Breast cancer brain metastasis (BCBM) is a major cause of breast cancer-related mortality, but the molecular mechanisms underlying its progression remain poorly understood. Here, we profiled the tumor immune microenvironment of BCBM at single-cell resolution and identified candidate regulators associated with brain metastatic progression. Single-cell RNA sequencing (scRNA-seq) was performed on brain metastatic tissue, adjacent tumor tissue, cerebrospinal fluid, and circulating tumor cells (CTCs) from seven patients, and bulk DNA sequencing was conducted on primary breast tumors, peripheral blood, and brain metastases from 47 patients. Analysis of 131,880 single cells identified 63 distinct cell clusters, including 12 tumor-associated macrophage (TAM) subtypes and 5 metastatic tumor cell (MTC) subtypes. Circulating TAMs displayed an M1-like inflammatory phenotype, whereas tissue-resident TAMs were predominantly M2-like. MTCs exhibited substantial transcriptional heterogeneity, and a neuro-related subtype showed adaptive upregulation of neuronal signaling pathways. Brain metastases also harbored a higher mutational burden than primary tumors, with recurrent mutations in GABRB3 and NRXN1 associated with poorer patient survival. Functional experiments further showed that loss of GABRB3 or NRXN1 impaired tumor growth and brain colonization in xenograft models. These findings nominate GABRB3 and NRXN1 as candidate regulators of brain metastatic fitness in BCBM and support their further evaluation as biomarkers and therapeutic targets.

This study aimed to evaluate the survival outcomes and identify prognostic factors in patients undergoing surgical resection for brain metastases (BMs), with a particular focus on the impact of surgical techniques and multimodal therapy sequences. We conducted a retrospective analysis of 228 consecutive patients who underwent neurosurgical resection for BMs at a single institution (2012–2020). Primary endpoints were median overall survival (mOS) and survival rates, analyzed via Kaplan-Meier methodology. Prognostic factors were assessed using univariate and multivariate Cox proportional hazards models, with subgroup analyses based on primary tumor origin. The cohort’s overall mOS was 14 months, with 1-, 2-, and 3-year survival rates of 60.3

Medulloblastoma (MB), a heterogeneous pediatric brain tumor, poses challenges in the treatment of tumor recurrence and dissemination. To characterize cellular diversity and genetic features, we comprehensively analyzed single-cell/nucleus RNA sequencing (sc/snRNA-seq), single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), and spatial transcriptomics profiles and identified distinct cellular populations in SHH (sonic hedgehog) and Group_3 subgroups, with varying proportions in local recurrence or dissemination. Local recurrence showed higher cycling tumor cell enrichment, whereas disseminated lesions had a relatively notable presence of differentiated subsets. Chromosomal alteration evaluation revealed distinct genetic subclones during MB progression, such as chr7q gain and chr11 loss in Group_3 disseminations. A subpopulation termed “high cellular plasticity (HCP)” emerged during MB progression and was associated with increased dividing potential and chromatin accessibility, contributing to recurrence. Inhibiting HCP-associated markers, like protein tyrosine phosphatase receptor type Z1 (PTPRZ1), efficiently suppressed MB progression in preclinical models. These findings address critical gaps in understanding the cellular diversity, chromosomal alterations, and biological dynamics of recurrent MB, offering potential therapeutic insights.

Cancer-testicular antigens (CTAs) have been considered as potential prognostic biomarkers and therapeutic targets due to their specific expression and roles in tumorigenesis and metastasis. Among these, the function and mechanism of SPANXB1 in breast cancer brain metastasis (BCBM) remain poorly understood. In this study, we investigated the role of SPANXB1 in BCBM. Our results demonstrated that SPANXB1 was highly expressed in brain-tropic breast cancer cells and brain metastasis samples. Functional assays revealed that SPANXB1 promoted breast cancer cell invasion, migration, vasculogenic mimicry (VM) formation, and blood-brain barrier (BBB) extravasation, thereby accelerating the process of brain metastasis. Mechanistically, SPANXB1 facilitated chromatin opening at the MMP1 promoter region via histone H3R17me2 modification and upregulated transcription factor YY1, leading to increased MMP1 expression. In vivo experiments further confirmed the role of SPANXB1 in enhancing brain metastasis. Notably, metformin effectively inhibited the expression of SPANXB1 and MMP1, thereby attenuating BCBM progression. The present study indicates the potential of SPANXB1 as a diagnostic and therapeutic target for BCBM. Additionally, our findings suggest metformin as a promising therapeutic strategy for this highly aggressive disease.

Two single-center Phase I trials evaluated safety (primary endpoint) and preliminary efficacy (secondary endpoint) of oncolytic adenovirus Ad-TD-nsIL12 in primary (Group A, NCT05717712) and progressive (Group B, NCT05717699) pediatric patients with IDH wild-type (WT) diffuse intrinsic pontine glioma (DIPG). Studies employed single-arm and 3 + 3 dose-escalation design. 9 patients were enrolled in Group A and 6 in Group B. Group A completed the dose escalation, and no severe adverse events were observed. Enrollment in Group B was halted after Group A completed escalation. All patients experienced drug-related adverse events. In Group A, three partial responses and five stable diseases were documented, with a median overall survival (mOS) of 10.3 months after the first virus and 11.3 months after onset. In Group B, three patients had stable diseases, and three had progressive disease, with an mOS of 6.4 months after the first virus and 12.7 months after onset. Both groups demonstrated improved mOS from onset compared to the DIPG patients in our center's retrospective study (mOS, 8.3 months). Both groups showed increased lymphocytes post-treatment, but only Group A decreased after radiotherapy. These trials confirmed the safety of Ad-TD-nsIL12 and provided preliminary efficacy evidence, offering insights for future clinical applications in DIPG.

BRG1 deficiency in patients with lung adenocarcinoma that has metastasized to the brain, termed BRG1-deficient brain metastasis lung adenocarcinoma, is an uncommon event. Prior to this study, these patients had not undergone extensive molecular and (epi)genetic analysis. We report a comprehensive clinical, histopathologic, and molecular assessment of 9 BRG1-deficient brain metastasis lung adenocarcinoma cohort (BRG1-deficient BM cohort) in comparison with a 16 BRG1-retained brain metastasis lung adenocarcinoma cohort (BRG1-retained BM cohort). Patients with BRG1-deficient BM exhibited a significantly increased risk of mortality. Molecular analysis revealed a high prevalence of mutations in SMARCA4 and TP53 genes within this group. DNA methylation molecular diagnostics showed a high rate of genomic instability and a markedly lower DNA methylation age in these patients. Functional enrichment analysis of differentially methylated genes suggested that hypomethylation genes were primarily associated with the negative regulation of neuron differentiation, G protein-coupled receptor signaling pathways, and cell differentiation. Conversely, hypermethylation was linked to the regulation of small GTPase mediated signal transduction, Rho protein signal transduction, DNA damage response, and apoptotic processes. This study investigated a rare subgroup of lung adenocarcinoma patients with brain metastasis characterized by BRG1 deficiency and a poor prognosis. Our study not only provides a comprehensive multi-omic data resource but also provides valuable biological insights into patients. The findings may serve as a valuable reference for the future pathological diagnosis of BRG1-deficient brain metastasis in lung adenocarcinoma patients.

2057 Background: Glioma is the most common primary central nervous system malignancy in adults, of which glioblastoma (GBM) accounts for more than 50% of the incidence and is the most aggressive subtype of glioma, with a median survival of about 15 months. Oncolytic virus emerges as a promising therapy for recurrent GBM (rGBM), but its safety and efficacy are not evaluated in patients with rGBM connecting to ventricular system. Methods: This is a dose-escalating trial to study the safety and efficacy of Ad-TD-nsIL12, a novel oncolytic adenovirus, in patients with rGBM connecting to the ventricular system. The assigned dose levels of Ad-TD-nsIL12 were 5x109vp, 1x1010vp, and 5x1010vp. Adverse events (AEs) associated with the virus were graded using the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0), and Grade ≥ 3 AEs identified the dose-limiting toxicity (DLT). Tumor response was evaluated based on the Response Assessment in Neuro-Oncology (RANO) criteria. Results: Eight patients, aged from 45 to 71 years, were enrolled between December 2019 and September 2022, with treatment doses ranging from 5x109 to 5x1010vp. Grade 3 seizure according to CTCAE occurred in two patients from Cohort 3 (5x1010vp) after AD-TD-nsIL12 injection. Minimal adverse events were observed at a treatment dose of 1x1010 vp, even after multiple injections. Complete response (CR) was demonstrated in one patient, partial response (PR) in one patient, stable disease (SD) in four patients and progressive disease (PD) in two patients. Immunohistochemical staining showed higher infiltrations of CD3+, CD4+ and CD8+ T cells and positivity of E1A and Hexon in virus post-treated tissues. Inflammation associated cytokines in the blood were not significantly elevated by Ad-TD-nsIL12. Conclusions: AD-TD-nsIL12 treatment was safe and effective in patients with rGBM and warrants examination in a phase II clinical study. Clinical trial information: ChiCTR2000032402.

Computed tomography (CT) scans and magnetic resonance imaging (MRI) are commonly utilized to detect brain gliomas and central nervous system inflammation diseases. However, there are instances where depending solely on medical imaging for a precise diagnosis may result in unsuitable medications or treatments. Pathological analysis is regarded as the definitive method for diagnosing brain gliomas or central nervous system inflammation diseases. To achieve this, a craniotomy or stereotaxic biopsy is necessary to collect brain tissue, which can lead to complications such as cerebral hemorrhage, neurological deficits, cerebrospinal fluid leaks, and cerebral edema. Consequently, the advancement of non-invasive or minimally invasive diagnostic techniques is currently a high priority. This study included samples from four glioma patients and five patients with central nervous system inflammatory diseases, comprising both serum and paired cerebrospinal fluid (CSF). A total of 40 human cytokines were identified in these samples. We utilized a receiver operating characteristic (ROC) analysis to assess the sensitivity and specificity for distinguishing central nervous system inflammation diseases and gliomas. Additionally, we examined the correlation of these factors between serum and CSF in the patients. Ultimately, the identified factors were validated using serum from patients with clinically confirmed gliomas and central nervous system inflammation diseases followed by detection and statistical analysis through ELISA. The levels of serum factors IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, CCL11, and VEGF were found to be significantly higher in gliomas compared with inflammatory diseases of the central nervous system (p < 0.05). Furthermore, a strong correlation was observed between the levels of CCL4 in serum and CSF, with a correlation coefficient of r = 0.92 (95% CI = 0.20–0.99, p = 0.027). We gathered more clinical samples to provide further validation of the abundance of CCL4 expression. A clinical study analyzing serum samples from 19 glioma patients and 22 patients with central nervous system inflammation diseases revealed that CCL4 levels were notably elevated in the inflammatory group compared with the glioma group (p < 0.001). These results suggest that assessing serum CCL4 levels may be useful in distinguishing those patients for clinical diagnostic purposes.

Malignant glioma is a highly fatal central nervous system malignancy with high recurrence rates. Oncolytic viruses offer potential treatment but need improvement in efficacy and safety. Here we describe a phase I, dose-escalating, single arm trial (ChiCTR2000032402) to study the safety of Ad-TD-nsIL12, an oncolytic adenovirus expressing non-secreting interleukin-12, in patients with recurrent high-grade glioma that connects with the ventricular system. Eight patients received intratumoral treatment via stereotaxis or an Ommaya reservoir, with doses ranging from 5 x 109 to 5 x 1010vp. The primary end point was to determine the maximal tolerated dose. Secondary endpoints included toxicity and anti-tumour ability. Minimal adverse events were observed at doses of 5 x 109 and 1 x 1010vp. Grade 3 seizure was observed in two patients from Cohort 3 (5 x 1010vp). Therefore, the maximum tolerated dose was determined to be 1 x 1010vp. Four patients developed hydrocephalus during follow-up. Among them, symptoms in two patients were relieved after placement of a ventriculo-peritoneal shunt, and the other two only showed ventriculomegaly on MRI scan without neurological deterioration. Complete response (according to Response Assessment in Neuro-Oncology Criteria) in one patient, a partial response in one patient and post-treatment infiltrations of CD4+ and CD8 + T cells into the tumour were documented during this trial. In conclusion, Ad-TD-nsIL12 has demonstrated safety and preliminary efficacy in patients with recurrent high-grade glioma. Oncolytic viruses have been tested in patients with malignant glioma, however clinical efficacy remains limited. Here the authors report the results of a phase I trial of Ad-TD-nsIL12, an oncolytic adenovirus expressing a mutant (non-secreting) form of IL12, in patients with high-grade glioma.

AIMS:Gliomas are the most common central nervous system malignancies, with limited therapeutic options and poor prognosis, which are primarily attributed to the "immune desert" microenvironment. Previously, we constructed a three-gene-deleted oncolytic adenovirus (Ad-TD) loaded with non-secreting interleukin-12 (nsIL-12), which could be amplified in tumor cells and induce immunity to suppress tumors. However, the effects of this oncolytic virus on gliomas and their immune microenvironment remain unclear. There is an urgent need for further research.MATERIALS AND METHODS:We constructed a Syrian hamster brain tumor model and demonstrated the efficacy and mechanism of the novel oncolytic virus in treating brain tumors through a series of in vitro and in vivo experiments. We investigated the efficacy and safety (the number of hamsters in each group is either 5 or 10) of the oncolytic virus treatment in Syrian hamsters using a virus-treated group, a control virus-treated group, and a blank control group.KEY FINDINGS:In vitro assays showed that Ad-TD-nsIL-12 could specifically proliferate in brain tumor cells which induce tumor cell apoptosis and intracellular expression of interleukin (IL)-12. Moreover, in vivo experiments demonstrated that Ad-TD-nsIL-12 could effectively inhibit the progression of brain tumors and prolong survival. Ad-TD-nsIL-12 significantly enhanced T-cell infiltration in the brain tumor microenvironment.SIGNIFICANCE:Ad-TD-nsIL-12 can inhibit glioma progression and increase T-cell infiltration in the tumor tissue, particularly infiltration by cytotoxic T cells (CD8+). Ad-TD-nsIL-12 can amplify and produce IL-12, inducing anti-glioma immune responses to inhibit tumor progression.

AIMS:Chordoma is a rare and aggressive bone tumor with high-recurrence and lack of effective treatment methods. Tumor associated macrophages (TAMs) are abundant in tumor microenvironment (TME) and polarize toward M2 in chordoma. It has been observed that the high proportion of M2 cells is associated with chordoma rapid progression. However, the mechanism of TAMs polarization and promotion to tumor progression in chordoma is still unclear. The is an urgent need for further research. MATERIALS AND METHODS:Flow cytometry and immunohistochemical staining was used to detect the degree of macrophages infiltration in chordoma. A co-culture model of chordoma cells and macrophages was established in vitro to investigate the effects of their interaction on cell function, cytokine secretion, and RNA transcriptome expression. KEY FINDINGS:In this study, we found M2 macrophage was predominantly abundant immune cell population in chordoma, and its proportion was associated with the degree of bone destruction. We demonstrated that interleukin 6 (IL-6) derived from chordoma cells could induce TAMs polarization by activating STAT3 phosphorylation, and TAMs could enhance chordoma cells migration and invasion through TNFα/NF-κB pathway. The interaction of chordoma cells and TAMs could promote the bone destruction-related factor Cathepsin B (CTSB) and inhibitory immune checkpoints expression. We also confirmed blocking IL-6/STAT3 pathway could significantly attenuate the M2 polarization of TAMs and decrease the secretion of TNFα. SIGNIFICANCE:This study illustrates the dynamics between chordoma cells and TAMs in promoting chordoma invasion and suggests that IL-6/STAT3 pathway is a potential therapeutic target to reduce TAM-induced chordoma invasion.

The pathological characteristics about MB25550 showed the classic (left) and SHH (right) subgroups.

Background: Gliomas are the most commonly-detected malignant tumors of the brain.They contain abundant long non-coding RNAs (lncRNAs), which are valuable cancer biomarkers.LncRNAs may be involved in genomic instability; however, their specific role and mechanism in gliomas remains unclear.LncRNAs that are related to genomic instability have not been reported in gliomas. Methods:The transcriptome data from The Cancer Genome Atlas (TCGA) database were analyzed.The co-expression network of genomic instability-related lncRNAs and mRNA was established, and the model of genomic instability-related lncRNA was identified by univariate Cox regression and LASSO analyses.Based on the median risk score obtained in the training set, we divided the samples into high-risk and low-risk groups and proved the survival prediction ability of genomic instability-related lncRNA signatures.The results were verified in the external data set.Finally, a real-time quantitative polymerase chain reaction assay was performed to validate the signature. Results:The signatures of 17 lncRNAs (LINC01579, AL022344.1,AC025171.5,LINC01116, MIR155HG, AC131097.3,LINC00906, CYTOR, AC015540.1,SLC25A21.AS1, H19, AL133415.1,SNHG18, FOXD3.AS1, LINC02593, AL354919.2and CRNDE) related to genomic instability were identified.In the internal data set and Gene Expression Omnibus (GEO) external data set, the low-risk group showed better survival than the high-risk group (p<0.001).In addition, this feature was identified as an independent risk factor, showing its independent prognostic value with different clinical stratifications.The majority of patients in the low-risk group had isocitrate dehydrogenase 1 (IDH1) mutations.The expression levels of these lncRNAs were significantly higher in glioblastoma cell lines than in normal cells. Conclusions:Our study shows that the signature of 17 lncRNAs related to genomic instability has prognostic value for gliomas and could provide a potential therapeutic method for glioblastoma.

supplementary figure legends.

H.E. staining for the intracranial xenograft derived from the MB25550 CSCs.

Daoy CSCs were treated with the indicated concentration of OTSSP167, DZNep and combination of both inhibitors for 42 h and proliferation was measured by MTT. No significant differences existed in the three therapeutic groups (p = 0.31).

Background: Gliomas are the most commonly-detected malignant tumors of the brain. They contain abundant long non-coding RNAs (lncRNAs), which are valuable cancer biomarkers. LncRNAs may be involved in genomic instability; however, their specific role and mechanism in gliomas remains unclear. LncRNAs that are related to genomic instability have not been reported in gliomas. Methods: The transcriptome data from The Cancer Genome Atlas (TCGA) database were analyzed. The co-expression network of genomic instability-related lncRNAs and mRNA was established, and the model of genomic instability-related lncRNA was identified by univariate Cox regression and LASSO analyses. Based on the median risk score obtained in the training set, we divided the samples into high-risk and low-risk groups and proved the survival prediction ability of genomic instability-related lncRNA signatures. The results were verified in the external data set. Finally, a real-time quantitative polymerase chain reaction assay was performed to validate the signature. Results: The signatures of 17 lncRNAs (LINC01579, AL022344.1, AC025171.5, LINC01116, MIR155HG, AC131097.3, LINC00906, CYTOR, AC015540.1, SLC25A21. AS1, H19, AL133415.1, SNHG18, FOXD3.AS1, LINC02593, AL354919.2 and CRNDE) related to genomic instability were identified. In the internal data set and Gene Expression Omnibus (GEO) external data set, the low-risk group showed better survival than the high-risk group (P < 0.001). In addition, this feature was identified as an independent risk factor, showing its independent prognostic value with different clinical stratifications. The majority of patients in the low-risk group had isocitrate dehydrogenase 1 (IDH1) mutations. The expression levels of these lncRNAs were significantly higher in glioblastoma cell lines than in normal cells. Conclusions: Our study shows that the signature of 17 lncRNAs related to genomic instability has prognostic value for gliomas and could provide a potential therapeutic method for glioblastoma.

A-B, Expression of MELK and EZH2 in pan-cancers from TCGA database. C, Correlation between MELK and EZH2 in SHH associated MB from GSE50765 dataset. D, Correlation between MELK and EZH2 mRNA relative expression in GBM from TCGA database.

Objective:To investigate the efficacy of surgical therapy of acoustic neuromas after failed gamma knife treatment.Methods:The clinical data of 23 patients with acoustic neuromas treated in the Department of Neurosurgery, Sanbo Brain Hospital of Capital Medical University from January 2010 to December 2021 were retrospectively analyzed. Twenty-three patients who were previously treated with gamma knife (experimental group) underwent surgical resection because of tumor progression. Functional nerve preservation was performed during the operation. Another 23 patients who had not been treated with gamma knife were defined as the control group with matching sex composition ratio, age, tumor size, texture and location. The patients were clinically followed up and the facial nerve function was evaluated by House-Brackmann (HB) grading system. The recurrence or progression of the tumor was evaluated by imaging follow-up. The baseline data, degree of surgical resection, postoperative complications, outcome of facial nerve function and recurrence were compared between the two groups.Results:The degree of adhesion between facial nerve and tumor in the experimental group was greater than that in the control group ( P<0.001). The total tumor resection rate in the experimental group (21.7%, 5/23) was lower than that in the control group (73.9%, 17/23)( χ2=12.54, P<0.001). The anatomic preservation rate of facial nerve was 100% (23/23) in both groups. Postoperative HB gradeⅠ-Ⅱ in the experimental group accounted for 73.9% (17/23), which was not significantly different from that in the control group (91.3%, 21/23) ( P=0.189). The median follow-up time was 57 months (range: 12-133 months) in the experimental group and 52 months (range: 14-120 months) in the control group. There was no tumor recurrence or progression in the two groups. At the last follow-up, the patients with HB grade Ⅰ-Ⅱ in the experimental group and control group accounted for 82.6% (19/23) and 95.7% (22/23) respectively, and there was no significant difference between the two groups ( P=0.412). Conclusions:The degree of surgical resection of advanced acoustic neuromas after failed gamma knife treatment is relatively low. Functional nerve preservation resection can optimize the results of facial nerve function and doesn′t increase the risk of tumor recurrence.

Table S1. Univariate and multivariate analysis by Cox proportional model.