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Neural development requires the orchestration of dynamic changes in gene expression to regulate cell fate decisions. This regulation is heavily influenced by epigenetics, heritable changes in gene expression not directly explained by genomic information alone. An understanding of the complexity of epigenetic regulation is rapidly emerging through the development of novel technologies that can assay various features of epigenetics and gene regulation. Here, we provide a broad overview of several commonly investigated modes of epigenetic regulation, including DNA methylation, histone modifications, noncoding RNAs, as well as epitranscriptomics that describe modifications of RNA, in neurodevelopment and diseases. Rather than functioning in isolation, it is being increasingly appreciated that these various modes of gene regulation are dynamically interactive and coordinate the complex nature of neurodevelopment along multiple axes. Future work investigating these interactions will likely utilize 'multi-omic' strategies that assay cell fate dynamics in a high-dimensional and high-throughput fashion. Novel human neurodevelopmental models including iPSC and cerebral organoid systems may provide further insight into human-specific features of neurodevelopment and diseases.

Despite the established efficacy of chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies, translating CAR T therapy to solid tumors has remained investigational. Glioblastoma, the most aggressive and lethal form of primary brain tumor, has recently been among the malignancies being trialed clinically with CAR T cells. Glioblastoma in particular holds several unique features that have hindered clinical translation, including its vast intertumoral and intratumoral heterogeneity, associated immunosuppressive environment, and lack of clear experimental models to predict response and analyze resistant phenotypes. Here, we review the history of CAR T therapy development, its current progress in treating glioblastoma, as well as the current challenges and future directions in establishing CAR T therapy as a viable alternative to the current standard of care. Tremendous efforts are currently ongoing to identify novel CAR targets and target combinations for glioblastoma, to modify T cells to enhance their efficacy and to enable them to resist tumor-mediated immunosuppression, and to utilize adjunct therapies such as lymphodepletion, checkpoint inhibition, and bi-specific engagers to improve CAR T persistence. Furthermore, new preclinical models of CAR T therapy are being developed that better reflect the clinical features seen in human trials. Current clinical trials that rapidly incorporate key preclinical findings to patient translation are emerging.

Immature dentate granule cells (imGCs) arising from adult hippocampal neurogenesis contribute to plasticity and unique brain functions in rodents(1,2) and they are dysregulated in multiple human neurological disorders(3–5). Little is known about molecular characteristics of adult human hippocampal imGCs and even their existence is under debate(1,6–8). Here we performed single-nucleus RNA-sequencing (snRNA-seq) aided by a validated machine learning-based analytic approach to identify imGCs and quantify their abundance in the human hippocampus during infant, child, adolescent, adult, and aging stages. We identified common molecular hallmarks of human imGCs across the lifespan and discovered age-dependent transcriptional dynamics unique to human imGCs that suggest changes in cellular functionality, niche interactions, and disease relevance, in contrast to those in mice9. We also found a decreased number of imGCs with altered gene expression in Alzheimer’s disease (AD). Finally, we demonstrated the capacity for neurogenesis in the adult human hippocampus with the presence of rare dentate granule cell fate-specific proliferating neural progenitors and with cultured surgical specimens. Together, our findings suggest the presence of a significant number of imGCs in the adult human hippocampus via low frequency de novo generation and protracted maturation, and our study reveals their molecular properties across the lifespan and in AD.

Successful treatment of glioblastoma (GBM) is hampered by primary tumor recurrence after surgical resection and poor prognosis, despite adjuvant radiotherapy and chemotherapy. In search of improved outcomes for this disease, quisinostat appeared as a lead compound in drug screening. A delivery system was devised for this drug and to exploit current clinical methodology: an injectable hydrogel, loaded with both the quisinostat drug and radiopaque gold nanoparticles (AuNP) as contrast agent, that can release these payloads as a response to radiation. This hydrogel grants high local drug concentrations, overcoming issues with current standards of care. Significant hydrogel degradation and quisinostat release were observed due to the radiation trigger, providing high in vitro anticancer activity. In vivo, the combination of radiotherapy and the radiation-induced delivery of quisinostat from the hydrogel, successfully inhibited tumor growth in a mice model bearing xenografted human GBM tumors with a total response rate of 67%. Long-term tolerability was observed after intratumoral injection of the quisinostat loaded hydrogel. The AuNP payload enabled precise image-guided radiation delivery and the monitoring of hydrogel degradation using computed tomography (CT). These exciting results highlight this hydrogel as a versatile imageable drug delivery platform that can be activated simultaneously to radiation therapy and potentially offers improved treatment for GBM.

This chapter discusses the incidence, pathology and classification, clinical features, and management of tumors of the central nervous system (CNS). Approximately 80,000 adult and 3600 childhood primary CNS tumors are estimated to be diagnosed in 2018. Most tumors of the CNS present symptomatically by mass effect on nearby normal brain tissue. In these cases, symptoms correspond to the neuro-anatomic location of the tumor. Certain hormone secreting tumors, such as those derived from the pituitary, may present more constitutionally due to hormonal changes. The new 2016 World Health Organization (WHO) Classification of CNS tumors represented a major change in the classification of brain tumors. Generally, tumors are classified as tumors of glial or non-glial origin. Novel molecular markers are now being used to distinguish glial tumors that otherwise are not distinguishable by pathology alone (such as in 1p19q in oligodendroglioma). Other markers, such as isocitrate dehydrogenase (IDH) mutations in glioblastoma, can also provide prognostic significance. The management of tumors of the CNS depends upon its histology. Tissue biopsy is generally required to make management decisions. Some malignant tumors such as glioblastoma require maximal resection followed by adjuvant chemoradiation. In contrast, non glial-tumors such as seminoma, germinoma and lymphoma respond well to non-surgical therapy. Novel therapies at the molecular level are now being developed and utilized to further improve patient outcomes.

Glioblastoma (GBM) is the most prevalent and lethal primary brain tumor, which carries a poor prognosis due to a high level of therapeutic resistance and rate of tumor recurrence. Underlying this aggressive behavior is extraordinary genomic and cellular heterogeneity, conferring numerous avenues for treatment escape and refractory growth. This work leverages single cell genomic analyses to link somatic mutations to transcriptomic signatures within the native tumor tissue. We implemented a three-stage pipeline to perform the following tasks: variant calling, machine learning (ML) classification, and biological pathway analysis. Somatic variants were called from smart-seq based scRNA-seq data and filtered based on predicted lethality, reference in cancer mutation databases e.g. COSMIC, and population prevalence. In the second stage, the genotyped cells and normalized gene expression profiles were pre-processed using differential gene expression, and input into ML classifiers trained on the task of predicting mutation status. The utilized ML models included a Support Vector Machine and Ridge Classifier and were selected based on performance and interpretability. The efficacy of the models was evaluated using the AUC-ROC metric across multiple dataset partitions. The output of this stage was a set of “important genes” identified using the learned ML model weights. In the third stage, the sets of identified genes were subsampled and analyzed using the g:Profiler API to identify significant biological pathways. The analysis was performed on 2 datasets containing 677 samples from 5 patients and 1091 samples from 4 patients. The EGFRvIII mutation was used to validate the performance of the methodology due to its prevalence in primary GBM tumors. The mutation was detected in 17 single-cell samples. The ML models both produced a mean AUC-ROC of 0.93 [0.92, 094], and the classifiers identified NPM1, TCEA1, PPIA, SUMO2, among other genes, to be of significance. The individual genes have functions involving cell cycle regulation and protein folding, while the identified biological pathways associated with gene subsets were linked to ribonuclease activity and transcription factor post-transcriptional modifications. The ML component was applied to an additional dataset containing cell samples from 4 patients and correctly identified the 2 patients positive for EGFRvIII. The larger analysis identified point mutations in EGFR, TP53, and ATRX, and the classifiers exhibited AUC-ROC metrics ranging from 0.94 [0.93, 0.94] to 0.99 [0.99, 1.0]. Overall, we developed and employed a pipeline to link somatic variants with their gene expression signatures at the single cell level, identified biological pathways for further examination and established a ML classifier that can be applied to high-throughput scRNA-seq methods that lack the transcript coverage of the variant loci. Citation Format: Christopher Streiffer, Daniel Zhang, Ryan Salinas, Steven Brem, Donald O'Rourke, Zev Binder, Han-Chiao Isaac Chen, Hongjun Song. Identifying the transcriptomic signatures of mutational heterogeneity in GBM using single cell genomics [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 2203.

Distinguishing neoplasm from normal brain parenchyma intraoperatively is critical for the neurosurgeon. 5-Aminolevulinic acid (5-ALA) has been shown to improve gross total resection and progression-free survival but has limited availability in the USA. Near-infrared (NIR) fluorescence has advantages over visible light fluorescence with greater tissue penetration and reduced background fluorescence. In order to prepare for the increasing number of NIR fluorophores that may be used in molecular imaging trials, we chose to compare a state-of-the-art, neurosurgical microscope (System 1) to one of the commercially available NIR visualization platforms (System 2). Serial dilutions of indocyanine green (ICG) were imaged with both systems in the same environment. Each system’s sensitivity and dynamic range for NIR fluorescence were documented and analyzed. In addition, brain tumors from six patients were imaged with both systems and analyzed. In vitro, System 2 demonstrated greater ICG sensitivity and detection range (System 1 1.5–251 μg/l versus System 2 0.99–503 μg/l). Similarly, in vivo, System 2 demonstrated signal-to-background ratio (SBR) of 2.6 ± 0.63 before dura opening, 5.0 ± 1.7 after dura opening, and 6.1 ± 1.9 after tumor exposure. In contrast, System 1 could not easily detect ICG fluorescence prior to dura opening with SBR of 1.2 ± 0.15. After the dura was reflected, SBR increased to 1.4 ± 0.19 and upon exposure of the tumor SBR increased to 1.8 ± 0.26. Dedicated NIR imaging platforms can outperform conventional microscopes in intraoperative NIR detection. Future microscopes with improved NIR detection capabilities could enhance the use of NIR fluorescence to detect neoplasm and improve patient outcome.

PURPOSE: Intraoperative molecular imaging with tumor-targeting fluorophores offers real-time detection of neoplastic tissue. The Second-Window-Indocyanine-Green (SWIG) technique relies on passive accumulation of indocyanine-green (ICG), a near-infrared fluorophore, in neoplastic tissues.In this study, we explore the ability of SWIG to detect neoplastic tissue and to predict postoperative Magnetic Resonance Imaging (MRI) findings intraoperatively. PROCEDURES: Retrospective data were collected from 36 patients with primary high-grade gliomas (HGG) enrolled as part of a larger trial between October 2014 and October 2018. Patients received systemic ICG infusions at 2.5–5mg/kg 24 h preoperatively. Near-infrared fluorescence was recorded throughout the case and from biopsy specimens. The presence/location of residual SWIG signal after resection was compared to the presence/location of residual gadolinium enhancement on postoperative MRI. The extent of resection was not changed based on near-infrared imaging. RESULTS: All 36 lesions demonstrated strong near-infrared fluorescence (signal-to-background = 6.8 ± 2.2) and 100% of tumors reaching the cortex were visualized before durotomy. In 78 biopsy specimens, near-infrared imaging demonstrated higher sensitivity and accuracy than white-light for diagnosing neoplastic tissue intraoperatively. Furthermore, near-infrared imaging predicted gadolinium enhancement on postoperative MRI with 91% accuracy, with visualization of residual enhancement as small as 0.3cm(3). Patients with no residual near-infrared signal after resection were significantly more likely to have complete resection on postoperative MRI (p-value < 0.0001). CONCLUSIONS: Intraoperative imaging with SWIG demonstrates highly sensitive detection of HGG tissue in real-time. Furthermore, post-resection near-infrared imaging correlates with postoperative MRI. Overall, our findings suggest that SWIG can provide surgeons with MRI-like results in real-time, potentially increasing resection rates.

Glioblastoma treatment options remain limited due to its aggressive and invasive nature. It is increasingly appreciated that molecular heterogeneity between tumors and within tumors likely contributes to the lack of therapeutic advances. To maintain the inherent heterogeneity of glioblastoma, we employed a novel method to rapidly culture glioblastoma organoids (GBOs) directly from neurosurgical resection. GBOs are routinely generated around two weeks following initial resection. Comprehensive histologic and sequencing analyses demonstrated similarity to primary tumors. Leveraging clinical molecular and sequencing data, selected GBOs were treated with radiation/temozolamide and targeted inhibitor therapies. The effect on proliferation was measured by the percentage of KI67+ cells and gene set enrichment (GSEA) analysis was performed to compare the pre-treated expression signature amongst responsive and non-responsive tumors. Treatment of organoids with radiation/temozolamide led to a decrease in the percentage of KI67+ cells in four of eight patient-derived organoid lines with some evidence of correlative radiographic response Gene sets associated with radiation response and TNF signaling were enriched in radiation/temozolamide sensitive GBOs. GBO response to EGFR inhibition via gefitinib treatment was specific to EGFR altered tumors, whose expression also enriched for EGF signaling pathway expression. Two GBOs had downstream NF1 mutations that responded to the MEK inhibitor trametinib. On GSEA, gene expression of NF1 mutated GBOs enriched for RAS signaling. One GBO line was found to have a PI3K mutation and responded dramatically to mTOR inhibition via everolimus. Dichotomous efficacy of MEK and mTOR inhibition was also noted by tumor-specific changes in GBO diameter following treatment. This novel culturing method of GBOs maintains intertumoral and intratumoral heterogeneity and allows for therapeutic testing within two weeks of neurosurgical resection. As clinical sequencing because increasingly prevalent, GBOs may become a valuable tool to functionally test mutation-specific treatment strategies in a patient-specific manner within a clinically relevant time frame.

Glioblastoma multiforme (GBM) is the most common primary and aggressive brain tumors in adults with extremely poor prognosis and limited treatment options. A major hallmark of GBM is the rapid and diffused infiltration of tumor cells into the surrounding healthy tissue that contribute to tumor recurrence and therapeutic resistance. However, existing in vitro cell culture or in vivo xenograft models inadequately recapitulate the inter-tumoral and intra-tumoral heterogeneity which are key features of GBM. For example, common oncogenic drivers of GBM such as epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation do not persist in traditional in vitro models due to selection pressures, thus requires exogenous overexpression. Alternatively, EGFR statuses can be maintained in xenografted mice, but implantation of the primary GBM cells into the flank is required to first establish the tumor prior to secondary injection into the brains. Recently, we have established a novel protocol for culturing GBM tissue as organoids (GBOs) directly from patient tumor resection that retain many distinct cell populations in vitro with high fidelity evidenced by histological, whole-exome, bulk and single cell RNA analyses. Compared to prolonged generation time of previously established in vitro and xenograft models, our methodology is robust for generating GBOs within 1–2 weeks from initial resection. In addition, these GBOs can be readily xenografted into the adult mouse brains as an intact organoid, exhibit rapid and aggressive infiltration phenotypes, and maintains driver mutation EGFRviii within as little as one month. Consequently, they can be used to test in vivo treatment efficacies in a timely fashion. The presence of diverse cell types in this GBO model offers a promising platform for not only understanding of tumor biology, but also more strategic development of new therapies.

Introduction: Gliosarcoma (GS) is a rare, malignant mixed tumor of the central nervous system with a median survival of approximately 13 months across multiple studies. Although the value of the extent of resection (EOR) has been confirmed as a prognostic survival factor in glioblastoma, no such association has been defined for GS. The goal of this study was to establish an association between EOR and survival and to determine if a threshold of resection exists for which a survival benefit is conferred in GS. Methods: The authors identified 11 patients with histologically confirmed GS between January 2005 and January 2015, treated at the Hospital of the University of Pennsylvania. Clinical, radiographic, and outcome data were retrospectively reviewed. Volumetric analysis was completed using semi-automated segmentation to measure the change in contrast-enhancing material based on preoperative T1-contrast (T1c) and postoperative T1 & T1c magnetic resonance imaging (MRI) scans. A log-rank test was completed to confirm an association between EOR and survival, and a series of Kaplan-Meier curves were constructed to determine an EOR threshold. Univariate Cox proportional hazards model (CPHM) followed by multivariate CPHM was also completed to evaluate associations between the prognostic clinical and immunohistochemistry variables under consideration. Results: Extent of resection categories were defined as gross total resection (GTR >95%), subtotal resection (STR 90%-95%), and partial resection (PR

The primary treatment for brain tumors often involves surgical resection for diagnosis, relief of mass effect, and prolonged survival. In neurosurgery, it is of utmost importance to achieve maximal safe resection while minimizing iatrogenic neurologic deficit. Thus, neurosurgeons often rely on extra tools in the operating room, such as neuronavigation, intraoperative magnetic resonance imaging, and/or intraoperative rapid pathology. However, these tools can be expensive, not readily available, time-consuming, and/or inaccurate. Recently, fluorescence-guided surgery has emerged as a cost-effective method to accurately visualize neoplastic areas in real-time to guide resection. Currently, 5-aminolevulinic-acid (5-ALA) remains the only fluorophore that has been approved specifically for fluorescence-guided tumor resection. Its use has demonstrated improved resection rates and prolonged progression-free survival. However, protoporphyrin-IX, the metabolic product of 5-ALA that accumulates in neoplastic cells, fluoresces in the visible-light range, which suffers from limited tissue penetration and significant auto-fluorescence. Near-infrared fluorescence, on the other hand, overcomes these problems with ease. Since 2012, researchers at our institution have developed a novel technique using indocyanine-green, which is a well-known near-infrared fluorophore used traditionally for angiography. This Second-Window-ICG (SWIG) technique takes advantage of the increased endothelial permeability in peritumoral tissue, which allows indocyanine-green to accumulate in these areas for intraoperative visualization of the tumor. SWIG has demonstrated utility in gliomas, meningiomas, metastases, pituitary adenomas, chordomas, and craniopharyngiomas. The main benefits of SWIG stem from its highly sensitive detection of neoplastic tissue in a wide variety of intracranial pathologies in real-time, which can help neurosurgeons both during surgical resections and in stereotactic biopsies. In this review of this novel technique, we summarize the development and mechanism of action of SWIG, provide evidence for its benefits, and discuss its limitations. Finally, for those interested in near-infrared fluorescence-guided surgery, we provide suggestions for maximizing the benefits while minimizing the limitations of SWIG based on our own experience thus far.

Immunotherapy, including chimeric antigen receptor (CAR) T cell therapy, has yielded major advancements for a number of hard-to-treat cancers; however, its transformative potential has yet to be realized in glioblastoma (GBM). Clinical studies of EGFRvIII targeted CAR T cell therapy have indicated that tumor heterogeneity, immune microenvironment, and adaptive responses to treatment may play important roles in limiting overall efficacy. We sought to examine comprehensively the dynamic molecular landscape of CAR T cell therapy in GBM using patient-derived GBM organoids (GBOs), a newly established laboratory model of inter- and intra- tumoral heterogeneity. Especially advantageous in these studies, GBOs preserve the intrinsic composition of somatic mutations, transcriptomic states, and non-neoplastic cells that contribute to the tumor microenvironment. Complementing the complexity of this biological system, we constructed an integrated single-cell multi-omics platform to interrogate gene expression, cell surface protein expression, somatic variants, and TCR sequences all from the same cell. Co-culture of GBOs and EGFRvIII targeted CAR T cells led to rapid T cell activation with concomitant upregulation of cytokine response gene programs in both antigen-positive and antigen-negative tumor cells. The adaptive tumor response also included expression of immune checkpoint receptor (ICR) ligands, such as PD-L1. At later time points, T cells transitioned into a dysfunctional or exhausted state, as characterized by increased cell surface expression of inhibitory receptors, such as PD-1, and decreased markers of effector activity despite the presence of antigen. Interestingly, CAR T cell therapy not only led to changes in immune-related pathways and tumor microenvironment, but it also induced shifts in tumor cell states with the depletion of an oligodendrocyte precursor cell-like state and corresponding enrichment in an astrocyte-like state. This finding suggests that EGFRvIII targeted CAR T cell therapy may leverage intrinsic cellular plasticity to induce differentiation-like effects in the surviving tumor cells.

Summary Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

SummaryWhile thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known. Here, we identify Pinky (Pnky) as a neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain. In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Pnky interacts with the splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates neurogenesis from embryonic and postnatal NSC populations.

Hyperprolactinemia occurs in patients with a prolactinoma and in those with a sellar mass compressing the pituitary stalk. Distinguishing these two diagnostic possibilities guides treatment with dopamine agonist therapy or surgical resection. We aimed to identify a simple, predictive algorithm to aid in the diagnosis of prolactinoma in patients with an elevated serum prolactin and a sellar mass. A case-control analysis of pathologically confirmed prolactinomas and non-endocrine secreting controls from the University of California, San Francisco was performed. From 2001 to 2011, this resulted in 177 patients with prolactinomas and 87 controls. Univariate and classification and regression tree (CART) analysis determined the significance of demographic variables, patient symptoms, laboratory values, and radiographic findings in distinguishing pathology. Additionally, a subset of patients with mildly elevated serum prolactin (25-125 ng/ml) was independently analyzed. Prolactinomas had a mean pre-operative prolactin of 858 ng/ml versus 17.57 ng/ml in controls (p0.01). One hundred and two (62.6%) of the prolactinomas were macroadenomas (size10mm) compared to 74 (92.5%) of the controls (p0.01). CART analysis identified preoperative prolactin (41.5 ng/ml), age (40.5 years), and size (17 mm) as being predictive of prolactinoma with a misclassification rate of 7.9% (21/264). Similar analysis on the subset of patients with mildly elevated serum prolactin (125 ng/ml) identified size (2.5 cm) and pre-operative prolactin (40 ng/ml) as key variables. These two factors correctly predicted 98.6% (69/70) of cases. Our model correctly classifies most patients with elevated serum prolactin and identifies those patients most amenable to surgical treatment.

Introduction Approximately 100,000 brain metastases are diagnosed annually in the United States. Our laboratory has pioneered a novel technique, second window indocyanine green (SWIG), which allows for real-time intraoperative visualization of brain metastasis through normal brain parenchyma and intact dura. Methods Thirteen patients with intraparenchymal brain metastases were administered indocyanine green (ICG) at 5 mg/kg the day before surgery. A near-infrared (NIR)– capable camera was used intraoperatively to identify the tumor and to inspect surgical margins. Neuropathology was used to assess the accuracy and precision of the fluorescent dye for identifying tumor. Results ICG was infused at 24.7 ± 3.45 hours before visualization. All 13 metastases fluoresced with an average signal-to-background ratio (SBR) of 6.62. The SBR with the dura intact was 67.2% of the mean SBR once the dura was opened. The NIR signal could be visualized through normal brain parenchyma up to 7 mm. For the 39 total specimens, the mean SBR for tumor specimens ( n = 28) was 6.9, whereas the SBR for nontumor specimens ( n = 11) was 3.7. The sensitivity, specificity, positive predictive value, and negative predictive value of NIR imaging for tumor was 96.4%, 27.3%, 77.1%, and 75.0%. Discussion SWIG relies on the passive accumulation of dye in abnormal tumor tissue via the enhanced permeability and retention effect. It provides strong NIR optical contrast, which can be used to localize tumors before dural opening. The use of SWIG for margin assessment remains limited by its lack of specificity (high false-positive rate); however, ongoing improvements in imaging parameters show great potential to reduce false-positive results.

SummaryThe epigenetic mechanisms that enable lifelong neurogenesis from neural stem cells (NSCs) in the adult mammalian brain are poorly understood. Here, we show that JMJD3, a histone H3 lysine 27 (H3K27) demethylase, acts as a critical activator of neurogenesis from adult subventricular zone (SVZ) NSCs. JMJD3 is upregulated in neuroblasts, and Jmjd3 deletion targeted to SVZ NSCs in both developing and adult mice impairs neuronal differentiation. JMJD3 regulates neurogenic gene expression via interaction at not only promoter regions but also neurogenic enhancer elements. JMJD3 localizes at neural enhancers genome-wide in embryonic brain, and in SVZ NSCs, JMJD3 regulates the I12b enhancer of Dlx2. In Jmjd3-deleted SVZ cells, I12b remains enriched with H3K27me3 and Dlx2-dependent neurogenesis fails. These findings support a model in which JMJD3 and the poised state of key transcriptional regulatory elements comprise an epigenetic mechanism that enables the activation of neurogenic gene expression in adult NSCs throughout life.

Glioblastoma exhibits enormous genetic, transcriptional, and cellular heterogeneity at the macroscopic level across regions of the tumor as well as at the microscopic level between neighboring cells, all of which present significant challenges towards creating a definitive treatment for this devastating disease. We have developed a method of generating glioblastoma organoids (GBOs) from fresh tissue obtained directly from surgical resection and maintaining them in a defined medium without bFGF/EGF. Whole exome sequencing revealed that GBOs maintain the genomic landscape of their parent tumors. Somatic and copy number variants are present in the GBOs at similar allele frequencies or copy ratios as in the parent tumor, suggesting that the relative proportions of clonal populations are largely maintained in the organoids. Bulk transcriptomic analysis demonstrated strong gene expression correlations between the parent tumor and corresponding GBOs through 12 weeks of culture. Some tumors were sampled at multiple different anatomic regions, and the corresponding GBOs maintained region-specific gene expression signatures and genomic variants. EGFRvIII, a tumor-specific variant targeted in a number of emerging therapies, also remains present in the GBOs at similar transcript frequencies, reflecting the native heterogeneity of the parent tumor. Finally, we used single cell transcriptomics to examine cellular heterogeneity and find that GBOs contain many different cell types that exhibit similar gene expression profiles as the matching cell type in the corresponding parent tumor. Notably, these GBOs retain neoplastic as well as non-neoplastic cells, such as tumor associated macrophages / microglia, T-cells, endothelial cells, stromal cells, and oligodendrocytes. These GBOs preserve complex tumor heterogeneity an in vitro environment, creating opportunities for extended manipulation, characterization, and functional study for mechanistic investigation and therapeutic testing.

INTRODUCTION: To improve surgical resection of brain tumors, our lab has pioneered a novel fluorescent dye technique, Second-Window Indocyanine-Green (SWIG), that relies on passive delivery and accumulation of indocyanine-green (ICG) in neoplastic tissue via the enhanced permeability and retention effect. We hypothesize that SWIG can provide early localization of brain metastasis prior to dural opening and can improve identification of surgical margins. METHODS: Subjects were prospectively enrolled in clinical trial after informed consent. Approximately 24 hours prior surgery, subjects were infused intravenously with 2.5mg/kg or 5mg/kg of ICG. Intraoperatively, a dedicated near-infrared (NIR) camera was used to detect ICG signal. After bone flap removal, the NIR imaging system was positioned above the presumed location of tumor. Additional NIR images were obtained after dural opening, corticectomy, and after conventional white-light surgical resection. RESULTS: We enrolled 50 patients with 51 total intraparenchymal brain metastases (23 lung, 7 breast, 8 GU/GI, 4 melanoma, and 7 others). Prior to dural opening, NIR signal was identified in 35 patients at an average depth of 4.3mm with SBR = 5.3 + 3.7. In the seven patients where NIR signal could not be identified prior to dural opening, tumor depth was an average of 8.4mm from cortical surface. Upon dural opening and tumor identification, all 51 tumors demonstrated strong NIR signal with SBR = 6.2 + 2.8. With white light alone, sensitivity/specificity/PPV/NPV for tumor detection was 83%, 94%, 98%, 57%. With NIR, sensitivity/specificity/PPV/NPV for tumor detection was 100%, 29%, 85%, 100%. DISCUSSION: NIR fluorophores are superior to visible light fluorophores in their depth of penetration. All contrast-enhancing brain metastasis accumulate ICG using our SWIG technique, and NIR fluorescence could be used to localize brain metastasis prior to dural opening. NIR fluorophores are likely to represent the next phase in tumor visualization given the rapid growth of fluorophores targeted to systemic cancers.

OBJECTIVE As odontoid process fractures become increasingly common in the aging population, a technical understanding of treatment approaches is critical. 3D image guidance can improve the safety of posterior cervical hardware placement, but few studies have explored its utility in anterior approaches. The authors present in a stepwise fashion the technique of odontoid screw placement using the Medtronic O-arm navigation system and describe their initial institutional experience with this surgical approach. METHODS The authors retrospectively reviewed all cases of anterior odontoid screw fixation for Type II fractures at an academic medical center between 2006 and 2015. Patients were identified from a prospectively collected institutional database of patients who had suffered spine trauma. A standardized protocol for navigated odontoid screw placement was generated from the collective experience at the authors' institution. Secondarily, the authors compared collected variables, including presenting symptoms, injury mechanism, surgical complications, blood loss, operative time, radiographically demonstrated nonunion rate, and clinical outcome at most recent follow-up, between navigated and nonnavigated cases. RESULTS Ten patients (three female; mean age 61) underwent odontoid screw placement. Most patients presented with neck pain without a neurological deficit after a fall. O-arm navigation was used in 8 patients. An acute neck hematoma and screw retraction, each requiring surgery, occurred in 2 patients in whom navigation was used. Partial vocal cord paralysis occurred after surgery in one patient in whom no navigation was used. There was no difference in blood loss or operative time with or without navigation. One patient from each group had radiographic nonunion. No patient reported a worsening of symptoms at follow-up (mean duration 9 months). CONCLUSIONS The authors provide a detailed step-by-step guide to the navigated placement of an odontoid screw. Their surgical experience suggests that O-arm–assisted odontoid screw fixation is a viable approach. Future studies will be needed to rigorously compare the accuracy and efficiency of navigated versus nonnavigated odontoid screw placement.

DNA is known to condense with multivalent cations and positively charged proteins. However, the properties and energetics of DNA superstructures, such as chromatin, are poorly understood. As a model system, we investigate histone H1 condensation of DNA with tethered particle motion and force-extension measurements. We show that after the addition of H1 to DNA, a concentration dependent lag time is followed by the DNA spontaneously condensing. The trigger for this condensation phase transition can be modeled as sufficient H1s having bound to the DNA, providing insight into the 30 nm fiber condensation upon H1 binding. Furthermore, optical tweezers force-extension measurements of histone H1 condensed DNA reveals a sequence of state transitions corresponding to the unwinding of superhelical turns. We determine the complete, experimental, multi-state free energy landscape for the complex using Crooks fluctuation theorem. The measured force-versus-extension and free energy landscape are compared to predictions from a simple, theoretical model. This work encourages the theoretical description of DNA/protein structure and energetics and their role in chromatin and other, more complex, systems.

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that theInk4a/Arf(also known asCdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passagein vitro, wild-type postnatal cortical astroglia become progressively resistant toDlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence ofInk4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced fromInk4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16Ink4aand p19Arfindicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient forInk4a/Arfalso exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate thatInk4a/Arfis a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

Author(s): Potts, Matthew B; Siu, Jason J; Price, James D; Salinas, Ryan D; Cho, Mathew J; Ramos, Alexander D; Hahn, Junghyun; Margeta, Marta; Oldham, Michael C; Lim, Daniel A | Abstract: BackgroundMixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons.ObjectiveTo identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons.MethodsWe performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology.ResultsTranscriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons.ConclusionOur results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

It is increasingly clear that while genetic alterations drive glioblastoma development, epigenetic dysregulation can also contribute to oncogenesis. Although there is clear evidence of tumors driven by specific mutations to histones or their modifiers, genomic studies have demonstrated that the majority of brain tumors are not molecularly classified by mutations in key epigenetic regulators. We hypothesized that some of the most common genetic mutations in glioblastoma can lead to chromatin-based transcriptional dysregulation. First, to investigate the transcriptional changes from specific genetic mutations, we performed RNA-sequencing analysis of a mouse tumor progression model with Ink4a/arf−/- cells derived from the subventricular zone (SVZ) which do not form tumors when intracranially injected into SCID mice, "primary" tumors produced by overexpression of EGFRviii in Ink4a/Arf −/- cells, and "secondary" tumors that models tumor recurrence via reinjection of primary tumor cells into the brains of other mice. Compared to Ink4a/Arf-/-, both primary and secondary tumors downregulated genes related to neuron differentiation and cell adhesion, while overexpressing homeobox genes related to cellular morphogenesis, including the HoxA locus. Secondary tumors, which carry worse survival, upregulate invasion-associated extracellular matrix and basement membrane genes. Intriguingly, ChIP-seq analysis revealed a clear loss of the repressive H3K27me3 mark at the HoxA locus and other homeodomain containing genes upon Ink4a/Arf inactivation. Subsequently, a subset of the genes became overexpressed upon tumorigenesis with a concomitant and progressive increase of the promoter activating H3K4me3 mark. Furthermore, IHC in mouse and a subset of human samples demonstrated heterogeneity of H3K27me3 staining compared to H3, while Western blot analysis also demonstrated a relative decrease in HK27me3 staining in mouse tumor samples compared to Ink4a/Arf−/-. These results support a model whereby Ink4a/Arf deficiency is sufficient to induce epigenetic changes via loss of H3K27me3, generating heterogeneity within a given tumor sample and allowing for EGFRviii driven oncogenic selection.