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Abstract Background Surgical resection is integral for the treatment of neuroblastoma, the most common extracranial solid malignancy in children. Safely locating and resecting primary tumor and remote deposits of disease remains a significant challenge, resulting in high rates of complications and incomplete surgery, worsening outcomes. Intraoperative molecular imaging (IMI) uses targeted radioactive or fluorescent tracers to identify and visualize tumors intraoperatively. GD2 was selected as an IMI target, as it is highly overexpressed in neuroblastoma and minimally expressed in normal tissue. Methods GD2 expression in neuroblastoma cell lines was measured by flow cytometry. DTPA and IRDye® 800CW were conjugated to anti-GD2 antibody to generate DTPA-αGD2-IR800. Binding affinity (Kd) of the antibody and the non-radiolabeled tracer were then measured by ELISA assay. Human neuroblastoma SK-N-BE(2) cells were surgically injected into the left adrenal gland of 3.5-5-week-old nude mice and the orthotopic xenograft tumors grew for 5 weeks. 111In-αGD2-IR800 or isotype control tracer was administered via tail vein injection. After 4 and 6 days, mice were euthanized and gamma and fluorescence biodistributions were measured using a gamma counter and ImageJ analysis of acquired SPY-PHI fluorescence images of resected organs (including tumor, contralateral adrenal, kidneys, liver, muscle, blood, and others). Organ uptake was compared by one-way ANOVA (with a separate analysis for each tracer/day combination), and if significant, Sidak’s multiple comparison test was used to compare the uptake of each organ to the tumor. Handheld tools were also used to detect and visualize tumor in situ, and to assess for residual disease following non-guided resection. Results 111In-αGD2-IR800 was successfully synthesized with 0.75-2.0 DTPA and 2–3 IRDye® 800CW per antibody and retained adequate antigen-binding (Kd = 2.39 nM for aGD2 vs. 21.31 nM for DTPA-aGD2-IR800). The anti-GD2 tracer demonstrated antigen-specific uptake in mice with human neuroblastoma xenografts (gamma biodistribution tumor-to-blood ratios of 3.87 and 3.88 on days 4 and 6 with anti-GD2 tracer), while isotype control tracer did not accumulate (0.414 and 0.514 on days 4 and 6). Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection. Conclusions We have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively. The tracer adequately binds to GD2, specifically accumulates in GD2-expressing xenograft tumors, and enables tumor visualization with a hand-held NIR camera. These results encourage the development of 111In-αGD2-IR800 for future use in children with neuroblastoma, with the goal of improving patient safety, completeness of resection, and overall patient outcomes.

Abstract Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor. Currently, there are few effective treatment options for GBM beyond surgery and chemo-radiation, and even with these interventions, median patient survival remains poor. While immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy against non-central nervous system cancers, ICI trials for GBM have typically had poor outcomes. TIGIT is an immune checkpoint receptor that is expressed on activated T-cells and has a role in the suppression of T-cell and Natural Killer (NK) cell function. As TIGIT expression is reported as both prognostic and a biomarker for anti-TIGIT therapy, we constructed a molecular imaging agent, [89Zr]Zr-DFO-anti-TIGIT (89Zr-αTIGIT), to visualize TIGIT in preclinical GBM by immunoPET imaging. PET imaging and biodistribution analysis of 89Zr-αTIGIT demonstrated uptake in the tumor microenvironment of GBM-bearing mice. Blocking antibody and irrelevant antibody tracer studies demonstrated specificity of 89Zr-αTIGIT with significance at a late time point post-tracer injection. However, the magnitude of 89Zr-αTIGIT uptake in tumor, relative to the IgG tracer was minimal. These findings highlight the features and limitations of using 89Zr-αTIGIT to visualize TIGIT in the GBM microenvironment.

Pediatric high-grade glioma (pHGG) including pediatric glioblastoma (pGBM) are highly aggressive pediatric central nervous system (CNS) malignancies. pGBM comprises approximately 3% of all pediatric CNS malignancies and has a 5-year survival rate of approximately 20%. Surgical resection and chemoradiation are often the standard of care for pGBM and pHGG, however, even with these interventions, survival for children diagnosed with pGBM and pHGG remains poor. Due to shortcomings associated with the standard of care, many efforts have been made to create novel immunotherapeutic approaches targeted to these malignancies. These efforts include the use of vaccines, cell-based therapies, and immune-checkpoint inhibitors. However, it is believed that in many pediatric glioma patients an immunosuppressive tumor microenvironment (TME) possess barriers that limit the efficacy of immune-based therapies. One of these barriers includes the presence of immunosuppressive myeloid cells. In this review we will discuss the various types of myeloid cells present in the glioma TME, including macrophages and microglia, myeloid-derived suppressor cells, and dendritic cells, as well as the specific mechanisms these cells can employ to enable immunosuppression. Finally, we will highlight therapeutic strategies targeted to these cells that are aimed at impeding myeloid-cell derived immunosuppression.

Abstract Neuroblastoma accounts for 15% of pediatric cancer deaths, despite multimodal therapy including surgical resection. Current neuroblastoma rodent models are insufficient for studying the impact of surgery and combination treatments, largely due to the small size of mouse models. Human neuroblastoma SK-N-BE(2) cells were injected into the left adrenal gland of 5–6-week-old RNU homozygous nude rats. Rats were either monitored by MRI until humane endpoint was reached or after 5 weeks underwent operative tumor resection, followed by monitoring for recurrence and survival. Following neuroblastoma cell implantation, the majority of tumors grew to greater than 5000 mm3 within 5.5–6.5 weeks, meeting the humane endpoint. Surgical resection was successfully done in 8 out of 9 rats, extending survival following tumor implantation from a median of 42 days to 78 days (p

Summary: Glioblastoma (GBM) is one of the most lethal central nervous systems (CNS) tumours in adults. As supplements to standard of care (SOC), various immunotherapies improve the therapeutic effect in other cancers. Among them, tumour vaccines can serve as complementary monotherapy or boost the clinical efficacy with other immunotherapies, such as immune checkpoint blockade (ICB) and chimeric antigen receptor T cells (CAR-T) therapy. Previous studies in GBM therapeutic vaccines have suggested that few neoantigens could be targeted in GBM due to low mutation burden, and single-peptide therapeutic vaccination had limited efficacy in tumour control as monotherapy. Combining diverse antigens, including neoantigens, tumour-associated antigens (TAAs), and pathogen-derived antigens, and optimizing vaccine design or vaccination strategy may help with clinical efficacy improvement. In this review, we discussed current GBM therapeutic vaccine platforms, evaluated and potential antigenic targets, current challenges, and perspective opportunities for efficacy improvement.

Summary: Understanding the glioblastoma (GBM) immune microenvironment and development of clinical treatment drugs rely on suitable preclinical GBM models. Here, we present a protocol to establish syngeneic orthotopic glioma mouse models. We also describe the steps to intracranially deliver immunotherapeutic peptides and monitor the treatment response. Finally, we show how to assess the tumor immune microenvironment with treatment outcomes.For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Brain tumors are one of the leading causes of cancer related death in both the adult and pediatric patient population. Gliomas represent a cohort of brain tumors derived from glial cell lineages which include astrocytomas, oligodendrogliomas and glioblastomas (GBMs). These tumors are known to grow aggressively and have a high lethality with GBM being the most aggressive tumor in this group. Currently, few treatment options exist for GBM outside of surgical resection, radiation therapy and chemotherapy. While these measures have been shown to marginally improve patient survival, patients, especially those diagnosed with GBM, often experience a recurrence of their disease. Following disease recurrence, treatment options become more limited as additional surgical resections can pose life threatening risk to the patient, patients may be ineligible for additional radiation, and the recurrent tumor may be resistant to chemotherapy. Immune checkpoint inhibitors (ICIs) have revolutionized the field of cancer immunotherapy as many patients with cancers residing outside the central nervous system (CNS) have experienced a survival benefit from this treatment modality. It has often been observed that this survival benefit is increased following neoadjuvant administration of immune checkpoint inhibitors as tumor antigen is still present in the patient which enables a more robust anti-tumor immune response. Interestingly, results for ICI-based studies for patients with GBM have been largely disappointing which is a stark contrast from the success this treatment modality has had in non-central nervous system cancers. In this review, we will discuss the various benefits of neoadjuvant immune checkpoint inhibition such as how this approach reduces tumor burden and allows for a greater induction of an anti-tumor immune response. Additionally, we will discuss several non-CNS cancers where neoadjuvant immune checkpoint inhibition has been successful and discuss why we believe this approach may provide a survival benefit for GBM patients. We hope this manuscript will foster future studies aimed at exploring whether this approach may be beneficial for patients diagnosed with GBM.

Gliomas have an extremely poor prognosis in both adult and pediatric patient populations as these tumors are known to grow aggressively and respond poorly to standard of care treatment. Currently, treatment for gliomas involves surgical resection followed by chemoradiation therapy. However, some gliomas, such as diffuse midline glioma, have more limited treatment options such as radiotherapy alone. Even with these interventions, the prognosis for those diagnosed with a glioma remains poor. Immunotherapy is highly effective for some cancers and there is great interest in the development of effective immunotherapies for the treatment of gliomas. Clinical trials evaluating the efficacy of immunotherapies targeted to gliomas have largely failed to date, and we believe this is partially due to the poor choice in pre-clinical mouse models that are used to evaluate these immunotherapies. A key consideration in evaluating new immunotherapies is the selection of pre-clinical models that mimic the glioma-immune response in humans. Multiple pre-clinical options are currently available, each one with their own benefits and limitations. Informed selection of pre-clinical models for testing can facilitate translation of more promising immunotherapies in the clinical setting. In this review we plan to present glioma cell lines and mouse models, as well as alternatives to mouse models, that are available for pre-clinical glioma immunotherapy studies. We plan to discuss considerations of model selection that should be made for future studies as we hope this review can serve as a guide for investigators as they choose which model is best suited for their study.

Oncolytic herpes simplex viruses (oHSVs) have demonstrated efficient lytic replication in human glioblastoma tumors using immunodeficient mouse models, but early-phase clinical trials have reported few complete responses. Potential reasons for the lack of efficacy are limited vector potency and the suppressive glioma tumor microenvironment (TME). Here we compare the oncolytic activity of two HSV-1 vectors, a KOS-strain derivative KG4:T124 and an F-strain derivative rQNestin34.5v.1, in the CT2A and GL261N4 murine syngeneic glioma models. rQNestin34.5v1 generally demonstrated a greater in vivo viral burden compared to KG4:T124. However, both vectors were rapidly cleared from CT2A tumors, while virus remained ensconced in GL261N4 tumors. Immunological evaluation revealed that the two vectors induced similar changes in immune cell recruitment to either tumor type at 2 days after infection. However, at 7 days after infection, the CT2A microenvironment displayed the phenotype of an untreated tumor, while GL261N4 tumors exhibited macrophage and CD4+/CD8+ T cell accumulation. Furthermore, the CT2A model was completely resistant to virus therapy, while in the GL261N4 model rQNestin34.5v1 treatment resulted in enhanced macrophage recruitment, impaired tumor progression, and long-term survival of a few animals. We conclude that prolonged intratumoral viral presence correlates with immune cell recruitment, and both are needed to enhance anti-tumor immunity.

Background: Malignant gliomas are deadly tumours with few therapeutic options. Although immunotherapy may be a promising therapeutic strategy for treating gliomas, a significant barrier is the CD11b+ tumour-associated myeloid cells (TAMCs), a heterogeneous glioma infiltrate comprising up to 40% of a glioma's cellular mass that inhibits anti-tumour T-cell function and promotes tumour progression. A theranostic approach uses a single molecule for targeted radiopharmaceutical therapy (TRT) and diagnostic imaging; however, there are few reports of theranostics targeting the tumour microenvironment. Methods: Utilizing a newly developed bifunctional chelator, Lumi804, an anti-CD11b antibody (αCD11b) was readily labelled with either Zr-89 or Lu-177, yielding functional radiolabelled conjugates for PET, SPECT, and TRT. Findings: 89Zr/177Lu-labeled Lumi804-αCD11b enabled non-invasive imaging of TAMCs in murine gliomas. Additionally, 177Lu-Lumi804-αCD11b treatment reduced TAMC populations in the spleen and tumour and improved the efficacy of checkpoint immunotherapy. Interpretation: 89Zr- and 177Lu-labeled Lumi804-αCD11b may be a promising theranostic pair for monitoring and reducing TAMCs in gliomas to improve immunotherapy responses. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Glioblastoma (GBM) remains an aggressive brain tumor with a high rate of mortality. Immune checkpoint (IC) molecules are expressed on tumor infiltrating lymphocytes (TILs) and promote T cell exhaustion upon binding to IC ligands expressed by the tumor cells. Interfering with IC pathways with immunotherapy has promoted reactivation of anti-tumor immunity and led to success in several malignancies. However, IC inhibitors have achieved limited success in GBM patients, suggesting that other checkpoint molecules may be involved with suppressing TIL responses. Numerous IC pathways have been described, with current testing of inhibitors underway in multiple clinical trials. Identification of the most promising checkpoint pathways may be useful to guide the future trials for GBM. Here, we analyzed the The Cancer Genome Atlas (TCGA) transcriptomic database and identified PD1 and TIGIT as top putative targets for GBM immunotherapy. Additionally, dual blockade of PD1 and TIGIT improved survival and augmented CD8+ TIL accumulation and functions in a murine GBM model compared with either single agent alone. Furthermore, we demonstrated that this combination immunotherapy affected granulocytic/polymorphonuclear (PMN) myeloid derived suppressor cells (MDSCs) but not monocytic (Mo) MDSCs in in our murine gliomas. Importantly, we showed that suppressive myeloid cells express PD1, PD-L1, and TIGIT-ligands in human GBM tissue, and demonstrated that antigen specific T cell proliferation that is inhibited by immunosuppressive myeloid cells can be restored by TIGIT/PD1 blockade. Our data provide new insights into mechanisms of GBM αPD1/αTIGIT immunotherapy.

Background: 23Na MRI correlates with tumor proliferation, and studies in pediatric patients are lacking. The purpose of the study: (1) to compare total sodium concentration (TSC) between pediatric glioma and non-neoplastic brain tissue using 23Na MRI; (2) compare tissue conspicuity of bound sodium concentration (BSC) using 23Na MRI dual echo relative to TSC imaging. Methods: TSC was measured in: (1) non-neoplastic brain tissues and (2) three types of manually segmented gliomas (diffuse intrinsic brainstem glioma (DIPG), recurrent supratentorial low-grade glioma (LGG), and high-grade glioma (HGG)). In a subset of patients, serial changes in both TSC and BSC (dual echo 23Na MRI) were assessed. Results: Twenty-six pediatric patients with gliomas (median age of 12.0 years, range 4.9–23.3 years) were scanned with 23Na MRI. DIPG treated with RT demonstrated higher TSC values than the uninvolved infratentorial tissues (p < 0.001). Recurrent supratentorial LGG and HGG exhibited higher TSC values than the uninvolved white matter (WM) and gray matter (GM) (p < 0.002 for LGG, and p < 0.02 for HGG). The dual echo 23Na MRI suppressed the sodium signal within both CSF and necrotic foci. Conclusion: Quantitative 23Na MRI of pediatric gliomas demonstrates a range of values that are higher than non-neoplastic tissues. Dual echo 23Na MRI of BCS improves tissue conspicuity relative to TSC imaging.

Abstract Background Sodium/hydrogen exchanger 1 (NHE1), encoded by the SLC9A1 gene (SoLute Carrier family 9A1) in humans, is the main H+ efflux mechanism in maintaining alkaline intracellular pH (pHi) and Warburg effects in glioma. However, to date, there are no clinical studies exploring pharmacological inhibition of NHE1 protein in cancer treatment. In this study, we investigated NHE1 expression in gliomas and its relationship with glioma clinical outcome. Methods The Chinese Glioma Genome Atlas (CGGA) dataset containing transcriptome sequencing data of 325 glioma samples and the Cancer Genome Atlas (TCGA) with 698 glioma mRNAseq data were analyzed in this study. Mouse SB28 and GL26 intracranial syngeneic glioma models in C57BL/6 J mice were established to investigate NHE1 expression and impact of NHE1 protein inhibition with its inhibitor HOE642 on tumorigenesis and anti-PD1 therapy. Tumor angiogenesis, immunogenicity, and progression were assessed by immunofluorescence staining and flow cytometric profiling. Results Analysis of SLC9A1 mRNA expression in two data sets, CGGA and TCGA, reveals significantly higher SLC9A1 mRNA levels in higher grade gliomas. The SLC9A1 mRNA expression was especially enriched in isocitrate dehydrogenase (IDH)1/2 wild-type glioblastoma (GBM) and in mesenchymal glioma subtypes. Worsened survival probabilities were correlated with the elevated SLC9A1 mRNA levels in gliomas. The underlying mechanisms include promoting angiogenesis, and extracellular matrix remodeling. Increased SLC9A1 mRNA expression was also associated with tumor-associated macrophage accumulation. NHE1 inhibitor HOE642 reduced glioma volume, invasion, and prolonged overall survival in mouse glioma models. Blockade of NHE1 protein also stimulated immunogenic tumor microenvironment via activating CD8 T-cell accumulation, increasing expression of interferon-gamma (Ifng), and sensitized animals to anti-PD-1 therapy. Conclusion Our findings strongly suggest that NHE1 protein emerges as a marker for tumorigenesis and prognosis in glioma. Blocking NHE1 protein is a novel strategy for adjuvant anti-cancer therapies.

Abstract Background Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. Results We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. Conclusions We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) receptors are frequently amplified and/or possess gain‐of‐function mutations in GBM. However, clinical trials of tyrosine‐kinase inhibitors have shown disappointing efficacy, in part due to intra‐tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single‐cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high‐resolution phylogenies of EGF‐driven and PDGF‐driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF‐driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor‐like cell type, expressing pro‐angiogenic factors. In contrast, phylogenetic analysis of an EGFR‐amplified tumor showed an up‐regulation of pro‐invasive genes. An in‐frame deletion in a specific dimerization domain of PDGF receptor correlates with an up‐regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In‐frame deletions in this domain are frequent in public GBM data.