Glioblastoma (GBM) is a highly aggressive and malignant brain tumor with limited therapeutic options and a poor prognosis. Despite current treatments, the invasive nature of GBM often leads to recurrence. A promising alternative strategy is to harness the potential of the immune system against tumor cells. Our previous data showed that the B_Vax (B cell–based vaccine) can induce therapeutic responses in preclinical models of GBM. In this study, we aimed to characterize the antigenic reactivity of B_Vax-derived Abs and evaluate their therapeutic potential. We performed immunoproteomics and functional assays in murine models and samples from patients with GBM. Our investigations revealed that B_Vax distributed throughout the GBM tumor microenvironment and then differentiated into Ab-producing plasmablasts. Proteomics analyses indicated that the Abs produced by B_Vax had unique reactivity, predominantly targeting factors associated with cell motility and the extracellular matrix. Crucially, these Abs inhibited critical processes such as GBM cell migration and invasion. These findings provide valuable insights into the therapeutic potential of B_Vax-derived Abs for patients with GBM, pointing toward a novel direction for GBM immunotherapy.

Aberrant metabolism is a hallmark of malignancies including gliomas. Intracranial microdialysis enables the longitudinal collection of extracellular metabolites within CNS tissues including gliomas and can be leveraged to evaluate changes in the CNS microenvironment over a period of days. However, delayed metabolic impacts of CNS injury from catheter placement could represent an important covariate for interpreting the pharmacodynamic impacts of candidate therapies. Intracranial microdialysis was performed in patient-derived glioma xenografts of glioma before and 72 h after systemic treatment with either temozolomide (TMZ) or a vehicle. Microdialysate from GBM164, an IDH-mutant glioma patient-derived xenograft, revealed a distinct metabolic signature relative to the brain that recapitulated the metabolic features observed in human glioma microdialysate. Unexpectedly, catheter insertion into the brains of non-tumor-bearing animals triggered metabolic changes that were significantly enriched for the extracellular metabolome of glioma itself. TMZ administration attenuated this resemblance. The human glioma microdialysate was significantly enriched for both the PDX versus brain signature in mice and the induced metabolome of catheter placement within the murine control brain. These data illustrate the feasibility of microdialysis to identify and monitor the extracellular metabolome of diseased versus relatively normal brains while highlighting the similarity between the extracellular metabolome of human gliomas and that of CNS injury.

Diseases of the central nervous system (CNS) manifest through a complex, dynamic network of intercellular communication. Glioblastoma (GBM), the most common and aggressive primary brain tumor, is emblematic of this complexity, with its rapid progression and dismal prognosis measured in months rather than years. GBM's biology is driven by intricate signaling molecule exchanges between neoplastic and stromal cells, underpinning aggressive disease progression. Here, we describe a detailed cerebral open-flow microperfusion (cOFM) protocol in GBM mouse models, enabling real-time longitudinal monitoring of tumor microenvironment dynamics within the interstitial fluid (ISF). Our approach describes the implantation of durable guide cannula head-mounts, their use for intracerebral glioma engraftment directly through the guide, and the collection of high-fidelity cOFM samples for metabolomic and proteomic LC-MS analyses. Crucially, cOFM overcomes the molecular size limitations of traditional microdialysis. Beyond GBM, cOFM methodology promises transformative insights into a spectrum of CNS disorders, including neurodegenerative, epileptic, and neuropsychiatric conditions, through its capacity to provide etiological and treatment-responsive biomarkers within their respective animal models.

Diseases of the central nervous system (CNS) manifest through a complex, dynamic network of intercellular communication. Glioblastoma (GBM), the most common and aggressive primary brain tumor, is emblematic of this complexity, with its rapid progression and dismal prognosis measured in months rather than years. GBM's biology is driven by intricate signaling molecule exchanges between neoplastic and stromal cells, underpinning aggressive disease progression. Here, we describe a detailed cerebral open-flow microperfusion (cOFM) protocol in GBM mouse models, enabling real-time longitudinal monitoring of tumor microenvironment dynamics within the interstitial fluid (ISF). Our approach describes the implantation of durable guide cannula head-mounts, their use for intracerebral glioma engraftment directly through the guide, and the collection of high-fidelity cOFM samples for metabolomic and proteomic LC-MS analyses. Crucially, cOFM overcomes the molecular size limitations of traditional microdialysis. Beyond GBM, cOFM methodology promises transformative insights into a spectrum of CNS disorders, including neurodegenerative, epileptic, and neuropsychiatric conditions, through its capacity to provide etiological and treatment-responsive biomarkers within their respective animal models.

Cerebrospinal fluid (CSF)-directed therapies are gaining importance in neuro-oncology, but long-term CSF access in preclinical murine models remains limited by anatomical constraints. This technical feasibility study evaluates the Transcranial Cisternal Port (TCP), a skull-anchored cannula that provides repeated access to the intrathecal space. The procedure is performed by: making an incision over the craniocervical junction and exposing the cisterna magna; performing a craniostomy on the interparietal bone and placing a guide cannula through it; verifying placement by threading a wire through the cannula and visualizing it within the cisterna magna; securing the cannula in place using surgical adhesive; skin closure and inserting a mandrin to maintain patency. The TCP was validated via Evans blue dye injection, and long-term outcomes were assessed over 3 weeks in 43 athymic nude mice. Evans blue dye distribution was observed throughout the cisterna magna, basal cisterns, and perivascular spaces. All animals recovered uneventfully without device-related complications. Functional retention was 93% at day 7, 86% at day 14, and 86% at day 21. Failure was related to intraluminal clogging (n = 4) and adhesive bonding of the dummy to the guide cannula (n = 2). Revision surgery was successful in all cases. The TCP provides a skull-anchored access route to the murine CSF, enabling reliable intrathecal delivery. Cannula patency remains a limitation in some animals. Future iterations might mitigate this by improving guide cannula design. Overall, these findings establish the TCP as a scalable, functionally robust platform for modeling intrathecal regimens in preclinical studies.