Lenvatinib is a first-line treatment for hepatocellular carcinoma (HCC), though drug resistance may develop with long-term use. to investigate the potential of pachymic acid to enhance lenvatinib’s anti-tumor efficacy. HepG2 and LM3 cells were exposed to pachymic acid and/or lenvatinib. Cell proliferation was evaluated using colony formation assays and flow cytometry analysis of apoptosis. Cell invasion was evaluated using wound healing assays and transwell assays. Glucose consumption, lactate production and glycolysis-related protein expression were measured to evaluate aerobic glycolysis status. Tumor-bearing experiments in nude mice were conducted to verify pachymic acid’s influence on HCC. Twenty µmol/L pachymic acid substantially inhibited the apoptosis rate (8.5
Gastric cancer (GC) remains a major malignancy of the digestive system, with a high burden in developing countries where early diagnosis is often limited. As a result, many patients are diagnosed at advanced stages, and despite comprehensive treatment strategies, recurrence rates remain high. These challenges underscore the urgent need for improved diagnostic and therapeutic approaches. Extracellular vesicles (EVs) have attracted increasing attention due to their critical roles in tumor progression and intercellular communication. Emerging evidence suggests that EVs are involved in key processes such as immune modulation, metastasis, and therapeutic resistance, highlighting their potential as biomarkers and therapeutic targets. In this review, we systematically summarize the biological functions of EVs in GC and critically evaluate their translational potential in diagnosis and therapy. We also discuss current challenges, including technical limitations, lack of standardization, and insufficient clinical validation, and propose future directions for advancing EV-based precision medicine.
Metabolic reprogramming characterized by enhanced glycolysis and lactate production plays a critical role in tumor progression and immune regulation. However, the cellular distribution and spatial organization of lactate-glycolysis activity within the lung adenocarcinoma (LUAD) microenvironment remain incompletely understood. We integrated single-cell RNA sequencing, spatial transcriptomics, and bulk transcriptomic datasets from TCGA and GEO cohorts to characterize lactate-glycolysis-associated metabolic heterogeneity in LUAD. AUCell-based scoring was used to quantify metabolic activity at single-cell resolution. Associations with immune infiltration and clinical outcomes were evaluated, and key findings were validated using spatial transcriptomics, quantitative PCR, and immunohistochemistry. Cells exhibiting high lactate-glycolysis activity were predominantly enriched in fibroblasts and neutrophils, indicating that this metabolic program extends beyond malignant cells. A metabolism-associated gene signature, comprising RPS2, GAPDH, and LDHA, was consistently correlated with immune remodeling and an unfavorable prognosis. Spatial transcriptomics further revealed the co-localization of GAPDH and LDHA with neutrophil-enriched regions. These findings were supported by experimental validation in clinical specimens. Our study reveals that lactate-glycolysis reprogramming in LUAD is spatially structured and closely associated with neutrophil-enriched immune remodeling. This metabolism-associated immune regulatory pattern provides insight into the organization of the tumor microenvironment and may have implications for metabolic- and immune-oriented therapeutic strategies in lung adenocarcinoma.
Lung cancer remains the leading cause of high cancer incidence and mortality worldwide. Among lung cancers, non-small cell lung cancer (NSCLC) is the predominant subtype accounting for majority of cancer deaths. The invasive and metastatic behavior of NSCLC largely contributes to its aggressive nature and therapeutic resistance. Farnesol (FAR), a naturally occurring sesquiterpene, has shown anticancer activity in several malignancies however, its mechanistic role in NSCLC invasion and metastasis remains not fully understood. In the present study, we investigated the effect of farnesol on A549 lung adenocarcinoma cells through in vitro approaches involving protein–protein/gene interactions, molecular analysis and 3D screening. Scratch assay and transwell invasion assays were performed to evaluate the effect of farnesol on A549 cell migration and invasion. Immunofluorescence staining analysis were employed to assess the anti-angiogenic potential of farnesol followed by western blotting and real-time PCR to validate the efficacy of farnesol in regulating invasion and metastasis associated markers. Farnesol significantly reduced the viability of A549 cells in a dose-dependent manner, with an IC₅₀ of 21.5 µg/mL inducing cell shrinkage and loss of cell density. Scratch and matrix-assisted transwell invasion assays demonstrated marked inhibition of migration and invasion with increased nonmigratory spaces in farnesol-treated group compared to control. Molecular analysis confirmed upregulation of E-cadherin, downregulation of VEGF, PCAF, COX2, and suppression of mesenchymal and matrix remodeling markers. Farnesol reduced spheroid diameter, dissociation of spheroid integrity and induced of apoptosis at concentrations above 125 µg/mL. Collectively, our findings suggest that farnesol potentially inhibits A549 cell migration, invasion and metastasis, highlighting farnesol as an effective therapeutic candidate for the treatment of lung adenocarcinoma. Schematic representation of the current study in elucidating the anticancer potential of FAR through 2D and 3D screening. 2D analysis showed FAR reduces A549 cell viability and migration, with molecular analyses confirmed the impact of FAR in the regulation of invasion and metastasis. In 3D spheroid, farnesol suppressed spheroid growth, reduced proliferation, and induced apoptosis. Additionally, farnesol inhibited HIF1A, COX2 and PCAF leading to reduced expression of MMP2/9 activity, VEGF, and ECM degradation preventing cadherin switching, EMT associated angiogenesis, invasion and metastasis. This highlights FARs therapeutic potential in targeting A549 cancer cell invasion and metastasis. Scratch and Matrigel-assisted transwell assays confirmed that farnesol suppressed the migration and invasion of A549 cells at 21.5 µg/mL. Farnesol inhibited the angiogenic potential of A549 cells via suppression of VEGF. Farnesol reduced the diameter, disrupted the morphology, and induced apoptosis in 3D A549 spheroids.
Copper and iron trigger distinct cuproptosis and ferroptosis as homeostasis is disrupted which would drive hepatocellular carcinoma (HCC) progression. Mitochondrial copper overload impairs protein lipoylation and iron-sulfur cluster stability which induces proteotoxic stress through accumulation of lipoylated TCA cycle enzymes. Elevated copper metabolism paradoxically increases their susceptibility to cuproptosis in HCC cells. Ferroptosis is characterized by iron-dependent lipid peroxidation resulting from insufficient antioxidant protection, particularly impairment of the GSH–GPX4 system that normally reduces lipid hydroperoxides. NRF2 and FSP1 serve as key regulators of HCC sensitivity by modulating antioxidant capacity and lipid metabolism. Dual targeting of these pathways offers a promising therapeutic strategy against HCC. Yet direct clinical evidence demonstrating therapeutic benefit from pharmacological induction of cuproptosis or coordinated cuproptosis–ferroptosis targeting in HCC is currently lacking. We explore the differential impacts of cuproptosis and ferroptosis on tumor progression and discuss the potential therapeutic implications of co-regulating these two cell death mechanisms in HCC.
Chromosomal instability (CIN) is associated with immunosuppression in the tumor microenvironment (TME), resulting in cancer progression, metastasis, and resistance to immune checkpoint blockade therapy. We previously established a tumor immune status scoring algorithm (TIMMUSCORA) in which the immune status in the TME is evaluated numerically from activation to suppression. In the present study, we clarified the relationships between structural variation-related parameters and immunological features by applying TIMMUSCORA to solid cancers. Whole genome sequencing (WGS) and gene expression profiling (GEP) data were obtained from 394 cancer patients, and CIN-related parameters, such as the tumor mutation burden (TMB), structural variant (SV), microsatellite instability (MSI) score, ploidy, homologous recombination deficiency score, and chromothripsis (CT) and whole genome duplication (WGD) scores, were assessed. The TIMMUSCORA system demonstrated that most CIN-related parameters contributed to the low TIMMUSCORA score implicating an immunosuppressive state. Comparisons of differentially expressed genes between WGD- or CT-positive and -negative tumors showed the down-regulation of B cell markers, the down-regulation of myeloid cell markers, and the up-regulation of the NKG2D gene. In addition, the following novel observations were verified; (1) TP53 and EGFR mutation events can be associated with WGD and low TIMMUSCORA scores, and (2) NK cell activation and cancer–testis antigen gene up-regulation might be associated with CT. These results suggest that TIMMUSCORA might be useful tool evaluating immune status of CIN-harboring tumors. In future, the specific mechanism for CIN-associated immunosuppression in the tumor can be explored and clarified.
Tumor-infiltrating myeloid cells occupy heterogeneous functional states that are not adequately represented by macrophage abundance or fixed polarization labels. We developed the myeloid damage response index (MDRi), a single-cell-informed framework comprising injury, resolution and antigen-presentation/interferon (APC/IFN) programs, together with a derived injury–resolution axis. MDRi was established in an immune checkpoint blockade-treated multi-cancer atlas containing 47,750 myeloid cells from 192 samples across eight cancer types and assessed separately in an independent multi-cancer myeloid dataset. The programs organized recurrent macrophage and monocyte states along related but non-identical functional dimensions. Non-overlapping antigen-presentation and interferon-response signatures provided gene-independent support for the APC/IFN dimension. Root-sensitivity analyses showed stable state connectivity but root-dependent pseudotime directionality, supporting transcriptional topology rather than a universal developmental sequence. Exploratory response analyses showed concurrent elevation of injury, resolution and APC/IFN scores in post-treatment non-responders, whereas cancer- and regimen-adjusted analyses did not support an independent predictive effect. Complementary T/NK-cell analyses indicated that response-associated immune differences were more evident in pretreatment samples. In primary-tumor TCGA cohorts summarized at the patient level, joint multivariable Cox models containing MDR injury, MDR resolution and MDR APC/IFN identified MDR injury as an adverse factor in selected cancers, whereas resolution and APC/IFN showed cancer- and endpoint-dependent associations. The derived injury–resolution axis was excluded from these joint models because it is mathematically dependent on injury and resolution. Benchmarking against immune-deconvolution and tumor-associated macrophage signatures demonstrated partial but non-uniform overlap. Nasopharyngeal carcinoma Visium and multi-cancer Xenium analyses further revealed platform- and cancer-dependent spatial distributions of MDRi-related programs. MDRi Explorer provides an open-source implementation for scoring, reference comparison and exploratory clinical analysis. MDRi therefore represents a reusable, hypothesis-generating framework for investigating context-dependent myeloid functional organization rather than a universal prognostic signature or clinically validated predictor.
Opoid dependency (OD) is associated with adverse clinical outcomes in hospitalized patients; nevertheless, the understanding of OD and its impact on cancer patients remains restricted. Alongside addressing the symptoms related to disease, hospitalized patients with OD also grapple with mental illness (MI) challenges and the risk of infectious consequences (ICs), which remain unrecognized in cancer patients. We utilized national inpatient database to examine patient and clinical characteristics among renal cell carcinomas (RCC), prostate cancers (PC) and cancers of the lip, oral cavity, and pharynx (CLOP). We used generalized linear models to evaluate the association of OD and outcome of burden of illness (BOI), specifically length of stay (LOS). We also evaluated the association between OD and emergency department (ED) referral/admission status, ICs (specifically septicemias), weight loss, fluid and electrolyte disorders (FED), status of MI screening, and anxiety and depressive disorders. Of the 88,105 RCC there were 610 OD patients; PC (209,410) with 1115 OD; CLOP (54,265) with 495 OD. PC patients with OD were associated with longer LOS (Coefficient, 1.59; 95
Cancer is increasingly recognized as a systemic disease whose biology is shaped by reciprocal interactions with its microenvironment, including the central and peripheral nervous systems. Recent evidence demonstrates that neural inputs can directly promote tumour growth through synaptic, paracrine, and neuroendocrine mechanisms, and that these inputs intersect with canonical drug-resistance pathways, creating new opportunities and challenges for precision oncology. Primary brain tumours such as gliomas form bona fide functional synapses with neurons, hijacking both excitatory (AMPA/NMDA) and inhibitory (GABA_A) inputs to sustain proliferation. Comparable mechanisms are now described in brain metastases and, remarkably, in small cell lung cancer (SCLC), where cortical and vagal neurons establish synaptic contacts with tumour cells. Beyond synaptic communication, paracrine neurotransmitter signalling, tumour innervation, autonomic balance (β-adrenergic versus vagal tone), and systemic stress responses jointly modulate tumour biology, immune surveillance, and therapeutic response. Convergent evidence further indicates that neural and stress-related signalling cooperates with classical resistance circuits—including p53–EGFR–ERK signalling, P-glycoprotein–mediated drug efflux, reactive oxygen species (ROS)–dependent redox programmes, and adipokine–Hsp90 axes—to attenuate the efficacy of chemotherapy, targeted agents, and immune checkpoint inhibitors. Neuron–tumour interactions represent a novel and clinically actionable dimension of cancer pathogenesis that extends well beyond gliomas. Targeting neuron–tumour synapses, neurotransmitter pathways, autonomic inputs, and the resistance circuits with which they intersect offers new therapeutic opportunities, but translation requires careful attention to specificity, neurological safety, and rational combination with cytotoxic, targeted, and immune therapies. Cancer is not driven solely by genetic mutations; it is also shaped by signals from the nervous system. This review synthesizes how nerves and brain activity directly influence tumour growth across multiple diseases—including gliomas, small cell lung cancer, breast cancer, pancreatic cancer, and prostate cancer—and how these neural signals converge with metabolic and drug-resistance pathways that limit the efficacy of conventional therapy. Recent discoveries show that some cancers form direct synapse-like connections with neurons and exploit neurotransmitters to fuel tumour progression and treatment escape. Understanding these interactions opens new therapeutic opportunities—such as targeting neural signalling, autonomic pathways, or neuro-modulated resistance mechanisms—and positions the nervous system as a previously underappreciated but clinically relevant driver of cancer behaviour.
Synthetic cobalt Schiff bases have recently emerged as promising metal-based anticancer candidates. In this study, we investigated the biological effects of a mononuclear octahedral cobalt(III) Schiff base complex [CoL3] (L = 2-((allylimino) methyl) − 6-methoxyphenol)) on the cell cycle progression and apoptosis of the HT-29 cells, a human colorectal cancer cell line. Cytotoxic activity of [CoL3] was evaluated using the MTT assay. Then, its impact on apoptosis was assessed via annexin V-FITC/propidium iodide (PI) staining. To elucidate the underlying mechanisms, we employed flow cytometry, real-time PCR, and Western blotting to analyze cell cycle distribution and the expression of apoptotic and cell cycle-related genes. Our results revealed that [CoL3] exhibited potent cytotoxicity with an IC50 of 3.47 µM and induced cell cycle arrest in the G1/S and G2/M transition phases in colorectal cancer cells (p < 0.001, p < 0.01). Treatment of colorectal cancer cells with [CoL3] resulted in a significant decrease in CDK4 and CDK6 expression, with mean values of 0.62 (p < 0.05) and 0.33 (p < 0.01), respectively. Additionally, the protein expression of CDK4 and CDK6 was downregulated, with mean protein intensities of 0.48 and 0.79, respectively (p < 0.001), supporting its role in G1 phase blockade. Moreover, [CoL3] downregulated BCL2 (p < 0.01), which was associated with increased early and late apoptosis in treated cells. Docking analyses further demonstrated favorable spontaneous binding of [CoL3] to CDK4 and CDK6, reinforcing its mechanistic involvement in cell cycle inhibition. Collectively, these findings indicate that [CoL3] induces cell-cycle arrest and promotes apoptosis in HT-29 colorectal cancer cells, potentially through the downregulation of CDK4/6 and BCL2.
Combination immunotherapy has shown encouraging activity across multiple solid tumors. We report the complete and consecutive cohort of patients with locally advanced oral squamous cell carcinoma (OSCC) treated at a single center within the prospective, multicenter, randomized phase 2 BelieveIT-201 trial (ASND0038), evaluating neoadjuvant intratumoral Toll-like receptor (TLR) 7/8 agonist (TransCon TLR7/8 Agonist) therapy combined with systemic immunotherapy. Patients with non-metastatic OSCC treated within the BelieveIT-201 trial (ASND0038) between April and December 2024 were included. Neoadjuvant therapy comprised two cycles of intratumoral TransCon TLR7/8 Agonist combined with either intravenous pembrolizumab or TransCon IL-2 β/γ according to 1:1 randomization, followed by surgical resection. The trial was terminated prematurely by the sponsor for reasons unrelated to safety or efficacy, which limited the cohort to six patients. Clinical, radiographic, pathological responses, and safety were assessed. Immunohistochemical analyses of paired pre- and post-treatment tumor samples evaluated immune cell infiltration (CD3, CD8, CD68, CD163). The individual patient was the unit of analysis, and given the small number of patients all analyses are descriptive; no inferential statistical testing was performed. Progression-free survival (PFS) and overall survival (OS) are reported as absolute event counts. Six patients were treated (median age 63 years; median follow-up 81 weeks). Three patients achieved a major clinical response, two showed partial response, and one had progressive disease. Pathologic evaluation revealed one complete response, one major response, and four non-responses. All patients underwent surgery; postoperative morbidity was substantial, with at least one grade III adverse event in every patient and one postoperative death. All three patients with a major clinical response developed sterile tumor-associated pseudoabscesses in spatial proximity to the injection site. Immunohistochemical analyses revealed remodeling of the tumor immune microenvironment, including increased T-cell infiltration, most pronounced in the tumor center. Within the first year, two of six patients experienced a progression-free survival event (n = 1 progressive disease, n = 1 death). After surgery none of the patients showed disease recurrence. Neoadjuvant intratumoral TransCon TLR7/8 Agonist-based combination immunotherapy was feasible in this small prospective cohort of patients with locally advanced OSCC and was accompanied by consistent remodeling of the tumor immune microenvironment. Because of the limited number of patients and the premature termination of the parent trial, no conclusions on efficacy or on comparative tolerability can be drawn.
T cell receptor (TCR)-like antibodies recognize intracellular antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. HLA-A*02:01 (HLA-A2) is the most frequent MHC class I allele worldwide, and most TCR-like antibodies have been designed to target peptides presented by HLA-A2. However, their therapeutic applicability may be limited by allele-restricted peptide presentation and the extensive polymorphism of MHC class I molecules. In this study, we developed a TCR-like antibody specific for the preferentially expressed antigen in melanoma (PRAME) peptide presented by HLA-A*24:02 (HLA-A24), the second most frequent MHC class I allele worldwide and the most common in East Asian populations. PRAME is a cancer-testis antigen expressed in various cancers but absent from normal adult tissues except the testes, making it an attractive target for TCR-like antibodies. An anti-PRAME301–309/HLA-A24 TCR-like scFv 2F7 was identified by phage display and reformatted into a bispecific T-cell–engaging antibody. In vitro studies using human peripheral blood mononuclear cells (PBMCs) and cancer cell lines demonstrated that the 2F7 bispecific TCR-like antibody (bsTLA) specifically bound to the PRAME301–309/HLA-A24 complex and redirected PBMC-derived T cells to kill target cells presenting this peptide-HLA complex.
Patients with advanced solid tumors who progress after standard therapies have limited treatment options. SDH-Combi is an autologous cellular immunotherapy integrating natural killer cells and tumor-primed T lymphocytes, designed to address tumor heterogeneity and immune escape through complementary immune mechanisms. This single-arm, phase 1 study evaluated SDH-Combi in patients with recurrent or progressive solid tumors. SDH-Combi was administered every 2 weeks for up to eight infusions. The primary endpoint was safety and tolerability in all treated patients (N = 9): Safety was assessed as treatment-emergent adverse events graded by CTCAE v5.0 with investigator-assigned causality, and tolerability as the proportion of patients completing all eight planned infusions, the relative dose intensity, and discontinuation due to toxicity. Secondary endpoints were objective response rate, disease control rate, progression-free survival, and overall survival, with tumor response evaluated according to RECIST v1.1. Quality of life (QoL), assessed with the EORTC QLQ-C30, was an exploratory endpoint. Nine patients received at least one infusion. For the primary endpoint, no grade ≥ 3 treatment-related adverse event occurred (0 of 9; 95
CD19-directed chimeric antigen receptor T cell (CAR-T) therapy is effective in relapsed or refractory B cell malignancies, but responses remain heterogeneous. Carbapenem-resistant Klebsiella pneumoniae (CRKP) colonization is common in heavily treated hematology patients. 1,5-Pentanediamine (PDA; cadaverine), a microbiota-associated metabolite produced by several Enterobacteriaceae, was detected in serum from CRKP-colonized patients. Its effects on CAR-T cells are unknown. Serum PDA was quantified by liquid chromatography–tandem mass spectrometry in 30 CRKP-colonized patients without documented bloodstream infection at sampling. Healthy-donor-derived CD19 CAR-T cells were exposed to PDA in vitro (0–12 mM; 9 mM for most functional assays). Metabolic activity, apoptosis, phenotype, target-cell killing, degranulation, cytokine secretion, and cytotoxic-molecule production were assessed. RNA sequencing, RT-qPCR, and PD-1 blockade were performed. PDA was detectable in all 30 patients. Without a non-colonized comparator cohort, these data demonstrate detectability but not CRKP-specific source attribution. At millimolar concentrations, PDA reduced CAR-T cell metabolic activity, increased apoptosis, upregulated activation and checkpoint-associated inhibitory markers, altered the CD4/CD8 distribution, and increased the regulatory T cell frequency. PDA-treated CAR-T cells showed reduced NALM-6 killing and lower cytokine, perforin, and granzyme B output despite preserved CD107a degranulation. Transcriptomic analysis showed enrichment of cell cycle, apoptosis, and stress-related pathways, suppression of immune pathways, and exhaustion-associated transcriptional features. PD-1 blockade alone did not restore function under the tested conditions. Acute millimolar PDA exposure produced a dysfunctional CAR-T cell state with impaired survival and effector output but preserved degranulation. Its physiological relevance requires validation in chronic low-dose models, controlled clinical cohorts, patient-derived CAR-T cells, and additional target-cell systems.
Clinical responses to immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) remain highly heterogeneous and PD-L1 expression alone has limited predictive performance. Emerging evidence suggests that therapeutic response depends not only on immune cell abundance but also on their spatial organization within the tumor microenvironment (TME). Here, we characterized the spatial architecture of the pre-treatment NSCLC TME and evaluated its association with response to ICIs in a real-world clinical cohort. Seventeen pre-treatment specimens (10 responders and 7 non-responders) were analyzed using 13-marker tissue-based cyclic immunofluorescence (t-CyCIF) panel with single-cell phenotyping. Cell density was assessed across all samples. Spatial analyses were performed on resection specimens with preserved architecture (4 responders and 3 non-responders). Spatial analyses included assessment of lymphoid organization, cellular neighborhoods, pairwise spatial relationships and higher-order tissue architecture. Global immune and tumor cell densities did not differ between groups. TLS-related metrics showed a trend toward higher values in responders, suggesting greater immune organization within the TME. Cellular neighborhood analysis further demonstrated enrichment of cytotoxic T cell- and vasculature-associated regions in responders, whereas non-responders displayed a predominantly tumor-centered spatial architecture. Responders also exhibited shorter vessel to cytotoxic T cell distances and greater perivascular enrichment of cytotoxic and PD-1⁺ cytotoxic T cells. Higher-order spatial analysis highlighted preferential organization of PD-1⁺ cytotoxic T cells within vascular-associated niches in responder tumors. These findings demonstrate that spatial organization of immune and vascular compartments provides information beyond cellular abundance alone and may serve as a complementary biomarker framework for predicting immunotherapy response in NSCLC.
Elevated expression of tumor necrosis factor α-induced protein 8 (TIPE) is observed in colorectal cancer (CRC) and is known to regulate decoy receptor 3 (DcR3) expression while promoting M2 polarization of macrophages. However, the precise role of DcR3 in macrophage M2 polarization remains to be elucidated. We employed bioinformatic analysis alongside in vitro and in vivo experimental approaches to investigate the regulatory mechanism by which TIPE influences macrophage M2 polarization in CRC. TIPE expression in CRC tissues positively correlates with M2 macrophage markers (CD206, CD163, IL-10). Functionally, TIPE promotes M2 polarization of macrophages by upregulating DcR3. Mechanistically, DcR3 binds to heparan sulfate proteoglycan 2 (HSPG2) in a heparan sulfate (HS)-dependent manner, leading to activation of the protein kinase Bα1 (AKT1) signaling pathway. AKT1 activation not only directly drives M2 polarization but also enhances macrophage chemotaxis and recruitment toward CRC cells by upregulating the chemokine CXCL8. This study reveals a novel mechanism through which TIPE regulates M2 macrophage polarization and function via DcR3, offering new perspectives for the diagnosis and therapeutic targeting of CRC.
Glioblastoma (GBM) is largely refractory to immune checkpoint blockade (ICB), which may be attributable in part to its immunosuppressive microenvironment and systemic T-cell dysfunction. Drug repurposing provides an efficient strategy for identifying potential immunomodulatory agents. The antibiotic clofoctol (Clo) has shown antitumor activity; however, its effects on the immune landscape of GBM remain poorly understood. In this study, we evaluated the therapeutic efficacy and potential immunomodulatory mechanisms of Clo using an orthotopic GL261 GBM model. Flow cytometric analysis showed that Clo treatment increased the intratumoral representation of CD8+ T cells and enhanced the expression of cytotoxic effector molecules, consistent with a more immunologically active tumor microenvironment. Analysis of bone marrow cell composition further suggested that Clo partially alleviated tumor-associated T-cell accumulation in the bone marrow and promoted their systemic redistribution. Notably, depletion of CD8+ T cells markedly attenuated the antitumor efficacy of Clo, indicating that CD8+ T cells contribute substantially to its therapeutic effects. In vitro co-culture experiments further showed that Clo pretreatment enhanced the cytotoxic activity of CD8+ T cells, accompanied by increased expression of granzyme B (GZMB), perforin (PRF1), and IFN-γ. Mechanistically, ITSA-MS identified BCL9 as a candidate Clo-responsive protein, while subsequent western blot analyses showed that Clo treatment was associated with increased phosphorylation of VAV1, AKT, and ERK. These findings suggest the potential involvement of BCL9-associated signaling in Clo-mediated T-cell activation, although the precise regulatory relationship requires further investigation. Moreover, combined treatment with Clo and anti-PD-1 antibody increased intratumoral CD8+ T cell abundance and effector activity, achieved greater tumor control, and prolonged survival compared with either treatment alone. Collectively, these findings suggest that Clo may exert complementary immunomodulatory effects by facilitating the systemic redistribution of CD8+ T cells and enhancing their cytotoxic function, supporting its further investigation as a potential combination strategy for improving the response of GBM to ICB.
Tertiary lymphoid structures (TLS) are pivotal modulators of anti-tumor immunity in solid tumors; however, their clinical significance in glioblastoma (GBM) remains under-characterized. This study aimed to evaluate the correlation between TLS spatial-quantitative characteristics and survival outcomes in GBM patients to refine prognostic stratification and immunotherapy optimization. A retrospective analysis was conducted on 62 patients with isocitrate dehydrogenase (IDH)-wildtype GBM treated at our institution (2016 – 2023). TLSs were identified via H E and CD3 / CD20 immunohistochemical (IHC) staining. A grading system was established based on the quantity and spatial distribution (intratumoral vs. peritumoral) of TLS. Survival outcomes were assessed using Kaplan–Meier curves and multivariate Cox regression models to identify independent prognostic factors. TLSs were present in 58.1
Glioblastoma remains one of the most lethal primary malignant brain tumors, and immunotherapy has produced limited clinical benefit despite major success in other cancers. This resistance reflects spatial and molecular heterogeneity, myeloid-dominant immune suppression, restricted lymphocyte infiltration, antigenic instability, blood–brain barrier-related constraints, corticosteroid exposure, and treatment-induced immune remodeling. Conventional preclinical models only partially reproduce these features, limiting their ability to predict patient-specific immunotherapy responses. Patient-derived glioblastoma organoids (GBOs) have emerged as experimentally tractable platforms that can preserve key features of parental tumors while enabling functional therapeutic testing. In this review, we discuss the rationale, technical evolution, applications, and translational challenges of GBOs in immuno-oncology, including tumor-immune modeling, checkpoint blockade, cellular therapies, myeloid modulation, vaccines, oncolytic virotherapy, multi-omic readouts, and artificial intelligence-assisted analysis. We place particular emphasis on oncolytic herpes simplex virus-based strategies and on the ability of GBOs to evaluate viral entry, replication, spatial spread, innate antiviral restriction, immunogenic cell death, and rational combinations. Finally, we outline staged, clinically integrated workflows while emphasizing immune-cell attrition, incomplete microenvironmental fidelity, resource requirements, and the need for prospective validation in small exploratory trials.
Background Whole-exome sequencing is a widely used technology to identify pathogenic variants in cancer. Although sequencing itself has become increasingly accessible, downstream analysis remains computationally complex, presenting a challenge for many researchers. Existing pipelines lack integrated support for somatic and germline variant detection and still require significant computational resources. Methods We developed GATES (GATK Automated Tool for Exome Sequencing), a lightweight pipeline that automates data preprocessing, variant calling, and variant annotation directly from raw paired-end FASTQ files through a simplified command-line interface. GATES implements the GATK Best Practices for somatic and germline variant detection and leverages Ensembl’s Variant Effect Predictor for functional annotation, outputting the results in a human-readable tab-separated values (TSV) file. We evaluated the pipeline’s performance using the SEQC-II benchmarking dataset and demonstrated its application using a clinical sample harboring known pathogenic germline and somatic variants. Results GATES was run on a standard laptop and performed end-to-end variant analysis for each sample within a few hours. In benchmarking with SEQC-II samples, germline and tumor-normal somatic variant calling modes demonstrated high concordance with their respective truth sets. Tumor-only somatic mode showed decreased accuracy, consistent with expected germline contamination. GATES demonstrated high performance across various hardware configurations and compared to the established nf-core/sarek pipeline. GATES further successfully performed somatic and germline analysis of a >100X clinical sample in under 7 hours. Importantly, the pipeline accurately distinguished the known KRAS p.G12V and KEAP1 p.S338L as somatic and germline, respectively. Conclusion By lowering the technical barriers to exome sequencing analysis, GATES provides a practical solution for pathogenic variant discovery for researchers both with and without computational expertise.
