect common and key genes in the inflammatory and angiogenesis pathways of GBM. Additionally, relevant long non-coding RNAs (lncRNAs) were recognized as important gene regulators. Consequently, real-time PCR and correlation analyses were used to investigate changes in gene and lncRNA expression levels and explain their relationship. RELA emerged as a common key gene in these biological processes. LINC01366 and LINC01433 were identified as putative RELA regulators in different metabolic pathways using computational assays. According to our findings, the expression levels of RELA, LINC01366 and LINC01433 were found to be significantly upregulated in GBM samples. Correlational studies revealed a significant positive relationship of gene expressions between LINC01366 and LINC01433, indicating that they may have a coordinated effect on GBM biology. Nevertheless, there was no significant correlation between these lncRNAs and RELA. The current study highlights the high expression of LINC01366 and LINC01433 in GBM and emphasizes the importance of studying lncRNAs as putative regulators in the pathophysiology of GBM. Further research is needed to clarify their specific functions, in particular the associated inflammatory and angiogenesis pathways.

Analysis of Connexin 43 and Spermine Co-localisation in Glioblastomas.

Gap junctions are channels between adjacent cells, contributing to the unhindered exchange of metabolites, second messengers, nucleotides, and other molecules. The functional status of gap junctions in brain tumours is underinvestigated. One avenue of research focuses on exploring the expression of polyamines and their co-localisation with the Connexin 43 (Cx43) in the growth zones of glioblastoma multiforme (GBM). The aim of this work was to analyse the expression of Cx43 and spermine in human GBM to reveal their roles in neuro-oncogenesis. Human GBM sample sections were used for the immunochemistry [glial fibrillary acidic protein (GFAP), Cx43, and spermine], confocal laser scanning microscopy, and electron immunohistochemistry. Immunofluorescent analysis revealed that the more extensive processes of GBM cells exhibit GFAP. All GBM samples (n=10) exhibited positive Cx43 signals in the form of variously sized dots and lines. Cx43 formed dotted lines around cell bodies with segmented transformed nuclei, which were also present in the gliovascular complexes. Furthermore, spermine was overexpressed in all tumour samples (cytoplasm and large and thin tumour processes), including the areas of Cx43 localisation. Merging the Cx43 and spermine signals showed co-expression in the same regions: the membranes of individual cells and individual points on processes in the tumour tissue. Therefore, we established the staining of the co-localisation of Cx43 and the polyamine spermine within glioblastoma, revealing that tumour processes housing the polyamine indeed form gap junctions, suggesting their potential joint interaction. This finding indicates that glioma cells can integrate into the surrounding neural networks, potentially serving as a mechanism to release glycolysis products, relying on gap junction activity facilitated by spermine. Cx43 exhibits sensitivity to polyamines, which play a role in opening gap junctional channels. Furthermore, polyamines have been observed to eliminate the blockades caused by hydrogen ions and calcium, which is crucial for cellular physiology.

RUNX1-PDIA5 Axis Promotes Malignant Progression of Glioblastoma by Regulating CCAR1 Protein Expression.

PDIA5 is responsible for modification of disulfide bonds of proteins. However, its impact on the malignant progression of glioblastoma multiforme (GBM) remains unknown. We analyzed the expression and prognostic significance of PDIA5 in cohorts of GBM and clinical samples. The PDIA5 protein was significantly overexpressed in GBM tissues, and higher expression of PDIA5 was statistically associated with a worse prognosis in patients with GBM. Transcriptional data from PDIA5 knockdown GBM cells revealed that downstream regulatory genes of PDIA5 were enriched in malignant regulatory pathways and PDIA5 enhanced the proliferative and invasive abilities of GBM cells. By constructing a PDIA5 CXXC motif mutant plasmid, we found CCAR1 was the vital downstream factor of PDIA5 in regulating GBM malignancy in vitro and in vivo. Additionally, RUNX1 bound to the promoter region of PDIA5 and regulated gene transcription, leading to activation of the PDIA5/CCAR1 regulatory axis in GBM. The RUNX1/PDIA5/CCAR1 axis significantly influenced the malignant behavior of GBM cells. In conclusion, this study comprehensively elucidates the crucial role of PDIA5 in the malignant progression of GBM. Downregulating PDIA5 can mitigate the malignant biological behavior of GBM both in vitro and in vivo, potentially improving the efficacy of treatment for clinical patients with GBM.

Development of a prognostic model for glioblastoma multiforme based on the expression levels of efferocytosis-related genes.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. The microenvironment of GBM is characterized by its highly immunosuppressive nature with infiltration of immunosuppressive cells and the expression levels of cytokines. Efferocytosis is a biological process in which phagocytes remove apoptotic cells and vesicles from tissues. Efferocytosis plays a noticeable function in the formation of immunosuppressive environment. This study aimed to develop an efferocytosis-related prognostic model for GBM. The bioinformatic methods were utilized to analyze the transcriptomic data of GBM and normal samples. Clinical and RNA-seq data were sourced from TCGA database comprising 167 tumor samples and 5 normal samples, and 167 tumor samples for which survival information was available. Transcriptomic data of 1034 normal samples were collected from the Genotype-Tissue Expression (GTEx) database as a control sample supplement to the TCGA database. In the end, 167 tumor samples and 1039 normal samples were obtained for transcriptome analysis. Efferocytosis-related differentially expressed genes (ERDEGs) were obtained by intersecting 7487 differentially expressed genes (DEGs) between GBM and normal samples along with 1189 hub genes. Functional enrichment analyses revealed that ERDEGs were mainly involved in cytokine-mediated immune responses. Moreover, 9 prognosis-related genes (PRGs) were identified by the least absolute shrinkage and selection operator (LASSO) regression analysis, and a prognostic model was therefore developed. The nomogram combining age and risk score could effectively predict GBM patients' prognosis. GBM patients in the high-risk group had higher immune infiltration, invasion, epithelial-mesenchymal transition, angiogenesis scores and poorer tumor purity. In addition, the high-risk group exhibited higher half maximal inhibitory concentration (IC50) values for temozolomide, carmustine, and vincristine. Expression analysis indicated that PRGs were overexpressed in GBM cells. PDIA4 knockdown reduced efferocytosis in vitro. In summary, the proposed prognostic model for GBM based on efferocytosis-related genes exhibited a robust performance.

Ultra High‐Plex Spatial Proteogenomic Investigation of Giant Cell Glioblastoma Multiforme Immune Infiltrates Reveals Distinct Protein and RNA Expression Profiles

AbstractBackgroundThe advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin‐fixed paraffin‐embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post‐translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”.MethodHere we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high‐plex digital quantitation of proteins (147‐plex) and RNA (whole transcriptome,>18,000‐plex) from a single FFPE sample. We demonstrated high concordance, R>0.85, and minor change in sensitivity (<11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies.ResultsWe observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in‐depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post‐translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods.ConclusionsWithin our dataset, we observed >2‐fold higher protein expression levels of phospho‐GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho‐Thr231 Tau was >2‐fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology.

LncRNA-associated competing endogenous RNA network analysis uncovered key lncRNAs involved in temozolomide resistance and tumor recurrence of glioblastoma.

Temozolomide (TMZ) is a common alkylating chemotherapeutic agent used to treat brain tumors such as glioblastoma multiforme (GBM) and anaplastic astrocytoma. GBM patients develop resistance to this drug, which has an unclear and complicated molecular mechanism. The competing endogenous RNAs (ceRNAs) play critical roles in tumorigenesis, drug resistance, and tumor recurrence in cancers. This study aims to predict ceRNAs, their possible involvement, and underlying molecular mechanisms in TMZ resistance. Therefore, we analyzed coding and non-coding RNA expression levels in TMZ-resistant GBM samples compared to sensitive GBM samples and performed pathway analysis of mRNAs differentially expressed (DE) in TMZ-resistant samples. We next applied a mathematical model on 950 DE long non-coding RNAs (lncRNAs), 116 microRNAs (miRNAs), and 7977 mRNAs and obtained 10 lncRNA-associated ceRNAs that may be regulating potential target genes involved in cancer-related pathways by sponging 25 miRNAs in TMZ-resistant GBM. Among these, two lncRNAs named ARFRP1 and RUSC2 regulate five target genes (IRS1, FOXG1, GNG2, RUNX2, and CACNA1E) involved in AMPK, AKT, mTOR, and TGF-β signaling pathways that activate or inhibit autophagy causing TMZ resistance. The novel lncRNA-associated ceRNA network predicted in GBM offers a fresh viewpoint on TMZ resistance, which might contribute to treating this malignancy.

HDAC1 mediates epithelial-mesenchymal transition and promotes cancer cell invasion in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most malignant tumors of the central nervous system, and its treatment has always been a difficult clinical problem. Here, we evaluated HDAC1 expression patterns and their effect on prognosis based on GBM cases from TCGA and CGGA databases. Expression was compared between GBM samples and normal controls. High HDAC1 expression was found to be an indicator of poor prognosis in glioblastoma. We also established a protein-protein interaction network to explore HDAC1-related interacting proteins, including the epithelial-mesenchymal transition (EMT)-related protein VIM, which is closely associated with HDAC1. Consistently, functional enrichment analysis showed that several GBM tissues with high HDAC1 were enriched in the expression of cancer markers, such as those involved in glycolysis, hypoxia, inflammation, and some signaling pathways. Next, this study analyzed the effect of HDAC1 on invasive ability and the EMT signaling pathway in GBM cells in vitro. The results showed that an HDAC1 inhibitor (RGFP109) could inhibit the EMT process in glioma cells in vitro, thereby affecting the invasion and migration of cells. Similar results were obtained based on in vivo studies. Our data suggest that HDAC1 has the potential to be a powerful prognostic biomarker, which might provide a basis for developing therapeutic targets for GBM.

IKBIP might be a potential prognostic biomarker for glioblastoma multiforme.

Due to the negative association between inhibitor of nuclear factor-kB kinase-interacting protein (IKBIP) and survival in gliomas, this study aimed to comprehensively analyze the potential function of IKBIP in glioblastoma multiforme (GBM). GBM samples were retrieved from The Cancer Genome Atlas and Chinese Glioma Genome Atlas as training and validation cohorts, respectively, and survival and Cox regression analyses were conducted. Based on clinical indicators and IKBIP, three prognostic models were established and then verified using the validation dataset. Infiltrating immune cell analysis and single-sample gene set enrichment analysis were also conducted to explore the underlying mechanisms. Finally, the key findings were validated through molecular biology experiments. Patients in the high IKBIP score group had poorer survival. Based on Cox regression and subgroup analyses, IKBIP was identified as an independent prognostic factor. Among the three models constructed, the model combining the IKBIP signature and clinical features displayed good performance in terms of discrimination, calibration, and model improvement capability in the training cohort. This model was also successfully validated in an external cohort from the CGGA. Further analysis revealed that many immune cells and related pathways were involved in the high-risk group. In vitro experiments revealed that the knockdown of IKBIP inhibited cell invasion and proliferation, and promoted their senescence. The prognostic value of IKBIP and its positive impact on the invasiveness of GBM were identified, indicating that IKBIP may serve as an underlying target for the treatment of GBM.

Synthetic miR-21 decoy circularized by tRNA splicing mechanism inhibited tumorigenesis in glioblastoma in vitro and in vivo models

Glioblastoma multiforme (GBM) is the deadliest primary central nervous system tumor. miRNAs (miRs), a class of non-coding RNAs, are considered pivotal post-transcriptional regulators of cell signaling pathways. miR-21 is a reliable oncogene that promotes tumorigenesis of cancer cells. We first performed an in silico analysis on 10 microarray datasets retrieved from TCGA and GEO databases to elucidate top differentially expressed miRs. Furthermore, we generated a circular miR-21 decoy, CM21D, using the tRNA-splicing mechanism in GBM cell models, U87 and C6. The inhibitory efficacy of CM21D with that of a linear form, LM21D, was compared under invitro conditions and an intracranial C6 rat glioblastoma model. miR-21 significantly overexpressed in GBM samples and confirmed in GBM cell models using qRT-PCR. CM21D was more efficient than LM21D at inducing apoptosis, inhibiting cell proliferation and migration, and interrupting the cell cycle by restoring the expression of miR-21 target genes at RNA and protein levels. Moreover, CM21D suppressed tumor growth more effectively than LM21D in the C6-rat GBM model (p<0.001). Our findings validate miR-21 as a promising therapeutic target for GBM. The introduced CM21D by sponging miR-21 reduced tumorigenesis of GBM and can be considered a potential RNA-base therapy to inhibit cancers.

NAGS, CPS1, and SLC25A13 (Citrin) at the Crossroads of Arginine and Pyrimidines Metabolism in Tumor Cells.

Urea cycle enzymes and transporters collectively convert ammonia into urea in the liver. Aberrant overexpression of carbamylphosphate synthetase 1 (CPS1) and SLC25A13 (citrin) genes has been associated with faster proliferation of tumor cells due to metabolic reprogramming that increases the activity of the CAD complex and pyrimidine biosynthesis. N-acetylglutamate (NAG), produced by NAG synthase (NAGS), is an essential activator of CPS1. Although NAGS is expressed in lung cancer derived cell lines, expression of the NAGS gene and its product was not evaluated in tumors with aberrant expression of CPS1 and citrin. We used data mining approaches to identify tumor types that exhibit aberrant overexpression of NAGS, CPS1, and citrin genes, and evaluated factors that may contribute to increased expression of the three genes and their products in tumors. Median expression of NAGS, CPS1, and citrin mRNA was higher in glioblastoma multiforme (GBM), glioma, and stomach adenocarcinoma (STAD) samples compared to the matched normal tissue. Median expression of CPS1 and citrin mRNA was higher in the lung adenocarcinoma (LUAD) sample while expression of NAGS mRNA did not differ. High NAGS expression was associated with an unfavorable outcome in patients with glioblastoma and GBM. Low NAGS expression was associated with an unfavorable outcome in patients with LUAD. Patterns of DNase hypersensitive sites and histone modifications in the upstream regulatory regions of NAGS, CPS1, and citrin genes were similar in liver tissue, lung tissue, and A549 lung adenocarcinoma cells despite different expression levels of the three genes in the liver and lung. Citrin gene copy numbers correlated with its mRNA expression in glioblastoma, GBM, LUAD, and STAD samples. There was little overlap between NAGS, CPS1, and citrin sequence variants found in patients with respective deficiencies, tumor samples, and individuals without known rare genetic diseases. The correlation between NAGS, CPS1, and citrin mRNA expression in the individual glioblastoma, GBM, LUAD, and STAD samples was very weak. These results suggest that the increased cytoplasmic supply of either carbamylphosphate, produced by CPS1, or aspartate may be sufficient to promote tumorigenesis, as well as the need for an alternative explanation of CPS1 activity in the absence of NAGS expression and NAG.

Abstract 4310: Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles

Abstract The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin-fixed paraffin-embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post-translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”. Here we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (147-plex) and RNA (whole transcriptome, &amp;gt; 18,000-plex) from a single FFPE sample. We demonstrated high concordance, R &amp;gt; 0.85, and minor change in sensitivity (&amp;lt;11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies. We observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post-translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Within our dataset, we observed &amp;gt;2-fold higher protein expression levels of phospho-GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho-Thr231 Tau was &amp;gt;2-fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology. Citation Format: Shilah Bonnett, Alyssa Rosenbloom, Giang Ong, Mark Conner, Daniel Newhouse, Felicia New, Hiromi Sato, Chi Pha, Saskia Ilcisin, John Lyssand, Gary Geiss, Joseph M. Beechem. Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4310.

Abstract 6442: Role of TWEAK-Fn14 axis in driving immunosuppression in glioblastoma multiforme

Abstract Cellular heterogeneity within glioblastoma multiforme (GBM) is a key driver of resistance to standard-of-care therapeutic approaches such as DNA damage (temozolomide, TMZ), angiogenesis inhibition (Bevacizumab, BM), radiation necrosis, and surgical resection. As such, understanding and targeting heterogeneous tumor-associated populations is a critical aim of ongoing GBM research. Central nervous system (CNS) resident microglia constitute significant percentages of primary GBM samples, often up to 50% of bulk tumor. Additionally, glioblastoma-associated microglia have been implicated in intra-tumoral immunosuppression, and are thus a key barrier to successful implementation of immunotherapies that seek to enhance T-cell mediated anti-tumoral cytotoxicity. As such, insight into targetable pathways that upregulate immunosuppressive cytokines within GBM-associated microglia is necessary for overcoming immuno-therapeutic failure. Here, we identify expression of the cytokine TWEAK (TNFSF12) and its receptor Fn14 (TNFRSF12A) within the myeloid compartment of primary GBM samples via scRNA-seq. Furthermore, we demonstrate TWEAK-mediated reprogramming of GBM-associated microglia towards an immunosuppressive phenotype capable of inhibiting CD8+ T cell activation and proliferation. These findings unveil a potent, targetable axis of immunosuppression within GBM and are thus an exciting step towards enhancing efficacy of existing T-cell based anti-cancer strategies for GBM patients. Citation Format: Angad Beniwal, Ryan Eghlimi, Kenneth D. Alexander, Mylan R. Blomquist, Nhan Tran. Role of TWEAK-Fn14 axis in driving immunosuppression in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6442.

Abstract 1604: Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2

Abstract Interleukin 13 receptor subunit alpha 2 (IL13RA2) is one of the two major receptors for the cytokine interleukin 13 (IL-13). While IL13RA1 has low affinity for IL-13 and it is expressed ubiquitously in humans, IL13RA2 has high binding affinity to IL-13 and its expression in normal tissues is mainly restricted to the testes. IL13RA2 was initially regarded as a decoy receptor because it has a short cytoplasmic tail with no known signaling motifs, and its binding to IL-13 does not lead to activation of the JAK/STAT6 pathway. However, recent studies demonstrated that IL-13-mediated IL13RA2 signaling occurs via STAT6-independent pathways. IL13RA2 is reported to be expressed at a high frequency in glioblastoma multiforme (GBM) as well as in other solid tumors including melanoma, renal cell carcinoma and adrenocortical carcinoma and is correlated with poor prognosis. ADCT-211 is an antibody-drug conjugate composed of HuCl47, a humanized IgG1 antibody directed against human IL13RA2, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG2000 (drug to antibody ratio ~ 1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-211 in human cancer cell lines and xenograft models, to determine its safety, tolerability and pharmacokinetic (PK) in the rat and to measure IL13RA2 expression by immunohistochemistry (IHC) in human tumor specimens. HuCl47 showed specific binding to recombinant IL13RA2, while it did not bind to IL13RA1. In vitro, ADCT-211 had highly potent and targeted cytotoxicity against various IL13RA2-expressing solid cancer cell lines. In vivo, a single dose of ADCT-211 at 10 mg/kg was able to completely eradicate tumors in the subcutaneous (sc) A375 human melanoma xenograft model and resulted in 8/8 tumor-free survivors (TFS) at the end of the study on day 57. In the sc U251 human glioblastoma xenograft model, a single dose of ADCT-211 at 3.75 mg/kg showed potent and durable antitumor activity and it resulted in 5/10 partial responders and 5/10 complete responders, 2 of which were TFS at the end of the study on day 51. In an MTD study in male rats, ADCT-211 was stable and tolerated up to 24 mg/kg single dose, with exposure data being indicative of a linear PK profile with a half-life of 12-19 days. Expression of membranous IL13RA2 was confirmed by IHC in a panel of primary and refractory GBM samples and malignant melanoma, highlighting them as potential indications for the clinical development of ADCT-211. In conclusion, ADCT-211 demonstrated potent and specific in vitro and in vivo anti-tumor activity and it was stable and well tolerated in the rat, warranting further development of ADCT-211 into the clinic in IL13RA2-expressing cancers. Citation Format: Francesca Zammarchi, Karin Havenith, Ben Leatherdale, Britanny Roberts, Lara Montolio, Afroze Patel, Narinder Janghra, Pedro Alves, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Lolke de Haan, Patrick H van Berkel. Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1604.

157 Identifying Spatial Transcriptomics Signaling Networks in Human Glioblastoma Using Graph-Based Machine Learning

INTRODUCTION: The median survival for glioblastoma multiforme (GBM) has only improved by 4.6 months in more than 100 years, underlying the need for innovative methods for understanding this devastating tumor. Spatial transcriptomics is a new method of whole-transcriptome assessment which allows for simultaneous visualization of mRNA expression in situ with histopathology. METHODS: We applied spARC to 25,517 Unique Molecular Identifiers from 10 surgically resected human IDH-mutant astrocytic gliomas (Grade II (n = 3) and Grade III (n = 3)) and GBMs (n = 4). spARC uses manifold learning, graph filters, data diffusion and diffusion filtration to enable discovery of gene-gene relationships, spatial co-localization of genes, and receptor-ligand interactions applicable across a variety of spatial transcriptomic methods. A Neuropathologist annotated whole-slide images blind to our computational analysis and we used in-situ hybridization and immunohistochemistry to demonstrate that spaRC imputation recovery of gene expression is superior to raw expression. RESULTS: We demonstrate that spARC can identify spatially restricted tumor niches of dense tumor, leading edge, infiltration/high tumor burden, hemoglobin, and vasculature with high specificity. These clusters can be identified with genes including CD44, VIM, LDHA (dense tumor); SNAP25, UCHL1, OLMF1 (leading edge); OLIG2, MBP, PDGFRA (Infiltrating/High Tumor Burden). In our assessment of ligand-receptor interactions we found that GBM samples were enriched for SPP1-CD44 signaling. We validated this using GBM TCGA data stratifying patients based on expression of SPP1 and CD44 genes and found that high expression conferred lower survival at 12 months. CONCLUSIONS: Spatial transcriptomics paired with graph-based machine learning methods applied to human GBMs is a powerful technique able to uncover signaling networks relevant to patient survival and may aid in developing new therapeutic strategies.