BACKGROUND:Glioblastoma (GBM) progression is driven by intricate interactions between neoplastic cells and immune populations within the tumor microenvironment (TME), yet the temporal organization of these processes remains insufficiently defined. While tumor-associated macrophages (TAMs), composed of microglia and monocyte-derived macrophages, constitute a major immune population in GBM, microglia play a prominent role in shaping local immunosuppressive niches, thereby limiting cytotoxic lymphocyte infiltration. However, static co-culture models constrain interrogation of dynamic tumor-immune signaling. METHODS:We utilized a programmable multi-inlet microfluidic platform that allows sequential, order-specific introduction of immune cells into GBM constructs, enabling controlled interrogation of MG- and natural killer (NK)-mediated interactions under defined temporal conditions. Bulk transcriptomic profiling and single-cell analyses were used to characterize tumor- and immune-associated pathways elicited by distinct temporal configurations. RESULTS:In triculture constructs containing identical populations of GBM cells, microglia, and NK cells, MG-first delivery preferentially activated STAT3-associated immunoregulatory transcriptional programs, whereas NK-first delivery induced cytotoxic and interferon-related gene expression profiles. IL12A expression was selectively increased under NK-first conditions and correlated with immune-active GBM states in patient datasets. STAT3 inhibition reduced MG-associated suppression, increased IL12A expression, and enhanced the response to temozolomide. CONCLUSIONS:This programmable spheroid platform not only recapitulates hallmark features of the GBM immune microenvironment but also uncovers temporally regulated pathways arising from sequential cellular interactions. By enabling controlled reconstruction of dynamic tumor-immune signaling, this system offers a versatile strategy for mechanistic investigation and provides a foundation for evaluating immune-modulatory approaches and microenvironment-targeted interventions in GBM and other malignancies.

Gliomas with mutant isocitrate dehydrogenase (IDH) are malignant brain tumours that typically arise in early to mid-adulthood and nearly always recur following treatment1,2. However, the genetic and cellular-state changes that drive IDH-mutant glioma progression under treatment remain incompletely understood. Here we integrated single-nucleus transcriptomic profiles, chromatin accessibility profiles and bulk DNA and RNA sequencing from 75 temporally separated gliomas across 35 patients comprising both the oligodendroglioma and astrocytoma IDH-mutant glioma tumour types. We show that malignant cell states transcriptionally resemble stages of normal glial-neuronal lineage development or a reactive mesenchymal-like state, mirroring states previously described in IDH wild-type glioblastoma3,4. Malignant cell states displayed distinct chromatin accessibility profiles that were comparable between both IDH-mutant glioma types. The abundance of less differentiated malignant cells increased with grade and with genetic alterations such as PDGFRA amplification. Longitudinal analysis highlighted two major malignant cell-state transition patterns. First, reduced lineage differentiation and increased proliferative malignant cells at recurrence were enriched in gliomas that acquired recurrence-associated genetic events. These included treatment-associated hypermutation, increased copy number changes and cell cycle alterations. Second, increased mesenchymal-like-state abundance occurred independently of acquired genetic alterations and instead coincided with elevated macrophage expression. Overall, our findings provide an integrative model that traces the cell intrinsic and extrinsic factors that shape cellular states during IDH-mutant glioma disease progression.

Cancer immunotherapy has substantially advanced cancer treatment, achieving durable responses in select malignancies. However, its widespread application is limited by significant challenges: low efficacy in many solid tumors, severe side effects, and immune evasion facilitated by the tumor microenvironment (TME). Nanotechnology offers a promising approach to address these obstacles. By employing nanoparticles (NPs), we can precisely deliver therapeutics to tumor sites, ensure controlled release to minimize side effects, and amplify the immune response, thereby substantially boosting the effectiveness of immunotherapy. This review comprehensively highlights the latest advancements in using nanotechnology to enhance cancer immunotherapy. This paper details various applications of nanotech in this field. It discusses smart nanoparticles that respond to TME signals to release drugs (e.g., checkpoint inhibitors) directly at the tumor, reducing systemic side effects and activating T-cells. We also explore how nanovaccines, which co-deliver tumor markers and immune boosters, can induce antigen-specific immune responses. Furthermore, mRNA-loaded nanoparticles can directly modify CAR T-cells inside the body, simplifying treatment and increasing efficacy. Strategies like using PLGA NPs to deliver immune enhancers such as IL-2 are also presented, which activate immune cells while minimizing systemic issues. The review also explains how nanoparticles can re-engineer the immunosuppressive TME to create an environment more conducive to immune action. We also emphasize that nanotechnology-enhanced adoptive therapies, particularly cytokine-induced killer (CIK) cell immunotherapy, hold great potential to improve tumor targeting, treatment persistence durability, and overall anticancer efficacy. Collectively, we highlight synergistic effects achieved by combining nanoparticles with other treatments like chemotherapy, radiation, photothermal/photodynamic therapy, and more, which can turn hard-to-treat tumors into susceptible targets. The integration of nanotechnology and immunotherapy holds the potential to meaningfully advance future cancer therapy.

Extrachromosomal DNA (ecDNA), a prevalent mechanism of gene amplification, drives oncogene overexpression and is associated with poor patient survival. Over 60% of primary glioblastoma (GBM) cases harbor ecDNA-mediated oncogene amplifications. To better understand the functional consequences of ecDNA amplification, we performed a comprehensive analysis of ecDNA distribution in tumors using longitudinally sampled multiomic data from 35 GBM patient specimens. This dataset integrates 68 whole-genome sequencing (WGS) datasets, 243, 459 transcriptomic and chromatin accessibility profiles derived from the same nuclei. We developed a single-nucleus ATAC-seq-based pipeline to classify ecDNA status at single-cell resolution. To validate the robustness of this pipeline, we utilized patient-derived neurospheres (n = 2), generating multiomic datasets and over 100 fluorescent in situ hybridization (FISH) images per cell line for structural validation. This approach enabled the identification of ecDNA-containing regions in each patient sample through WGS data and the inference of single-cell-level copy number variations. In total, we classified the ecDNA status of over 86, 345 malignant cells. Our findings demonstrated that ecDNA copy numbers remain stable during tumor progression, with no significant changes in the proportion of ecDNA-containing cells over time. Notably, we observed that oncogenes amplified on ecDNA are associated with distinct tumor cell states. By applying non-negative matrix factorization and a random forest machine learning algorithm, we extracted gene programs linked to ecDNA that are enriched for cell-cycle regulation and chromatin regulating. These results provide new insights into the regulatory landscape of ecDNA-driven glioblastoma and highlight its potential role in shaping tumor cell identity and progression. Dacheng Zhao, Kevin Anderson, Amit Gujar, Megan Costa, Djamel Nehar-Belaid, Frederick Varn, Elise Courtois, Paul Gabrie, Paul Robson, Hyo-Eun Moon, Anna Golebiewska, Sun Ha Paek, Simone Niclou. Integrated multiomic analysis to infer longitudinal patterns of ecDNA in IDH-wildtype integrated multiomic analysis to infer longitudinal patterns of ecDNA in IDH wild-type glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7504.

Astrocytomas and oligodendrogliomas are slow-growing and treatment-sensitive IDH-mutant gliomas diagnosed at ages 30-50. Local tumor regrowth and treatment resistance is inevitable resulting in 3-10 year astrocytoma and up to >20 years oligodendroglioma survival. We sought to identify genetic changes associated with tumor evolution in response to therapy through multi-timepoint whole-genome/whole-exome sequencing of 206 IDH-mutant glioma patient samples collected through the Glioma Longitudinal Analysis (GLASS) Consortium. We validated known genomic markers of tumor progression, including hypermutation and CDKN2A homozygous deletion, and discovered novel genetic alterations that distinguish the response to treatment in astrocytomas compared to oligodendrogliomas. Point mutations in PIK3CA , PIK3R1 , and NOTCH1 were newly acquired in recurrent oligodendrogliomas and associated with increased mutation rates. Focal oncogene amplifications, together with CDKN2A homozygous deletions, were associated with an increase in recurrence-specific chromosomal imbalances in astrocytomas. Mutational signature analysis revealed additional differences and detected enrichment for the SBS11, and SBS119 mutational signatures after temozolomide treatment in both IDH-glioma subtypes, whereas astrocytomas showed increased ID8 signatures after radiotherapy. These signatures suggest that the genomes of oligodendroglioma and astrocytoma adapt to the selective pressures of tumor progression and treatment in different ways. However, in both IDH-mutant glioma subtypes we observed a convergence of acquired driver gene alterations with genome-wide changes and worse patient outcomes, signaling selection of treatment-refractory clones. By identifying new prognostic markers and delineating the genomic divergence of oligodendrogliomas and astrocytomas after diagnosis, our results suggest that different DNA damage response mechanisms are engaged following chemo- and radiation therapy.

Superparamagnetic iron oxide nanoparticles (SPIONs) are promising contrast agents for imaging-guided cancer therapies. However, challenges such as the requirement for a high alternating magnetic field (AMF), dosage limitations, and suboptimal imaging contrast have hindered their practical applications. Methods: First, the optimal doping ratio of Mn and Zn in MnxZn1-xFe2O4 nanoparticles synthesized using a modified high-temperature thermal decomposition method (mHTTD) was determined. Then, the magnetic and physical properties of the optimal 7-nm Mn0.5Zn0.5Fe2O4 SPIONs were systematically and comprehensively characterized via hysteresis measurements, dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray absorption fine structure (XAFS) spectroscopy, and X-ray absorption near edge structure (XANES) spectroscopy. Next, the stability, biosafety, biocompatibility, and theranostic performance of 7-nm Mn0.5Zn0.5Fe2O4 SPIONs in magnetic hyperthermia therapy (MHT) were evaluated by in vivo and in vitro studies involving mouse models, magnetic resonance imaging (MRI), and bioassays. The results were then compared with those for conventional SPIONs. Results: Under an AMF of 140 Oe at 100 kHz, 7-nm Mn0.5Zn0.5Fe2O4 SPIONs demonstrated significantly higher heat production than conventional SPIONs. Following surface modification with methoxy-PEG-silane, PEGylated 7-nm Mn0.5Zn0.5Fe2O4 SPIONs showed excellent monodispersity and magnetic properties, with an exceptionally high T2 relaxivity (r2). Conclusions: The high in vitro and in vivo theranostic performance of PEGylated 7-nm Mn0.5Zn0.5Fe2O4 SPIONs as efficient and stable contrast agents for treating glioblastoma, encompassing strengthened magnetic hyperthermia, activated anti-tumor immunity, and remarkable T2 contrast enhancement, underscores the potential of precisely designed ferrites to concurrently enhance the T2 contrast and magnetocaloric properties for optimal theranostic outcomes. Our study provides a compelling rationale for the development of tailored magnetic nanoprobes for improved glioblastoma theranostics.

Superparamagnetic iron oxide nanoparticles (SPIONs) are promising contrast agents for imaging-guided cancer therapies. However, challenges such as the requirement for a high alternating magnetic field (AMF), dosage limitations, and suboptimal imaging contrast have hindered their practical applications. Methods: First, the optimal doping ratio of Mn and Zn in MnxZn1-xFe2O4 nanoparticles synthesized using a modified high-temperature thermal decomposition method (mHTTD) was determined. Then, the magnetic and physical properties of the optimal 7-nm Mn0.5Zn0.5Fe2O4 SPIONs were systematically and comprehensively characterized via hysteresis measurements, dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray absorption fine structure (XAFS) spectroscopy, and X-ray absorption near edge structure (XANES) spectroscopy. Next, the stability, biosafety, biocompatibility, and theranostic performance of 7-nm Mn0.5Zn0.5Fe2O4 SPIONs in magnetic hyperthermia therapy (MHT) were evaluated by in vivo and in vitro studies involving mouse models, magnetic resonance imaging (MRI), and bioassays. The results were then compared with those for conventional SPIONs. Results: Under an AMF of 140 Oe at 100 kHz, 7-nm Mn0.5Zn0.5Fe2O4 SPIONs demonstrated significantly higher heat production than conventional SPIONs. Following surface modification with methoxy-PEG-silane, PEGylated 7-nm Mn0.5Zn0.5Fe2O4 SPIONs showed excellent monodispersity and magnetic properties, with an exceptionally high T2 relaxivity (r2). Conclusions: The high in vitro and in vivo theranostic performance of PEGylated 7-nm Mn0.5Zn0.5Fe2O4 SPIONs as efficient and stable contrast agents for treating glioblastoma, encompassing strengthened magnetic hyperthermia, activated anti-tumor immunity, and remarkable T2 contrast enhancement, underscores the potential of precisely designed ferrites to concurrently enhance the T2 contrast and magnetocaloric properties for optimal theranostic outcomes. Our study provides a compelling rationale for the development of tailored magnetic nanoprobes for improved glioblastoma theranostics.

The evolution of isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) after standard-of-care therapy remains poorly understood. Here we analyzed matched primary and recurrent GBMs from 59 patients using single-nucleus RNA sequencing and bulk DNA sequencing, assessing the longitudinal evolution of the GBM ecosystem across layers of cellular and molecular heterogeneity. The most consistent change was a lower malignant cell fraction at recurrence and a reciprocal increase in glial and neuronal cell types in the tumor microenvironment (TME). The predominant malignant cell state differed between most matched pairs, but no states were exclusive or highly enriched in either time point, nor was there a consistent longitudinal trajectory across the cohort. Nevertheless, specific trajectories were enriched in subsets of patients. Changes in malignant state abundances mirrored changes in TME composition and baseline profiles, reflecting the co-evolution of the GBM ecosystem. Our study provides a blueprint of GBM's diverse longitudinal trajectories and highlights the treatment and TME modifiers that shape them.

In isocitrate dehydrogenase wildtype glioblastoma (GBM), cellular heterogeneity across and within tumors may drive therapeutic resistance. Here we analyzed 121 primary and recurrent GBM samples from 59 patients using single-nucleus RNA sequencing and bulk tumor DNA sequencing to characterize GBM transcriptional heterogeneity. First, GBMs can be classified by their broad cellular composition, encompassing malignant and nonmalignant cell types. Second, in each cell type we describe the diversity of cellular states and their pathway activation, particularly an expanded set of malignant cell states, including glial progenitor cell-like, neuronal-like and cilia-like. Third, the remaining variation between GBMs highlights three baseline gene expression programs. These three layers of heterogeneity are interrelated and partially associated with specific genetic aberrations, thereby defining three stereotypic GBM ecosystems. This work provides an unparalleled view of the multilayered transcriptional architecture of GBM. How this architecture evolves during disease progression is addressed in the companion manuscript by Spitzer et al.

To understand the role of extrachromosomal DNA (ecDNA) amplifications in cancer progression, we detected and classified focal amplifications in 8,060 newly diagnosed primary cancers, untreated metastases and heavily pretreated tumors. The ecDNAs were detected at significantly higher frequency in untreated metastatic and pretreated tumors compared to newly diagnosed cancers. Tumors from chemotherapy-pretreated patients showed significantly higher ecDNA frequency compared to untreated cancers. In particular, tubulin inhibition associated with ecDNA increases, suggesting a role for ecDNA in treatment response. In longitudinally matched tumor samples, ecDNAs were more likely to be retained compared to chromosomal amplifications. EcDNAs shared between time points, and ecDNAs in advanced cancers were more likely to harbor localized hypermutation events compared to private ecDNAs and ecDNAs in newly diagnosed tumors. Relatively high variant allele fractions of ecDNA localized hypermutations implicated early ecDNA mutagenesis. Our findings nominate ecDNAs to provide tumors with competitive advantages during cancer progression and metastasis. A pan-cancer genomic analysis finds an increase of extrachromosomal DNA (ecDNA) in treated and metastatic tumors compared to primary, untreated samples, as well as ecDNA features enriched in advanced disease.

Glioblastoma is one of the most malignant primary brain cancer. Despite surgical resection with modern technology followed by chemo-radiation therapy with temozolomide, resistance to the treatment and recurrence is common due to its aggressive and infiltrating nature of the tumor with high proliferation index. The median survival time of the patients with glioblastomas is less than 15 months. Till now there has been no report of molecular target specific for glioblastomas. Early diagnosis and development of molecular target specific for glioblastomas are essential for longer survival of the patients with glioblastomas. Development of biomarkers specific for glioblastomas is most important for early diagnosis, estimation of the prognosis, and molecular target therapy of glioblastomas. To that end, in this study, we have conducted a comprehensive proteome study using primary cells and tissues from patients with glioblastoma. In the discovery stage, we have identified 7429 glioblastoma-specific proteins, where 476 proteins were quantitated using Tandem Mass Tag (TMT) method; 228 and 248 proteins showed up and down-regulated pattern, respectively. In the validation stage (20 selected target proteins), we developed quantitative targeted method (MRM: Multiple reaction monitoring) using stable isotope standards (SIS) peptide. In this study, five proteins (CCT3, PCMT1, TKT, TOMM34, UBA1) showed the significantly different protein levels (t-test: p value ≤ 0.05, AUC ≥ 0.7) between control and cancer groups and the result of multiplex assay using logistic regression showed the 5-marker panel showed better sensitivity (0.80 and 0.90), specificity (0.92 and 1.00), error rate (10 and 2%), and AUC value (0.94 and 0.98) than the best single marker (TOMM34) in primary cells and tissues, respectively. Although we acknowledge that the model requires further validation in a large sample size, the 5 protein marker panel can be used as baseline data for the discovery of novel biomarkers of the glioblastoma.

Abstract Despite standard treatment, IDH-mutant gliomas inevitably recur. Previous studies suggest that therapeutic resistance may result from a combination of intratumoral cellular heterogeneity, epigenetic evolution, and acquired genetic alterations. However, how these multilayered molecular features interact to influence the evolutionary paths of IDH-mutant gliomas remains incompletely understood. To chart this evolution, 75 glioma samples were longitudinally collected from 35 patients (n = 13 oligodendroglioma, n = 22 astrocytoma) and profiled using single nucleus RNA sequencing, ATAC sequencing, and bulk DNA sequencing. Analysis of 331,016 nuclei (snRNA) revealed eight tumor microenvironment cell types and five pan-IDH-mutant malignant cellular states. The malignant states were distributed along a cellular hierarchy of stem-like populations (neural progenitor cell-like, oligodendrocyte progenitor cell-like, and undifferentiated) and more differentiated populations (mesenchymal-like and astrocyte-like) with cycling cells enriched in the stem-like populations. Joint open chromatin accessibility data (snATAC) was available for a subset of malignant cells (71,088 nuclei) and state-specific differentially accessible peaks supported that these states are epigenetically encoded. Across both subtypes, higher tumor grade was associated with reduced malignant cell differentiation and increased cycling populations. Between the two time points, we identified that there was a longitudinal increase in the cycling, undifferentiated, and mesenchymal-like populations with a corresponding decrease in the astrocyte-like population. Longitudinal genetic analysis revealed that 19 of 35 tumors acquired at least one of the following genetic alterations: treatment-associated hypermutation, cell cycle alteration, or large changes in copy number alterations. Importantly, tumors that acquired these key genetic alterations demonstrated significant shifts towards reduced differentiation and increased cycling populations while those tumors that did not had more stable malignant profiles. Collectively, our results suggest a common cellular hierarchy across IDH-mutant gliomas with a shift towards reduced differentiation, increased cycling populations during disease progression that is driven by acquired genetic alterations.

Glioblastoma (GBM), the most lethal primary brain cancer, exhibits intratumoral heterogeneity and molecular plasticity, posing challenges for effective treatment. Despite this, the regulatory mechanisms underlying such plasticity, particularly mesenchymal (MES) transition, remain poorly understood. In this study, we elucidate the role of the RNA-binding protein ELAVL2 in regulating aggressive MES transformation in GBM. We found that ELAVL2 is most frequently deleted in GBM compared to other cancers and associated with distinct clinical and molecular features. Transcriptomic analysis revealed that ELAVL2-mediated alterations correspond to specific GBM subtype signatures. Notably, ELAVL2 expression negatively correlated with epithelial-to-mesenchymal transition (EMT)-related genes, and its loss promoted MES process and chemo-resistance in GBM cells, whereas ELAVL2 overexpression exerted the opposite effect. Further investigation via tissue microarray analysis demonstrated that high ELAVL2 protein expression confers a favorable survival outcome in GBM patients. Mechanistically, ELAVL2 was shown to directly bind to the transcripts of EMT-inhibitory molecules, SH3GL3 and DNM3, modulating their mRNA stability, potentially through an m6A-dependent mechanism. In summary, our findings identify ELAVL2 as a critical tumor suppressor and mRNA stabilizer that regulates MES transition in GBM, underscoring its role in transcriptomic plasticity and glioma progression.

Abstract The evolution of cellular heterogeneity in IDH-wildtype glioblastoma (GBM) after standard-of-care therapy remains poorly understood. To address it, we assembled a longitudinal cohort of 121 primary and recurrent GBM specimens from 59 patients, with extensive clinical annotations, and profiled it by single-nucleus RNA-sequencing and bulk tumor DNA sequencing. In most cases, longitudinal samples diverged in their composition of cell types and cell states. However, almost all theoretical trajectories were observed in our cohort such that the overall distribution of cell types and cell states was comparable between primary and recurrent samples. The most consistent longitudinal effect (66% of patients) was a lower malignant cell fraction at recurrence and a reciprocal increase in proportions of glio-neuronal TME cell types; in some cases, this was further accompanied by a coordinated shift of malignant cells towards neuronal-like states. MGMT methylation and radiation-related small deletion phenotypes were linked to particular trajectories, with depletion of mesenchymal-like cells and enrichment of hypoxia-related malignant cells, respectively. Importantly, changes in malignant states were also associated with specific changes in TME composition. In summary, our analysis highlights diverse longitudinal GBM trajectories that are shaped by treatment response and TME interactions.

0.9% saline-treated control group. The cell were recorded in 2 h after seeding on 6-well plate equipped with x10 objective lens.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

The heterogeneity within tumors has long been associated with therapy failure and disease progression. Recent advances in single-cell RNA-sequencing technologies have enabled us to dissect the cellular diversity in glioblastoma (GBM). However, how these cellular programs change longitudinally under therapy remains poorly understood. To address this question, we collected and profiled a large-scale longitudinal cohort of 59 matched IDH-wildtype GBM sample pairs provided by 7 centers worldwide by single-nucleus RNA-sequencing (snRNA-seq) and whole-exome/whole-genome sequencing. The majority of patients (51 patients) in this cohort received standard-of-care (temozolomide and radiation) following initial tumor resection. Leveraging this large-scale snRNS-seq dataset of 457,442 cells, we detected novel cellular states and associations between malignant and tumor microenvironment (TME) cells, and then performed longitudinal analyses. The recurrent samples showed significantly lower malignant cell fraction (p=0.002) and reciprocal increase in proportions of glio-neuronal TME cell types (oligodendrocytes, neurons and astrocytes). The TME composition, malignant cell state proportions and baseline expression programs were retained at recurrence more than expected by chance, but overall were not well conserved between primary and recurrent samples. A subset of pairs (12/59 pairs) enriched with glio-neuronal TME at recurrence showed a significant transcriptomic shift and was associated with better clinical course (p=0.02). The tumors that acquired radiation-related small deletion phenotype underwent a transition towards MES/Hypoxia phenotype (0/9 at primary, 6/9 at recurrence, p=0.02). We defined pairs as likely responders or non-responders to treatment based on the MGMT methylation status of the primary tumor sample. This uncovered diverging evolutionary trajectories in cellular programs between the two groups. Strikingly, the changes in malignant state frequency and baseline malignant expression profile were strongly associated with specific changes in the TME composition. Our findings based on high-resolution longitudinal snRNA-seq analyses highlight the diverse evolutionary trajectories in GBM that are shaped by TME changes and treatments.

Patient-derived xenograft (PDX) models, which can retain the characteristics of original tumors in an in vivo-mimicking environment, have been developed to identify better treatment options. However, although original tumors and xenograft tissues mostly share oncogenic mutations and global gene expression patterns, their detailed mutation profiles occasionally do not overlap, indicating that selection occurs in the xenograft environment. To understand this mutational alteration in xenografts, we established 13 PDX models derived from 11 brain tumor patients and confirmed their histopathological similarity. Surprisingly, only a limited number of somatic mutations were shared between the original tumor and xenograft tissue. By analyzing deleteriously mutated genes in tumors and xenografts, we found that previously reported brain tumor-related genes were enriched in PDX samples, demonstrating that xenografts are a valuable platform for studying brain tumors. Furthermore, mutated genes involved in cilium movement, microtubule depolymerization, and histone methylation were enriched in PDX samples compared with the original tumors. Even with the limitations of the heterogeneity of clinical lesions with a heterotropic model, our study demonstrates that PDX models can provide more information in genetic analysis using samples with high heterogeneity, such as brain tumors.

Supplementary Figure S1. Characterization of cancer spheroids. Supplementary Figure S2. Characteristics of spheroids based on morphologic classification. Supplementary Figure S3. Validation of spheroid morphology-dependent characteristics. Supplementary Figure S4. Validation of PLS-DA model. Supplementary Figure S5. Network models describing cellular processes associated with spheroid subtypes. Supplementary Figure S6. Drug penetration test in spheroids using bimolecular fluorescence complementation (BiFC). Supplementary Figure S7. Molecular signature of GBM patient-derived round type spheroid. Supplementary Figure S8. Xenograft model to validate JAK-STAT pathway for drug sensitivity.

Abstract Glioblastoma (GBM), the most lethal type of primary brain cancer, is characterized by cellular and molecular plasticity, which leads to intratumoral heterogeneity and hinders effective treatment. However, the regulation of such plasticity, including mesenchymal (MES) transition, is poorly understood. Here, we demonstrate that the RNA-binding protein ELAVL2 regulates aggressive MES transformation in GBM. ELAVL2 was most frequently deleted in GBM compared to other cancers and associated with distinct clinical and molecular features. ELAVL2-mediated transcriptomic alterations were indicative of GBM subtype signatures. Expression of ELAVL2 negatively correlated with that of epithelial-to-mesenchymal transition (EMT)-related genes, and its loss promoted the EMT process and chemo-resistance. Tissue microarray analysis revealed that high ELAVL2 protein expression level confers a favorable survival in GBM patients. On a molecular level, ELAVL2 regulated the expression of EMT-inhibitory molecules SH3GL3 and DNM3. Overall, these findings demonstrate ELAVL2 as a critical tumor suppressor that regulates MES transition in GBM, highlighting its role in transcriptomic plasticity and glioma progression.