Brain metastasis (BM) in renal cell carcinoma (RCC) remains poorly understood and often resistant to immune checkpoint inhibitors. We generated a large single-nucleus RNA-seq data of RCC BM, profiling 14 BM samples alongside matched extracranial metastases and primary tumors. Tumor cells in BM displayed neuronal infiltration, neural-like adaptation, and marked remodeling of the microenvironment, including expansion of immunosuppressive myeloid cells and depletion of antigen-presenting dendritic cells. Tumor, immune, and stromal cells exhibited metabolic rewiring characterized by fatty-acid metabolism, oxidative phosphorylation, and MYC-driven programs. CD8 T cells showed terminal exhaustion and impaired proliferative capacity, and tertiary lymphoid structures were absent. Spatial profiling of 12 BM samples (13,128 cells) validated key cellular interactions, while ligand-receptor analysis revealed immunoregulatory circuits between tumor, stromal, and immune cells. These findings define BM-specific adaptations that promote immune evasion and resistance, revealing therapeutic vulnerabilities in RCC BM.

Abstract Amongst solid cancers, melanoma brain metastases (MBM) have the highest likelihood of metastasizing to the brain. The use of immune checkpoint inhibitors (ICI) has emerged as first line therapy; however, intracranial progression and acquired resistance highlight the need for novel immunotherapeutic strategies for this patient population. Oncolytic viral (OV) therapy leverages tumor cell replication machinery to selectively replicate and kill tumor cells while promoting anti-tumor immunity. The oncolytic adenovirus Delta-24-RGDOX (RGDOX) has been shown to function as a TME modulator in both preclinical and clinical settings. Here, we investigated the anti-tumor efficacy of RGDOX combined with ICIs in synchronous extracranial—intracranial (s.c.-i.c.) and intracranial—intracranial (i.c.-i.c.) murine melanoma models (B16F10 and D4M). Using this approach, we inoculated the s.c. (s.c.-i.c. model) or i.c. (i.c.-i.c.) tumor with RGDOX and monitored for viral induced cytotoxicity within the infected tumor while assessing for the simultaneous generation of intracranial antitumor immunity against the uninfected tumor. The combinatorial strategy of RGDOX with PD-1/CTLA-4 inhibition significantly reduced tumor growth within the infected and distant intracranial tumor while also improving survival in tumor bearing mice. Through a series of immune depletion experiments, therapeutic efficacy was determined to be mediated through CD8+ T cells. Additionally, eradication of subcutaneous tumor led to immunologic memory preventing intracranial tumor formation. Immunophenotyping and spatial profiling demonstrated that treatment boosted anti-tumor immunity in both infected and uninfected intracranial tumors through enhanced T-cell activity, and increased infiltration of both dendritic cells and inflammatory macrophages. In contrast, immunosuppressive populations were reduced in both infected tumors and uninfected intracranial tumors following treatment. These findings provide the foundation for our trial—Dose Ranging, Toxicity Seeking, Phase 1 Trial of Oncolytic Adenoviral Therapy for Melanoma Intracranial and Extracranial Metastases (NCT07444606)—and suggest oncolytic viral therapy is a promising therapeutic approach for the generation of antitumor immunity against MBM.

Abstract Introduction: Glioblastoma (GBM) is characterized by profound molecular heterogeneity and resistance to current therapies, underscoring the need for innovative strategies that target noncanonical oncogenic mechanisms. Emerging evidence indicates that structured RNA elements, particularly precursor microRNAs, represent functionally critical and pharmacologically tractable regulators of tumor progression. Among these, miR-10b is consistently overexpressed in GBM and contributes to tumor aggressiveness through repression of key tumor suppressor pathways. Methods: Integrative analysis of TCGA datasets and independent patient-derived samples identified miR-10b as a highly upregulated microRNA in GBM. To interrogate the therapeutic potential of RNA structure-directed inhibition, patient-derived glioblastoma stem-like cells were evaluated in 2D cultures and in 3D human cerebral organoids derived from induced pluripotent stem cells. An RNA structure-targeting inhibitor designed to bind the precursor hairpin of miR-10b and disrupt its maturation was applied to GBM models. Molecular and phenotypic effects were assessed by RT-qPCR, immunoblotting, immunofluorescence, and high-content imaging. Single-cell RNA sequencing (scRNA-seq) was used to define transcriptomic remodeling and tumor-neuron interactions within the organoid context. Results: RNA structure-targeted inhibition selectively reduced mature miR-10b levels by blocking precursor processing, leading to reactivation of the tumor suppressors PTEN and HOXD10. This molecular reprogramming resulted in significant suppression of GBM cell proliferation and invasive behavior. In human cerebral organoids, treatment markedly limited tumor expansion while preserving neuronal viability and synaptic marker expression. scRNA-seq analysis revealed attenuation of oncogenic signaling pathways and restoration of differentiation-associated transcriptional programs, without perturbation of global miRNA biogenesis or neuronal cell states. Discussion: These findings demonstrate that structured RNA intermediates represent actionable therapeutic targets in glioblastoma. Targeting miRNA precursor architecture enables selective suppression of oncogenic networks while sparing normal neural tissue, addressing a central challenge in brain cancer therapy. Conclusions: RNA structure-targeted inhibition effectively suppresses glioblastoma growth in human cerebral organoids by disrupting miR-10b maturation and restoring tumor suppressor signaling. This work establishes RNA structural elements as a novel therapeutic vulnerability in GBM and supports their further exploration in translational neuro-oncology. Citation Format: Maria-Ancuta Jurj, Michael A. Attathikhun, Sanjay Kumar Singh, Meng Chen, Venkata Narayana Vidadala, Frederick Lang, Gabriele Varani, George A. Calin. RNA structure-targeted inhibition suppresses glioblastoma growth in human cerebral organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB296.

Diffuse midline glioma (DMG)/diffuse intrinsic pontine glioma (DIPG) are deadly childhood gliomas with median survival less than 12 months. D24, a genetically engineered adenovirus specific for glioma, has anti-tumoral effect in a phase 1 study when given by direct intratumoral injection. Our preliminary work has shown endovascular super-selective intra-arterial delivery of D24 packaged in mesenchymal stem cells (MSC-D24) is feasible and safe in patients. We hypothesize that endovascular intra-arterial delivery of MSC-D24 will be safe and effective for treatment of DMG/DIPG. In vitro killing assays were used to assess the tumoricidal effects of MSC-D24 against DIPG using TP54, DIPG 36, and SF8628 cell lines (from 102 to 105 cells). Transwell migration assays were performed to test homing of MSC (105 cells) to the same cell lines. To assess safety of infusion in the vertebrobasilar circulation, a rabbit survival model (7 days) was used and 0.4 mL of 107 MSC-D24 cells were infused into the basilar artery. Clinical examination, histology, angiography, and MRI were used to assess stroke and other complications after infusion. The efficacy of MSC-D24 against will be tested in-vivo using a mice xenograft model of DIPG. Transwell killing assays showed a dose dependent tumoricidal effects of MSC-D24 against all 3 DIPG cell lines. MSCs successfully homed to TP54 in the migration assay. Rabbits (n=3) did not exhibit neurologic deficits after the MSC-D24 intra-arterial infusion and MRI, histology, and angiography post-infusion did not show any strokes or other evidence of tissue injury. Bioluminescent imaging of DIPG bearing mice showed tumor reduction and improved survival. Endovascular vertebrobasilar intra-arterial infusion of MSC-D24 is safe and the oncolytic virus appears effective against DIPG in both in vitro and in vivo models. The results support a phase 1 trial of MSC-D24 for patients with DMG/DIPG.

Advanced solid tumors pose significant clinical challenges due to a heterogeneous and immunosuppressive tumor microenvironment, which limits the efficacy of approved therapies. Based on promising clinical trial results with the oncolytic adenovirus (OA) Delta-24-RGD in glioma patients, we previously demonstrated that Delta-24-RGDOX, engineered to express the immune co-stimulatory molecule OX40 ligand (OX40L), enhances local and systemic anti-tumor immunity via an in situ autovaccination effect in immunocompetent mice bearing orthotopic intracranial (i.c.) gliomas or disseminated subcutaneous (s.c.)/i.c. melanomas. To further enhance therapeutic efficacy, we developed Delta-24-RGDOX-IL15, a novel OA co-expressing IL-15 and OX40L. IL-15 is known to activate NK and T cells and support the persistence of CD8⁺ memory T cells. Delta-24-RGDOX-IL15 maintained robust viral replication and oncolytic activity comparable to its predecessors and more effectively stimulated T cell responses, including CAR T cells, against cancer cells expressing corresponding tumor-associated antigen. In C57BL/6 mice bearing syngeneic GD2-expressing tumors, including i.c. gliomas or disseminated s.c./i.c. melanomas, intratumoral delivery of Delta-24-RGDOX-IL15 into the i.c. glioma or s.c. melanoma significantly reduced microglia and myeloid cell frequency while increasing T cell frequency and activation of NK, dendritic, and T cells within the tumor-bearing brain hemisphere from the mice of the two mouse models. Notably, when combined with murine GD2 CAR T cells, Delta-24-RGDOX-IL15 enhanced CAR T cell accumulation in both treated and distant, untreated brain tumors, leading to long-term survival in 20% of mice with s.c./i.c. melanomas. Currently, we are optimizing the treatment regimen to achieve better efficacy in both mouse models. Together, these findings demonstrate the potent immunostimulatory and therapeutic activity of Delta-24-RGDOX-IL15 and its potential to synergize with CAR T cell therapy in treating advanced primary or metastatic brain tumors.

Malignant gliomas pose a significant therapeutic challenge. Remarkably, complete tumor regressions have been observed in a subset of patients with recurrent malignant gliomas treated with Delta-24-RGD oncolytic adenovirus in phase 1 and 2 clinical trials. Radiographical pseudo-progressions preceded tumor regressions, suggesting the involvement of an anti-tumor immune response. To evaluate the role of T cells, we confirmed CD3+ T cell infiltration in tumors obtained during a phase 1 clinical trial (NCT00805376) and further investigated the T cell landscape using T cell receptor (TCR) sequencing. Notably, tumors resected two weeks post-treatment (n=9) exhibited significantly reduced TCR diversity compared to pre-treatment tumors (n=6, p = 0.036). An independent dataset of peripheral blood from pediatric DIPG patients treated with Delta-24-RGD (NCT03178032) confirmed the decrease in TCR diversity post-treatment (n=10, p = 0.043). Notably, patients who retained higher TCR diversity post-treatment demonstrated prolonged survival (p = 0.022), strongly suggesting a link between immune repertoire retention and clinical outcomes. Further analysis of pre- and post-treatment samples revealed limited overlap in T cell clonotypes, with only 20% of the post-treatment clonotypes detected in pre-treatment tumors. The degree of overlap correlated with patient survival (p = 0.039). Using GLIPH2 clustering, we identified 617 specificity groups among 2,737 tumor-derived CDR3b sequences, with CDR3 motifs including RHAG, KSFS and S%SPKET shared with an independent glioma dataset, potentially indicating glioma-reactive T cells. In summary, our findings provide for the first time a clinically relevant assessment of T cell dynamics in oncolytic-adenovirus-treated glioma patients. Based on our clinical data revealing the significance of TCR diversity and repertoire overlap, we demonstrate that immune profiling is a potential biomarker for therapy response and survival, paving the way for more precise and effective immunotherapy strategies for glioma.

Chordomas are locally aggressive neoplasms accounting for approximately 20% of primary spinal tumors. Patients experience high rates of disease progression and limited treatment options, highlighting an unmet clinical need and the importance of identifying novel therapeutic strategies for chordomas. Oncolytic viral (OV) therapy uses genetically modified viruses which selectively replicate in tumor cells, mediate tumor cell lysis, and initiate a pro-inflammatory response within the infected tumor microenvironment (TME). This study evaluates the anti-chordoma effect of the oncolytic adenovirus Delta-24-RGD. The efficacy of Delta-24-RGD was assessed using in vitro approaches, ex vivo bone scaffolds, and in vivomurine models. Additionally, we explored the underlying mechanism of OV cytotoxicity, immunogenic cell death (ICD), brachyury modulation, and reshaping of the TME towards a pro-inflammatory state. Delta-24-RGD achieved viral infectivity, oncolysis and ICD across multiple human chordoma cell lines and ex vivo bone scaffold models. Viral infection was associated with concomitant brachyury downregulation within both infected and uninfected chordoma cells in vitro. In vivo murine xenograft models of human CH22 and U-CH1 chordoma treated with Delta-24-RGD resulted in tumor volume reduction and enhanced overall survival. RNA-Seq analysis followed by Ingenuity Pathway Analysis predicted activation of tumor-associated extracellular matrix remodeling and host immune response pathways. Analyzing a human chordoma tissue microarray, the immunosuppressive macrophage marker CD163 was associated with shortened recurrence-free survival. Using macrophage/chordoma co-culture, OV infection reduced CD163 expression, induced a concomitant pro-inflammatory macrophage polarization, and enhanced cytotoxicity against chordoma cells. This synergistic cytotoxic effect was further validated in co-cultures of chordoma cell lines with healthy donor and chordoma patient-derived peripheral blood mononuclear cells (PBMCs). Lastly, histological evaluation of patient-derived chordoma tumor slice cultures confirmed infectivity of Delta-24-RGD, underscoring the clinical potential of this oncolytic virus. These studies provide a framework for future clinical translation amongst patients with recurrent or metastatic chordoma.

Glioblastoma (GBM) is the most common primary brain tumor in adults and associated with abysmal patient outcomes. While it is well-known that glutamatergic neurons can form functional synapses with GBM cells to enhance tumor growth, less is known about the effects of the activity of other neuronal types on GBM biology. Here, we examined the effect of acetylcholine on the growth of GBM cells belonging to different molecular subtypes. We also explored ways to modulate acetylcholine produced by cholinergic neurons in mouse basal forebrain to further study the effect on the pathophysiology of GBM. Patient-derived GBM cell lines were exposed to varying concentrations of neurotransmitters (acetylcholine & glutamate) and assessed for cell viability using CCK-8 and EdU assays. Atropine, an antagonist of muscarinic receptors, was used to analyze the effect of cholinergic receptor inhibition on acetylcholine-mediated GBM cell growth in vitro and on the survival of mice bearing human GBM xenografts in vivo. Cholinergic neurons in the mouse basal forebrain were targeted for modulation of acetylcholine production by means of chemogenetics, as well as an attention-related behavior paradigm such as a novel object recognition test (NORT). We also examined possible dysregulation of cholinergic signaling in a preneoplastic glioma mouse model using RNA-seq analysis. RNA sequencing analyses revealed altered expression of cholinergic receptors in preneoplastic glioma mouse models, suggesting a potential role of cholinergic signaling in glioma formation. GBM cell growth was increased upon incubation with acetylcholine. Consistently, pharmacologic inhibition of muscarinic receptors prolonged the survival of mice bearing human GBM xenografts, suggesting a potential role of cholinergic signaling in glioma growth. Last, we tested the possibility of targeting cholinergic neurons in mouse basal forebrain using AAV-mediated chemogenetics and attention-associated behavior tasks. Our findings suggest that GBM cells, irrespective of molecular subtype, may leverage cholinergic signaling mechanisms to support their pathophysiology. Future studies will investigate the role of cholinergic neuron activity in GBM initiation and growth. Khushboo Irshad, Aishani S. Gargapati, Sanjay Singh, Khalil Ali Ahmad Kasm, Kechen Ban, Benjamin E. Aghoghovwia, Rajasekaran Mahalingam, Momo K. Harris, Sama Alnassiry, Renae Bertrand, Alexia T. Mendoza, Valeria S. Vega Sánchez, Nathan Bui, Truc Kuo, Frederick Lang, Yuan Pan. Role of acetylcholine in glioblastoma pathophysiology: Emerging insights [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB020.

Amongst solid cancers, melanoma brain metastases (MBM) have the highest likelihood of metastasizing to the brain. The use of immune checkpoint inhibitors (ICI) for metastatic melanoma has emerged as first line therapy; however, intracranial progression and acquired resistance highlight the need for novel immunotherapeutic strategies for this patient population. Oncolytic viral (OV) therapy leverages tumor cell replication machinery to selectively replicate and kill tumor cells while promoting anti-tumor immunity through modulation of the tumor microenvironment (TME). The oncolytic adenovirus Delta-24-RGDOX (RGDOX) has been shown to function as a TME modulator in both preclinical and clinical settings. Here, we investigated the anti-tumor efficacy of RGDOX combined with ICIs in synchronous extracranial and intracranial (s.c.-i.c.) murine melanoma models (B16F10 and D4M). Using this approach, we inoculated the s.c. tumor with Delta-24-RGDOX and monitored for viral induced cytotoxicity within the infected tumor while assessing for the simultaneous generation of intracranial antitumor immunity. The combinatorial strategy of Delta-24-RGDOX with PD-1/CTLA-4 inhibition significantly reduced both infected s.c. and distant i.c. tumor growth while also improving survival in tumor bearing mice. Through a series of immune depletion experiments, therapeutic efficacy was determined to be mediated through CD8+ T cells. Additionally, eradication of subcutaneous tumor led to immunologic memory preventing intracranial tumor formation. Immunophenotyping and spatial profiling approaches, including full-spectrum flow cytometry and COMET multiplex immunofluorescence analysis, demonstrated the combination of RGDOX with ICI boosted anti-tumor immunity in both s.c. and i.c. tumors through enhanced T-cell activity and increased infiltration of dendritic cells and anti-tumor M1 macrophages. In contrast, immunosuppressive populations, such as myeloid-derived suppressor cells and M2 macrophages, were markedly reduced in both s.c. and i.c. tumors in the combination treatment group. These findings suggest the clinical translation of RGDOX with PD-1/CTLA-4 inhibition is a promising therapeutic approach for the generation of antitumor immunity against MBM.

There is a paucity of studies comparing conventional surgical resection (CSR) and stereotactic radiosurgery (SRS) for single brain metastases (BMs). This study evaluates local treatment failure (LTF) in patients with single, treatment-naïve BMs measuring 1–3 cm in diameter, eligible for either SRS or CSR. We identified patients treated between 1998–2024 with either upfront single-fraction SRS or CSR with adjuvant or neoadjuvant radiation. Fine and Gray’s method was used to analyze predictors of LTF, and propensity score-matched analysis (PSMA) ensured cohort comparability between SRS and CSR by primary tumor type, systemic disease extent, and tumor volume. Among 645 patients (523 SRS; 122 CSR + radiation), CSR was associated with significantly lower 1- and 2-year cumulative LTF rates (4% and 8%) compared to SRS (19% and 23%; p=0.0001). Median tumor volume pre-matching was 7.45 cm³ for CSR and 1.80 cm³ for SRS (p<0.0001). On multivariate analysis, predictors of increased LTF included SRS (HR=5.1, 95% CI: 2.6–9.8; p<0.0001), melanoma (vs renal cell carcinoma, HR=2.2, 95% CI: 1.2-4.3; p=0.018), colorectal cancer (vs RCC, HR=3.4, 95% CI: 1.4-7.8; p=0.005), breast cancer (vs RCC, HR=2.4, 95% CI: 1.1-5.2; p=0.037), greater tumor volume (HR= 1.2, 95% CI: 1.0-1.4 per fold increase; p=0.032), and eloquent location (vs non-eloquent, HR=1.8, 95% CI: 1.1-2.8; p=0.014). In 84 matched pairs, CSR remained associated with lower 1- and 2-year LTF rates (5% and 9%) vs SRS (28% and 37%; HR=0.2; p<0.0001). Among patients with tumor volume ≤5.71 cm³ (2.2 cm diameter; n=69), SRS was significantly associated with local recurrence (HR= 13.5, 95% CI: 3.2-56.2; p=0.0004). In conclusion, for single BMs measuring 1–3 cm, CSR was associated with significantly lower LTF compared to SRS, particularly for lesions <2.2 cm or those from melanoma, breast, or colorectal primaries. These findings support the consideration of CSR to optimize local control.

Recent clinical trials have demonstrated that oncolytic virotherapy using adenoviruses can extend survival in a subset of brain tumor patients by promoting T-cell infiltration into otherwise immunologically “cold” tumors (NCT00805376, NCT03178032, and NCT02798406). However, the early detection and elimination of the virus by the host immune system likely limit the therapeutic efficacy of this approach. To address this challenge, we investigated the molecular pathways and sensors responsible for recognizing adenoviruses during infection, with the ultimate goal of modulating this process to improve viroimunotherapy clinical efficacy. RNA-seq analyses revealed the upregulation of Non-Pou Domain-Containing Octamer-Binding (NONO) in intracranial glioma models treated with the oncolytic adenovirus Delta-24-RGD. We demonstrate that NONO is essential for initiating the cGAS-STING pathway and modulating innate immune responses against adenovirus in normal cells and glioma models. Immunoprecipitation and mass spectrometry studies indicate the formation of complexes between NONO and adenovirus DNA-binding protein (DBP), which facilitate cGAS activation. Given that high-grade brain tumors and corresponding cell models often harbor homozygous deletions of downstream interferon effector genes, we further examined the role of the immunotransmitter 2’3’-cyclic GMP-AMP (cGAMP) in local immune activation. Our results show that tumor-derived cGAMP is sufficient to activate local immune cells and that NONO is required for its production following virotherapy. In vivo experiments reveal that downregulation of NONO enhances viral persistence and significantly prolongs overall survival in athymic mice treated with Delta-24-RGD. Collectively, our results identified NONO as a novel sensor of adenoviral DNA and capsid proteins, which is essential for the secretion of cGAMP and activation of innate immune cells. In addition, this data suggests that targeting the NONO-related pathway may guide the development of the next generation of oncolytic viruses to improve the clinical results obtained using viroimmunotherapy and other immunotherapy modalities.

When operating on gliomas near critical language regions, surgeons risk either leaving residual tumor or inducing permanent postoperative language deficits (PLDs). Despite the advent of intraoperative mapping techniques, subjective judgments frequently determine important surgical decisions. We aim to inform data-driven surgery by constructing a non-invasive mapping approach that quantitatively predicts the impact of individual surgical decisions on long-term language function. This study included 79 consecutive patients undergoing resection of language-eloquent gliomas. Patients underwent preoperative navigated transcranial magnetic stimulation (TMS) language mapping to identify language-positive sites (“TMS points”) and their associated white matter tracts (“TMS tracts”) as well as formal language evaluations pre-and postoperatively. The resection of regions identified by preoperative mapping was correlated with permanent postoperative language deficits (PLDs). Resected tract segments (RTS) were normalized to MNI space for comparison with normative data. The resection of TMS points did not predict PLDs. However, a TMS point subgroup defined by white matter connectivity significantly predicted PLDs (OR = 8.74, p  < 0.01) and demonstrated a canonical distribution of cortical language sites at a group level. TMS tracts recapitulated normative patterns of white matter connectivity defined by the Human Connectome Project. Subcortical resection of TMS tracts predicted PLDs independently of cortical resection (OR = 60, p  < 0.001). In patients with PLDs, RTS showed significantly stronger co-localization with normative language-associated tracts compared to RTS in patients without PLDs ( p  < 0.05). Resecting patient-specific co-localizations between TMS tracts and normative tracts in native space predicted PLDs with an accuracy of 94% (OR = 134, p  < 0.001). Prospective application of this data in a patient with glioblastoma precisely predicted the results of intraoperative language mapping with direct subcortical stimulation. Long-term postoperative language deficits result from resecting patient-specific white matter segments. We integrate these findings into a personalized tool that uses TMS language mappings, diffusion tractography, and population-level connectivity to preoperatively predict the long-term linguistic impact of individual surgical decisions.

Abstract BACKGROUND Diffuse infiltration is an aggressive feature of high-grade gliomas with survival implications. The contribution of crosstalk between non-neoplastic and neoplastic cells to tumor infiltration remains largely understudied due to the lack of profiling techniques that retain spatial information. Spatial multi-omic profiling is a promising approach to comprehensively analyze transcript-omics, prote-omics and metabol-omics on the same tissue section while preserving information about the spatial organization of cells. Integration of these spatial studies allows for inferring the consequences of complex cell-cell communication underlying tumor infiltration. We hypothesize that the glioma edge is enriched with pro-infiltration ligands-receptors driving tumor infiltration. Strategies that disrupt these ligand-receptor networks may suppress glioma infiltration and improve clinical outcomes. METHODS We utilized a glioma tissue micro-array (TMA) to establish the neoplastic and non-neoplastic heterogeneity in the GBM infiltration edge. Each TMA slide consists of pathologist annotated tumor core and edge samples of Glioblastoma (IDH Wildtype, WHO Grade 4; n=10), Diffuse Astrocytoma (IDH Mutant, WHO Grade 3; n=3), Oligodendroglioma (IDH mutant, WHO Grade 2; n=5) and 2 non-brain control samples. We performed spatial multi-omics profiling on adjacent sections of the TMA using 10x Xenium spatial transcriptomics, imaging mass cytometry (IMC) and mass spectrometry imaging (MSI). Integration, visualization, and quantification of the spatial data was done on VisioPharm. RESULTS In GBM, we identified candidate mRNA transcripts and proteins for ligands enriched at the infiltrating edge (compared to tumor core; p<0.0001) that correlated with a poor progression free survival (PFS; r2=0.22). These candidate ligands were also significantly enriched at the edge of oligodendroglioma WHO Grade 2 and astrocytoma WHO Grade 3. CONCLUSIONS Spatial multi-omics profiling on a TMA consisting of Glioma WHO grades 2-4 identified differentially expressed and targetable pro-tumor infiltration ligands associated with lower PFS in GBM. Functional studies to uncover the role of metabolites enriched at the glioma edge for the expression of the identified pro-infiltration ligands are ongoing.