Development of a human glioblastoma model using humanized DRAG mice for immunotherapy.

Glioblastoma (GBM) is the most common and lethal primary brain tumor. The development of alternative humanized mouse models with fully functional human immune cells will potentially accelerate the progress of GBM immunotherapy. We successfully generated humanized DRAG (NOD.Rag1KO.IL2RγcKO) mouse model by transplantation of human DR4+ hematopoietic stem cells (hHSCs), and effectively grafted GBM patient-derived tumorsphere cells to form xenografted tumors intracranially. The engrafted tumors recapitulated the pathological features and the immune cell composition of human GBM. Administration of anti-human PD-1 antibodies in these tumor-bearing humanized DRAG mice decreased the major tumor-infiltrating immunosuppressive cell populations, including CD4+PD-1+ and CD8+PD-1+ T cells, CD11b+CD14+HLA-DR+ macrophages, CD11b+CD14+HLA-DR-CD15- and CD11b+CD14-CD15+ myeloid-derived suppressor cells, indicating the humanized DRAG mice as a useful model to test the efficacy of GBM immunotherapy. Taken together, these results suggest that the humanized DRAG mouse model is a reliable preclinical platform for studying brain cancer immunotherapy and beyond.

TP53-PTEN-NF1 depletion in human brain organoids produces a glioma phenotype in vitro.

Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids.

Multiomics analyses reveal DARS1-AS1/YBX1-controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance.

The glioblastoma (GBM) stem cell-like cells (GSCs) are critical for tumorigenesis/therapeutic resistance of GBM. Mounting evidence supports tumor-promoting function of long noncoding RNAs (lncRNAs), but their role in GSCs remains poorly understood. By combining CRISPRi screen with orthogonal multiomics approaches, we identified a lncRNA DARS1-AS1-controlled posttranscriptional circuitry that promoted the malignant properties of GBM cells/GSCs. Depleting DARS1-AS1 inhibited the proliferation of GBM cells/GSCs and self-renewal of GSCs, prolonging survival in orthotopic GBM models. DARS1-AS1 depletion also impaired the homologous recombination (HR)-mediated double-strand break (DSB) repair and enhanced the radiosensitivity of GBM cells/GSCs. Mechanistically, DARS1-AS1 interacted with YBX1 to promote target mRNA binding and stabilization, forming a mixed transcriptional/posttranscriptional feed-forward loop to up-regulate expression of the key regulators of G1-S transition, including E2F1 and CCND1. DARS1-AS1/YBX1 also stabilized the mRNA of FOXM1, a master transcription factor regulating GSC self-renewal and DSB repair. Our findings suggest DARS1-AS1/YBX1 axis as a potential therapeutic target for sensitizing GBM to radiation/HR deficiency-targeted therapy.

Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some patients with GBM; however, there is a need for noninvasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cell activation. Therefore, we aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Herein, we performed CD69 immunostaining on human and mouse T cells following in vitro activation and post immune checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequencing (scRNA-seq) data from patients with recurrent GBM receiving ICI. Radiolabeled CD69 Ab PET/CT imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TIL) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from patients with ICI-treated recurrent GBM as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for patients with GBM. Significance: Immunotherapy may hold promise for the treatment of some patients with GBM. There is a need to assess therapy responsiveness to allow the continuation of effective treatment in responders and to avoid ineffective treatment with potential adverse effects in the nonresponders. We demonstrate that noninvasive PET/CT imaging of CD69 may allow early detection of immunotherapy responsiveness in patients with GBM.

Tumor Niches: Perspectives for Targeted Therapies in Glioblastoma.

Significance: Glioblastoma (GBM), the most common and lethal primary brain tumor with a median survival rate of only 15 months and a 5-year survival rate of only 6.8%, remains largely incurable despite the intensive multimodal treatment of surgical resection and radiochemotherapy. Developing effective new therapies is an unmet need for patients with GBM. Recent Advances: Targeted therapies, such as antiangiogenesis therapy and immunotherapy, show great promise in treating GBM based upon increasing knowledge about brain tumor biology. Single-cell transcriptomics reveals the plasticity, heterogeneity, and dynamics of tumor cells during GBM development and progression. Critical Issues: While antiangiogenesis therapy and immunotherapy have been highly effective in some types of cancer, the disappointing results from clinical trials represent continued challenges in applying these treatments to GBM. Molecular and cellular heterogeneity of GBM is developed temporally and spatially, which profoundly contributes to therapeutic resistance and tumor recurrence. Future Directions: Deciphering mechanisms of tumor heterogeneity and mapping tumor niche trajectories and functions will provide a foundation for the development of more effective therapies for GBM patients. In this review, we discuss five different tumor niches and the intercellular and intracellular communications among these niches, including the perivascular, hypoxic, invasive, immunosuppressive, and glioma-stem cell niches. We also highlight the cellular and molecular biology of these niches and discuss potential strategies to target these tumor niches for GBM therapy. Antioxid. Redox Signal. 39, 904-922.

Protocol to assess the antitumor efficacy of an immunotherapeutic peptide in syngeneic orthotopic glioma mouse models.

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.

A neurodevelopmental epigenetic programme mediated by SMARCD3-DAB1-Reelin signalling is hijacked to promote medulloblastoma metastasis.

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin-DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

Development of a human glioblastoma model using humanized DRAG mice for immunotherapy.

Glioblastoma (GBM) is the most common and lethal primary brain tumor with high mortality rates and a short median survival rate of about 15 months despite intensive multimodal treatment of maximal surgical resection, radiotherapy, and chemotherapy. Although immunotherapies have been successful in the treatment of various cancers, disappointing results from clinical trials for GBM immunotherapy represent our incomplete understanding. The development of alternative humanized mouse models with fully functional human immune cells will potentially accelerate the progress of GBM immunotherapy. In this study, we developed a humanized DRAG (NOD.Rag1KO.IL2RγcKO) mouse model, in which the human hematopoietic stem cells (HSCs) were well-engrafted and subsequently differentiated into a full lineage of immune cells. Using this humanized DRAG mouse model, GBM patient-derived tumorsphere lines were successfully engrafted to form xenografted tumors, which can recapitulate the pathological features and the immune cell composition of human GBM. Importantly, the administration of anti-human PD-1 antibodies in these DRAG mice bearing a GBM patient-derived tumorsphere line resulted in decreasing the major tumor-infiltrating immunosuppressive cell populations, including CD4 + PD-1 + and CD8 + PD-1 + T cells, CD11b + CD14 + HLA-DR + macrophages, CD11b + CD14 + HLA-DR - CD15 - and CD11b + CD14 - CD15 + myeloid-derived suppressor cells, indicating the humanized DRAG mouse model as a useful model to test the efficacy of immune checkpoint inhibitors in GBM immunotherapy. Together, these results suggest that humanized DRAG mouse models are a reliable preclinical platform for brain cancer immunotherapy and beyond.

Phagocytosis of Glioma Cells Enhances the Immunosuppressive Phenotype of Bone Marrow-Derived Macrophages.

Tumor-associated macrophages (TAM) play a crucial role in immunosuppression. However, how TAMs are transformed into immunosuppressive phenotypes and influence the tumor microenvironment (TME) is not fully understood. Here, we utilized single-cell RNA sequencing and whole-exome sequencing data of glioblastoma (GBM) tissues and identified a subset of TAMs dually expressing macrophage and tumor signatures, which were termed double-positive TAMs. Double-positive TAMs tended to be bone marrow-derived macrophages (BMDM) and were characterized by immunosuppressive phenotypes. Phagocytosis of glioma cells by BMDMs in vitro generated double-positive TAMs with similar immunosuppressive phenotypes to double-positive TAMs in the GBM TME of patients. The double-positive TAMs were transformed into M2-like macrophages and drove immunosuppression by expressing immune-checkpoint proteins CD276, PD-L1, and PD-L2 and suppressing the proliferation of activated T cells. Together, glioma cell phagocytosis by BMDMs in the TME leads to the formation of double-positive TAMs with enhanced immunosuppressive phenotypes, shedding light on the processes driving TAM-mediated immunosuppression in GBM. SIGNIFICANCE: Bone marrow-derived macrophages phagocytose glioblastoma cells to form double-positive cells, dually expressing macrophage and tumor signatures that are transformed into M2-like macrophages and drive immunosuppression.

Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma.

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM's natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A-FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow-derived macrophages through activation of the FOSL2-ANXA1-FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration. SIGNIFICANCE: GBM progression could be induced by hypoxia via the HIF1A-FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow-derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration. This article is highlighted in the In This Issue feature, p. 2711.

Polarized macrophages promote gestational beta cell growth through extracellular signal-regulated kinase 5 signalling.

AIM: To show that depletion of pancreatic macrophages impairs gestational beta cell proliferation and leads to glucose intolerance. MATERIALS AND METHODS: Genetic animal models were applied to study the effects of depletion of pancreatic macrophges on gestational beta-cell proliferaiton and glucose response. The crosstalk between macrophages and beta-cells was studied in vivo using beta-cell-specific extracellular-signal-regulated kinase 5 (ERK5) knockout and epidermal growth receptor (EGFR) knockout mice, and in vitro using a co-culture system. RESULTS: Beta cell-derived placental growth factor (PlGF) recruited naïve macrophages and polarized them towards an M2-like phenotype. These macrophages then secreted epidermal growth factor (EGF), which activated extracellular signal-regulated kinase 5 (ERK5) signalling in beta cells to promote gestational beta cell proliferation. On the other hand, activation of ERK5 signalling in beta cells likely, in turn, enhanced the production and secretion of PlGF by beta cells. CONCLUSIONS: Our study shows a regulatory loop between macrophages and beta cells through PlGF/EGF/ERK5 signalling cascades to regulate gestational beta cell growth.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

Noninvasive Method for Predicting the Expression of Ki67 and Prognosis in Non-Small-Cell Lung Cancer Patients: Radiomics.

Purpose: In this study, we aimed to develop and validate a noninvasive method based on radiomics to evaluate the expression of Ki67 and prognosis of patients with non-small-cell lung cancer (NSCLC). Patients and Methods. A total of 120 patients with NSCLC were enrolled in this retrospective study. All patients were randomly assigned to a training dataset (n = 85) and test dataset (n = 35). According to the preprocessed F-FDG PET/CT image of each patient, a total of 384 radiomics features were extracted from the segmentation of regions of interest (ROIs). The Spearman correlation test and least absolute shrinkage and selection operator (LASSO), after normalization on the features matrix, were applied to reduce the dimensionality of the features. Furthermore, multivariable logistic regression analysis was used to propose a model for predicting Ki67. The survival curve was used to explore the prognostic significance of radiomics features. Results: A total of 62 Ki67 positive patients and 58 Ki67 negative patients formed the training set and test training dataset and test dataset. Radiomics signatures showed good performance in predicting the expression of Ki67 with AUCs of 0.86 (training dataset) and 0.85 (test dataset). Validation and calibration showed that the radiomics had a strong predictive power in patients with NSCLC survival, which was significantly close to the effect of Ki67 expression on the survival of patients with NSCLC. Conclusion: Radiomics signatures based on preoperative F-FDG PET/CT could distinguish the expression of Ki67, which also had a strong predictive performance for the survival outcome.

Pre-clinical models for evaluating glioma targeted immunotherapies.

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.

A resource of high-quality and versatile nanobodies for drug delivery.

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs "Duraleukin" and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.

TIGIT and PD-1 Immune Checkpoint Pathways Are Associated With Patient Outcome and Anti-Tumor Immunity in Glioblastoma.

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.

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma.

More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.

Therapy-Induced Transdifferentiation Promotes Glioma Growth Independent of EGFR Signaling.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFß/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFß secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFß-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. SIGNIFICANCE: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.