Intracranial Implantation Research Articles

Hybrid fabrication of multimodal intracranial implants for electrophysiology and local drug delivery.

New fabrication approaches for mechanically flexible implants hold the key to advancing the applications of neuroengineering in fundamental neuroscience and clinic. By combining the high precision of thin film microfabrication with the versatility of additive manufacturing, we demonstrate a straight-forward approach for the prototyping of intracranial implants with electrode arrays and microfluidic channels. We show that the implant can modulate neuronal activity in the hippocampus through localized drug delivery, while simultaneously recording brain activity by its electrodes. Moreover, good implant stability and minimal tissue response are seen one-week post-implantation. Our work shows the potential of hybrid fabrication combining different manufacturing techniques in neurotechnology and paves the way for a new approach to the development of multimodal implants.

EXTH-81. STING ACTIVATION PROMOTES ROBUST IMMUNE RESPONSE AND TUMOR REGRESSION IN GLIOBLASTOMA MODELS

Abstract The immunosuppressive tumor microenvironment in glioblastoma presents a major barrier to effective application of immunotherapeutic approaches in this disease. Although immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease glioblastoma remains largely refractory to current immunotherapeutic approaches. The cGAS-STING cytoplasmic double stranded DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties, but has not been well investigated in glioblastoma to date. Here we investigated the presence of STING pathway components in glioblastoma patient specimens and cell lines by Western blotting and immunostaining. The functionality of the pathway was determined by ELISA, and immune infiltrates, and animal survival were investigated in mouse glioblastoma models after treatment with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature, and appears to be activated as evidenced by elevated phosphoTBK1 staining. We show that human GBM cells do not respond agonists, but that GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils and NK populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants the further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, CAR T cells, or immune checkpoint blockade.

CBIO-16. DOSE INTENSIFIED TEMOZOLOMIDE IMPROVES INFILTRATION OF CD70 CAR T CELLS INTO THE GBM TUMOR MICROENVIRONMENT

Abstract CD70 CAR T cells developed by our group have anti-tumor efficacy in syngeneic murine GBM model and are currently being developed for first-in-human testing. The objective of this study was to evaluate the impact of temozolomide (TMZ) in various dosing strategies on the expansion, persistence and function of the CD70 CAR T cells after infusion. C57BL/6 mice underwent intracranial implantation of KR-158 overexpressing CD70 tumor cells. Tumor bearing animals were treated with standard dose (SD) (50 mg/kg x 5 days), metronomic dose (MD) (25mg/kg x 10 days), or dose intensified (DI) (75mg/kg x 5 days) of TMZ, followed by 10×106 CAR T cells infusion. Peripheral blood was collected to monitor the persistence of CAR T cells in the systemic blood circulation. Six weeks post treatments, spleens and tumors were collected and CAR T cells abundance and function were measured. TMZ preconditioning resulted in the expected lymphopenia in animals by generation of dose dependent lymphopenia. Circulating CD70 CAR T cells peaked in the systemic blood 2 weeks after infusion (3-fold, p< 0.0001). Markers of T cell exhaustion including PD-1 and TOX expression on CAR T cells were not different between the three TMZ treatment groups compared to control. Six weeks post treatment, CD70 CAR T cells were found to be highly infiltrated within the tumor microenvironment in the DI TMZ group compared to the other groups (4-fold,p < 0.0001). Tumor infiltrating CD70 CAR T cells in the DI TMZ group did not have an increase in PD-1 and TOX expression which was seen in the MD and SD TMZ groups. DI TMZ preconditioning results in greater CD70 CAR T cell trafficking to the tumor without T cell exhaustion compared to lower doses of TMZ. The affects of TMZ on the immune microenvironment to enhance CAR T cell regimens warrants further study.

Severity Assessment of Complex and Repeated Intracranial Surgery in Rats

Introduction: Evidence-based grading of the impact of intracranial surgery on rat’s well-being is important for ethical and legal reasons. We assessed the severity of complex and repeated intracranial surgery in a 6-hydroxydopamine (6-OHDA) Parkinson’s rat model with subsequent intracranial electrode implantation and in an intracranial tumor model with subsequent resection. Methods: Stereotactic surgery was performed in adult male rats with the same general anesthesia and perioperative pain management. In Parkinson’s model, Sprague Dawley rats received unilateral injection of 6-OHDA (n = 11) or vehicle (n = 7) into the medial forebrain bundle as first operation (1st OP). After four weeks, neural electrodes were implanted in all rats as second operation (2nd OP). For tumor formation, BDIX/UlmHanZtm rats (n = 8) received frontocortical injection of BT4Ca cells as 1st OP, followed by tumor resection as 2nd OP after one week. Multiple measures severity assessment was done two days before and four days after surgery in all rats, comprising clinical scoring, body weight, and detailed behavioral screening. To include a condition with a known burden, rats with intracranial tumors were additionally assessed up to a predefined humane endpoint that has previously been classified as “moderate”. Results: After the 1st OP, only 6-OHDA injection resulted in transient elevated clinical scores, a mild long-lasting weight reduction, and motor disturbances. After the second surgery, body weight was transiently reduced in all groups. All other parameters showed variable results. Principal component analysis showed a separation from the preoperative state driven by motor-related parameters after 6-OHDA injection, while separation after electrode implantation and more clearly after tumor resection was driven by pain-related parameters, although not reaching the level of the humane endpoint of our tumor model. Conclusion: Overall, cranial surgery of different complexity only transiently and rather mildly affects rat’s well-being. Multiple measures assessment allows the differentiation of model-related motor disturbances in Parkinson’s model from potentially pain-related conditions after tumor resection and electrode implantation.

Investigating visuo-spatial neglect and visual extinction during intracranial electrical stimulations: The role of the right inferior parietal cortex

Both visuo-spatial neglect and visual extinction may occur following right-brain damage. So far, studies on brain-damaged patients have not provided definite evidence about which lesion patterns may lead to the association or dissociation of these deficits. This study was set out to address this issue using Intracranial Electrical Stimulation (IES) in a group of nine patients affected by refractory epilepsy. Cerebral regions associated with visuo-spatial neglect and visual extinction were stimulated, including the right frontal, temporal, and posterior parietal areas. During IES, patients with intracranial implantation involving at least one of these cortical regions were administered with a manual line bisection task (N = 9) to assess visuo-spatial neglect, and a computerized task (N = 8) assessing visual extinction. Results showed that parietal IES induced a rightward bias at the manual bisection task, together with a general improvement in reaction times at bilateral and unilateral visual stimuli detection at the extinction task. The occurrence of visual extinction did not vary across stimulations. By adopting a complementary approach to anatomo-clinical correlation studies, our work corroborates the notion that lesions to the right inferior parietal lobule play a pivotal role in the pathogenesis of visuo-spatial neglect. Importantly, our results also suggest that temporarily interfering with the activity of this region is not sufficient per se to generate visual extinction, which instead may involve a broader and/or different network, possibly extending beyond the cerebral regions considered here, posing important theoretical and clinical implications.

KS01.5.A Allergic airway inflammation impacts tumor take and delays experimental glioblastoma progression

Abstract BACKGROUND Numerous epidemiological studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanistic explanations behind these phenomena remain unexplored. Our objective was to set up a preclinical model and investigate the mechanisms underlying such protection to improve our understanding of the crosstalk between immune system and brain tumor development. MATERIAL AND METHODS A mouse model of allergic airway inflammation (AAI) induced by repeated nasal instillation of House Dust Mite extract was initiated before intracranial implantation of GL261 glioma cells, in both immunocompetent (C57BL/6) and immunodeficient (RAG-KO) mice. Tumor take and tumor growth were monitored by MRI. Central (microglia) and peripheral (spleen, bone marrow) immune cells were characterized by flow cytometry. The response of microglia was further assessed by RNA sequencing. Impact of candidate genes on patient survival was characterized by Cox regression analysis using data from TCGA and CGGA. RESULTS Following AAI induction in C57BL/6 mice, engraftment of GL261 cells in the brain was delayed and tumor growth rate was reduced. This correlated with an increase in survival of the mice and was accompanied by increased effector memory T-cells in the circulation. Of note, the survival benefit was lost in RAG-KO mice devoid of adaptive immunity. At the level of the brain, we observed enhanced secretion of TNFα and IL6 in microglia ex vivo. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state. We identified an allergy-related microglia gene signature that is associated with improved prognosis of glioma patients. CONCLUSION Our results demonstrate that AAI limits both tumor take and GBM progression in mice, providing a preclinical model to study the role of allergic inflammation in GBM susceptibility and prognosis, respectively. At the functional level, we identify a potentiation of microglial and adaptive anti-tumoral immunity. Further investigations are warranted to shed light on the reciprocal crosstalk between microglial reprogramming and peripheral immunity in the context of allergies and brain tumors.

TOP2B Enzymatic Activity on Promoters and Introns Modulates Multiple Oncogenes in Human Gliomas.

The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

Abstract 2806: Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo

Abstract While the use of bioluminescent proteins for molecular imaging is a powerful technology to support our knowledge of complex processes, with the advent of near infrared-labeling capabilities optical imaging can be pursuit with deeper tissue penetration, a better signal to background ratio and a quicker image acquisition without the need to inject substrates in preclinical oncology mouse models. This is specifically important in orthoptic brain tumors, when the substrate concentration in the tumor tissue might be influenced by the blood-brain barrier. In our recent study, we validated the feasibility of stable or transient transfection of human brain tumor cell lines and patient derived xenografts to determine tumor load in an intracranial setting. As a proof-of principle, we stably transfected the human glioblastoma cell line U87MG with an iRFP713 construct and followed tumor growth of intracranially implanted NSG mice. As a follow-up, five PDX of pediatric brain tumors (two Ependymoma, two Medulloblastoma, one high grade Glioblastoma) were transiently transfected with the same construct 24h before intracranial implantation. Tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany) and animals were examined for neurological symptoms every day. When the overall condition of the mice got deteriorated, animals were taken down and brains fixed in formalin. The tumor load and localization was confirmed by H&E and human-specific IHC for LamininB. The comparison between non-transfected and transfected lines of one specific tumor model, revealed that the tumor growth was very similar in both settings. The tumor take rate was as well not affected. In the subsequent analyses we could show that the tumor load linearly correlated with the imaging signal. Moreover, the iRFP713 signal was stable post-resection of the tumor. Thus, it was possible to correlate the tumor load quantification via iRFP713-Protein expression and LamininB-IHC within one slide. The transient transfection was successful for the five tested models. When termination criteria were reached, over 90% of the tumor tissue were expressing the iRFP713 protein. The more general applicability for larger cohorts of mice (> 20) and drug testing experiments will be evaluated soon. We believe the superior optical properties of iRFP713 will be a valuable asset to overcome some of the complications inherent to imaging live animals and a powerful tool for preclinical drug development in clinical relevant mouse models of brain cancer. Citation Format: Kanstantsin Lashuk, Jacqueline Bersano, Dorothee Lenhard, Eva Oswald, Kerstin Klingner, Julia Schüler. Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2806.

Abstract 2854: Adaptation of colorectal cancer cells to the brain microenvironment: the role of IRS2

Abstract Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with rising incidence. Yet, molecular mechanisms supporting the formation of these lesions from CRC are unknown. We aimed to explore drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of insulin receptor substrate 2 (IRS2) gene amplification was noted in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. We subsequently constructed an in vitro system mimicking the brain microenvironment using cultured human astrocytes or their conditioned media (CM). Under these conditions using InSphero system, IRS2-overexpressed CRC cells survived better and formed larger 3D spheres. IRS2-silenced CRC cells showed a mirror image. Moreover, in an intracranial CRC surgical implantation BMs mouse model, IRS2-overexpressed CRC cells generated larger brain lesions, while silencing IRS2 in IRS2-amplified CRC cells dramatically decreased tumor outgrowth and survival. Interestingly, transcriptomic analysis revealed significant enrichment of the oxidative phosphorylation pathway by IRS2. Indeed, IRS2-expressing CRC cells showed increased mitochondrial activity using Seahorse extracellular flux analysis and glycolysis-independent viability. Furthermore, IRS2-expressing cells showed enhanced AKT phosphorylation and inhibition of PI3K or AKT using Alpelisib or iAKT, respectively, slightly decreased their proliferation in CM, suggesting that mechanisms additional to the AKT pathway may mediate IRS2 activity. The Wnt/β-catenin pathway was among the most significantly enriched pathways in the transcriptome. Indeed, IRS2-expressing cells had increased transcriptional activity of the β-catenin. In addition, iAKT or Alpelisib, and most significantly the IRS2 inhibitor (NT219), decreased the transcriptional activity of β-catenin in IRS2-expressing CRC cells, suggesting relevance of IRS2 in activating β-catenin. Moreover, NT219 demonstrated significant and dose-dependent inhibition of CRC cells viability. These data reveal, for the first time, the unique genomic profile of CRC BMs and imply the IRS2 role in promoting CRC BMs. These effects may be mediated, at least in part, by modulation of the AKT and β-catenin pathway. Given the molecular signature described, the approach to patients with BMs may be significantly impacted by agents such as NT219. Citation Format: Inbal Greenberg, Anat Klein, Rachel Grossman, Ethan Sokol, Eilam Yeini, Paula Ofek, Ronit Satchi-Fainaro, Bertrand Liang, Hadas Reuveni, Tami Rubinek, Ido Wolf. Adaptation of colorectal cancer cells to the brain microenvironment: the role of IRS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2854.

Electrocorticography and stereo EEG provide distinct measures of brain connectivity: implications for network models.

Brain network models derived from graph theory have the potential to guide functional neurosurgery, and to improve rates of post-operative seizure freedom for patients with epilepsy. A barrier to applying these models clinically is that intracranial EEG electrode implantation strategies vary by centre, region and country, from cortical grid & strip electrodes (Electrocorticography), to purely stereotactic depth electrodes (Stereo EEG), to a mixture of both. To determine whether models derived from one type of study are broadly applicable to others, we investigate the differences in brain networks mapped by electrocorticography and stereo EEG in a cohort of patients who underwent surgery for temporal lobe epilepsy and achieved a favourable outcome. We show that networks derived from electrocorticography and stereo EEG define distinct relationships between resected and spared tissue, which may be driven by sampling bias of temporal depth electrodes in patients with predominantly cortical grids. We propose a method of correcting for the effect of internodal distance that is specific to electrode type and explore how additional methods for spatially correcting for sampling bias affect network models. Ultimately, we find that smaller surgical targets tend to have lower connectivity with respect to the surrounding network, challenging notions that abnormal connectivity in the epileptogenic zone is typically high. Our findings suggest that effectively applying computational models to localize epileptic networks requires accounting for the effects of spatial sampling, particularly when analysing both electrocorticography and stereo EEG recordings in the same cohort, and that future network studies of epilepsy surgery should also account for differences in focality between resection and ablation. We propose that these findings are broadly relevant to intracranial EEG network modelling in epilepsy and an important step in translating them clinically into patient care.

Virtual Surgical Planning for Intracranial Intraosseous Meningioma Reconstruction.

To assess the use of custom-made intracranial implants and three-dimensional cutting guides to direct the intracranial, intraorbital, and temporal reconstruction process for intraosseous meningioma. A retrospective analysis was conducted on 6 patients who were operated on by the senior author for intraosseous meningioma between 2017 and 2020. Three-dimensional models of the maxillofacial skeleton were created from preoperative virtual planned reconstruction and postoperative computed tomography scan images in the Mimics and 3-Matics software. Orbital reconstruction and temporal implant accuracy assessments were performed through the Materialise Mimics software. Orbital cone volume had a mean discrepancy between the planned and actual orbital volume of 1.5% ± 1.6%. The reconstructed postoperative orbital volume was within 1.3% ± 2.0% of the unaffected orbit. Temporal bone reconstructions had a mean implant accuracy of 81.0%. Our results show that the postextirpative intracranial applications of virtual surgical planning are particularly suited for high fidelity reconstructions such as orbital reconstructions, as well as temporal reconstructions with intraoperative adjustments. Custom implants and virtual three-dimensional planning is particularly ideal and promising for intraosseous meningiomas given the involvement of complex intracranial and intraorbital bony structures.

Abstract 1467: BLU-945, a fourth-generation, potent and highly selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with intracranial activity, demonstrates robust in vivo antitumor activity in models of osimertinib-resistant non-small cell lung cancer (NSCLC)

Abstract Background: EGFR-activating mutations (L858R and ex19del) are major drivers (15-47%) of lung adenocarcinoma. EGFR TKI development has changed the treatment paradigm for EGFR-mutant (EGFR+) NSCLC. However, tumors often develop acquired resistance mutations to current TKIs leading to clinical progression. EGFR+/T790M is the most prevalent acquired resistance mutation after first- and second-generation TKI treatment. Osimertinib, a third-generation TKI which targets EGFR+/T790M, can overcome this resistance, but faces further resistance within 10-18 months. EGFR+/C797S is the most significant on-target resistance mechanism to osimertinib, leading to EGFR+/T790M/C797S double resistant mutants. There are currently no approved targeted therapies for patients with EGFR+/T790M/C797S NSCLC. BLU-945 is a potent and highly selective inhibitor of the EGFR+/T790M/C797S and EGFR+/T790M mutations, with in vivo activity in subcutaneous and intracranial EGFR-mutant tumor models. Method: In vivo antitumor activity of BLU-945 was evaluated in engineered triple mutant cell line-derived xenograft (CDX) models and osimertinib-resistant patient-derived xenograft (PDX) models of NSCLC. Plasma and tumor samples were collected for future pharmacokinetic and pharmacodynamic analyses. In vivo activity was also evaluated in an intracranial implantation model using luciferase-expressing YU-1097 patient-derived-cells harboring EGFRex19del/T790M/C797S resistance mutations, with the tumor burden of intracranial lesions measured by bioluminescence imaging. Results: Oral administration of BLU-945 to tumor-bearing mice showed potent EGFR pathway inhibition and significant single-agent antitumor activity at well-tolerated doses in the engineered osimertinib-resistant EGFRL858R/T790M/C797S CDX model, and an EGFRex19del/T790M/C797S PDX model. In addition, combination of BLU-945 with osimertinib showed enhanced antitumor activity compared with single-agent treatment in the EGFRex19del/T790M/C797S PDX model. Pharmacodynamic assays revealed treatment with BLU-945 resulted in marked inhibition of EGFR, AKT and ERK phosphorylation in the EGFRL858R/T790M/C797S CDX and EGFRex19del/T790M/C797S PDX models. BLU-945 also demonstrated potent activity in a patient-derived model of EGFRex19del/T790M/C797S implanted intracranially. Conclusion: BLU-945 is a potent, selective, and orally available fourth-generation EGFR inhibitor with robust antitumor activity in osimertinib-resistant NSCLC models grown subcutaneously and intracranially. BLU-945 shows activity as a single agent and in combination with other EGFR inhibitors. Clinical development of BLU-945 is expected in 2021. Citation Format: Sun Min Lim, Chae Won Park, Zhuo Zhang, Rich Woessner, Tom Dineen, Faith Stevison, John Hsieh, Meredith Eno, Doug Wilson, John Campbell, Caitlin Utt, Faris Albayya, Nicolas Lamontagne, Marion Dorsch, Klaus Hoeflich, Byoung Chul Cho, Stefanie Schalm. BLU-945, a fourth-generation, potent and highly selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with intracranial activity, demonstrates robust in vivo antitumor activity in models of osimertinib-resistant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1467.

Spectral power and theta-gamma coupling in the basolateral amygdala related with methamphetamine conditioned place preference in mice

The basolateral amygdala (BLA) plays a crucial role in conditioned place preference (CPP) for addictive drugs. However, neural signaling associated with methamphetamine (METH) craving and seeking remained to be investigated. This study characterized local field potential (LFP) oscillatory patterns in the BLA and conditioned place preference induced by METH-related context. Male Swiss albino ICR mice were deeply anesthetized for LFP intracranial electrode implantation in the BLA. Control and METH groups received sessions to learn to associate saline-paired and METH-paired compartments of the CPP apparatus with saline and METH injections, respectively, for 10 days. LFP signals and exploring behavior were recorded simultaneously during pre- and post-conditioning phases. Time spent in METH-paired compartment was normalized and expressed as CPP scores. Fast Fourier Transform (FFT) algorithm was used to analyze LFP powers of 8 discrete frequency ranges (delta, theta, alpha, beta, gamma I-IV). During post-conditioning phase of METH CPP with METH cues, statistical analysis revealed that METH group significantly increased time spent in METH-paired compartment. Significant suppressions of theta and alpha powers were observed. Phase-amplitude cross frequency coupling analyses confirmed significant increases in maximal modulation index (MI), frequency for phase of slow wave and MI of theta-gamma II coupling. Taken together, LFP oscillation in the BLA was sensitive in association with METH CPP. These research findings might suggest the underlying mechanisms of drug reward learning and adaptive changes in the BLA in acquisition of METH CPP and dependence.

Clinical safety of intracranial EEG electrodes in MRI at 1.5 T and 3 T: a single-center experience and literature review.

Intracranial electroencephalography (EEG) can be a critical part of presurgical evaluation for drug resistant epilepsy. With the increasing use of intracranial EEG, the safety of these electrodes in the magnetic resonance imaging (MRI) environment remains a concern, particularly at higher field strengths. However, no studies have reported the MRI safety experience of intracranial electrodes at 3 T. We report an MRI safety review of patients with intracranial electrodes at 1.5 and 3 T. One hundred and sixty-five consecutive admissions for intracranial EEG monitoring were reviewed. A total of 184 MRI scans were performed on 135 patients over 140 admissions. These included 118 structural MRI studies at 1.5 T and 66 functional MRI studies at 3 T. The magnetic resonance (MR) protocols avoided the use of high specific energy absorption rate sequences that could result in electrode heating. The intracranial implantations included 114 depth, 15 subdural, and 11 combined subdural and depth electrodes. Medical records were reviewed for patient-reported complications and radiologic complications related to these studies. Pre-implantation, post-implantation, and post-explantation imaging studies were reviewed for potential complications. No adverse events or complications were seen during or after MRI scanning at 1.5 or 3 T apart from those attributed to electrode implantation. There was also no clinical or imaging evidence of worsening of pre-existing implantation-related complications after MR imaging. No clinical or radiographic complications are seen when performing MRI scans at 1.5 or 3 T on patients with implanted intracranial EEG electrodes while avoiding high specific energy absorption rate sequences.

Metabolic changes and anti-tumor effects of a ketogenic diet combined with anti-angiogenic therapy in a glioblastoma mouse model

The ketogenic diet (KD) is a high fat and low carbohydrate diet that produces ketone bodies through imitation of starvation. The combination of KD and Bevacizumab (Bev), a VEGF inhibitor, is considered to further reduce the supply of glucose to the tumor. The metabolite changes in U87 glioblastoma mouse models treated with KD and/or Bev were examined using gas chromatography-mass spectrometry. The combination therapy of KD and Bev showed a decrease in the rate of tumor growth and an increase in the survival time of mice, although KD alone did not have survival benefit. In the metabolome analysis, the pattern of changes for most amino acids are similar between tumor and brain tissues, however, some amino acids such as aspartic acid and glutamic acid were different between tumors and brain tissues. The KD enhanced the anti-tumor efficacy of Bev in a glioblastoma intracranial implantation mouse model, based on lowest levels of microvascular density (CD31) and cellular proliferation markers (Ki-67 and CCND1) in KD + Bev tumors compared to the other groups. These results suggested that KD combined with Bev may be a useful treatment strategy for patients with GBM.

Cell Therapy for Chronic TBI: Interim Analysis of the Randomized Controlled STEMTRA Trial.

ObjectiveTo determine whether chronic motor deficits secondary to traumatic brain injury (TBI) can be improved by implantation of allogeneic modified bone marrow–derived mesenchymal stromal/stem cells (SB623).MethodsThis 6-month interim analysis of the 1-year double-blind, randomized, surgical sham–controlled, phase 2 Stem Cell Therapy for Traumatic Brain Injury (STEMTRA) trial (NCT02416492) evaluated safety and efficacy of the stereotactic intracranial implantation of SB623 in patients with stable chronic motor deficits secondary to TBI. Patients in this multicenter trial (n = 63) underwent randomization in a 1:1:1:1 ratio to 2.5 × 106, 5.0 × 106, or 10 × 106 SB623 cells or control. Safety was assessed in patients who underwent surgery (n = 61), and efficacy was assessed in the modified intent-to-treat population of randomized patients who underwent surgery (n = 61; SB623 = 46, control = 15).ResultsThe primary efficacy endpoint of significant improvement from baseline of Fugl-Meyer Motor Scale score at 6 months for SB623-treated patients was achieved. SB623-treated patients improved by (least square [LS] mean) 8.3 (standard error 1.4) vs 2.3 (standard error 2.5) for control at 6 months, the LS mean difference was 6.0 (95% confidence interval 0.3–11.8, p = 0.040). Secondary efficacy endpoints improved from baseline but were not statistically significant vs control at 6 months. There were no dose-limiting toxicities or deaths, and 100% of SB623-treated patients experienced treatment-emergent adverse events vs 93.3% of control patients (p = 0.25).ConclusionsSB623 cell implantation appeared to be safe and well tolerated, and patients implanted with SB623 experienced significant improvement from baseline motor status at 6 months compared to controls.ClinicalTrials.gov Identifier:NCT02416492.Classification of EvidenceThis study provides Class I evidence that implantation of SB623 was well tolerated and associated with improvement in motor status.

Robot-assisted versus stereotactic frame-based stereoelectroencephalography in medically refractory epilepsy

AimTo explore the difference between robot assisted (RA) and stereotactic frame based (SF) stereoelectroencephalography (SEEG) in patients with medically refractory epilepsy. MethodsWe undertook a retrospective review of 33 SEEG cases at our center, of which 14 were SF performed from March to October 2018 and 19 were RA performed from November 2018 to December 2019. Detailed review of medical histories and operative records as well as imaging and trajectory plans was carried out for each patient, and the results related to each technique compared. A multiple linear regression model was used to test for variables that significantly influenced placement error. ResultsCompared to the SF group, the RA group had a higher mean number of electrodes per patient (10.7 ± 2.8 versus 6.4 ± 0.8, P < 0.0001) and a significantly shorter mean operative time (127.3 ± 40.7 versus 152.7 ± 13.6 min, P = 0.033). For the RA group, the intracranial implantation length was positively correlated with target point error (p = 0.000), depth error (p = 0.043), and two-dimensional (2D) radial error (p = 0.041). Conversely, skull thickness was negatively correlated with the TP error (p = 0.004), depth error (p = 0.037) and 2D radial error (p = 0.000). We also analyzed the mean entry point, target point, depth and 2D radial errors, the complication rates, and the results of epileptogenic zone (EZ) localization and Engel class. The results showed no difference in these aspects between the SF group and the RA group. ConclusionThis study suggests that, compared to stereotactic frame based SEEG, robot assisted SEEG is significantly more efficient and comparable in safety and effectiveness.

STEM-07. IDENTIFICATION OF A NOVEL CSC SIGNALING AXIS CONTAINING JAM-A/SERPINB3/TGF-BETA

Abstract Despite therapeutic interventions including surgery, radiation, and chemotherapy, multiple clones of treatment resistant cells repopulate the tumor, resulting in recurrence and a high rate of mortality in glioblastoma. Effective targeting of these cancer stem cells (CSCs) has been limited by our incomplete understanding of the intracellular signaling mechanisms maintaining the CSC state. Our laboratory previously identified junctional adhesion molecule-A (JAM-A) on CSCs and, through functional studies, demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. We determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the intermediate signaling network is yet to be fully elucidated. Therefore, our goal was to delineate the pathway downstream of JAM-A. To achieve this, we immunoprecipitated JAM-A from GBM CSCs and performed mass spectrometry leading to the identification of the serine/cysteine protease inhibitor SerpinB3. While SerpinB3 has a functional role in lung adenocarcinoma, breast, pancreas, as well as in hepatocellular carcinoma, there is limited information in GBM or CSCs, and its relationship to JAM-A is yet to be elucidated. Using in vitro CSC functional assays, we observed that SerpinB3 is necessary for the maintenance of CSCs. Additionally, in an intracranial implantation model of GBM the knockdown of SerpinB3 extended the survival of mice. Knockdown of SerpinB3 in multiple GBM CSCs models also led to decreased expression of TGF-β, a known regulator of the CSC state. We also observed that SerpinB3 stabilizes JAM-A expression and further studies are investigating the mechanism of this stabilization, identifying the specific interaction sites of JAM-A and Serpin B3, and determining how TGF-β alters the association through positive and negative feedback mechanisms. Taken together, these data highlight the role of SerpinB3 in GBM CSCs and provides a novel target to inhibit the JAM-A-mediated CSC maintenance as a next-generation GBM therapy.

IMMU-24. TEMOZOLOMIDE INDUCED IMMUNE DYSFUNCTION REQUIRES THE PRESENCE OF INTRACRANIAL TUMORS

Abstract Temozolomide was recently shown to cause peripheral and intra-tumoral T cell dysfunction in a dosing schedule dependent fashion. Standard dose (SD) temozolomide (TMZ) resulted in T cell dysfunction precluding response to immune checkpoint inhibition that was avoided with a metronomic dosing (MD) schedule. Building on these studies, we investigated the TMZ-induced immune changes in tumor and non-tumor bearing models to understand the interaction of an intracranial tumor on host immunity. C57BL/6 mice underwent intracranial implantation of GL-261 tumor cells. Tumor bearing animals and naïve animals with no tumor were treated with standard dose (50 mg/kg x 5 days) or metronomic dose (25mg/kg x 10 days) of TMZ. Peripheral blood and spleens were collected for flow cytometry, ELISA and luciferase killing assay. Tumor bearing animals treated with SD TMZ demonstrated an increase in circulating myeloid derived suppressor cells (MDSCs), an upregulation of exhaustion markers on endogenous host CD8 T cells (TIM3, LAG3) and a decrease in IFN-gamma secretion from adoptively transferred T cells (tested via ELISA). The cell killing capability of adoptively transferred T cells was not reduced after exposure to a TMZ treated host. Non-tumor bearing animals treated with SD TMZ did not demonstrate an increase in circulating MDSCs or exhaustion markers on endogenous T cells. IFN-gamma secretion from adoptively transferred T cells was still reduced in these animals. The host immune dysfunction induced by TMZ is dependent on the presence of an intracranial malignancy. The mechanisms causing these changes are under active investigation.

RBIO-03. HETEROGENEITY OF HUMAN PATIENT-DERIVED XENOGRAFTS GROWTH RATES RESPONSES TO THE RADIATED MICROENVIRONMENT

Abstract BACKGROUND Radiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumors are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. METHODS Female athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected patient-derived xenograft (PDX) GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. RESULT Of nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196). However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM 39, 123 whose rate of growth was not impacted by the radiated brain. CONCLUSION These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.