Glioblastoma is the most common malignant primary brain tumor in adults. Despite treatment consisting of surgical resection followed by radiotherapy and adjuvant chemotherapy, survival remains poor at a rate of 26.5% at 2 years. Recent successes in using immunotherapies to treat a number of solid and hematologic cancers have led to a growing interest in harnessing the immune system to target glioblastoma. Several studies have examined the efficacy of various immunotherapies, including checkpoint inhibitors, vaccines, adoptive transfer of lymphocytes, and oncolytic virotherapy in both pre-clinical and clinical settings. However, these therapies have yielded mixed results at best when applied to glioblastoma. While the initial failures of immunotherapy were thought to reflect the immunoprivileged environment of the brain, more recent studies have revealed immune escape mechanisms created by the tumor itself and adaptive resistance acquired in response to therapy. Several of these resistance mechanisms hijack key signaling pathways within the immune system to create a protumoral microenvironment. In this review, we discuss immunotherapies that have been trialed in glioblastoma, mechanisms of tumor resistance, and strategies to sensitize these tumors to immunotherapies. Insights gained from the studies summarized here may help pave the way for novel therapies to overcome barriers that have thus far limited the success of immunotherapy in glioblastoma.

Recent longitudinal studies of glioblastoma (GBM) have demonstrated a lack of apparent selection pressure for specific DNA mutations in recurrent disease. Single-cell lineage tracing has shown that GBM cells possess a high degree of plasticity. Together this suggests that phenotype switching, as opposed to genetic evolution, may be the escape mechanism that explains the failure of precision therapies to date. We profiled 86 primary-recurrent patient-matched paired GBM specimens with single-nucleus RNA, single-cell open-chromatin, DNA and spatial transcriptomic/proteomic assays. We found that recurrent GBMs are characterized by a shift to a mesenchymal phenotype. We show that the mesenchymal state is mediated by activator protein 1. Increased T-cell abundance at recurrence was prognostic and correlated with hypermutation status. We identified tumor-supportive networks of paracrine and autocrine signals between GBM cells, nonmalignant neuroglia and immune cells. We present cell-intrinsic and cell-extrinsic targets and a single-cell multiomics atlas of GBM under therapy.

While the poor prognosis of glioblastoma arises from the invasion of a subset of tumor cells, little is known of the metabolic alterations within these cells that fuel invasion. We integrated spatially addressable hydrogel biomaterial platforms, patient site-directed biopsies, and multiomics analyses to define metabolic drivers of invasive glioblastoma cells. Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site-directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Among oncologic ROS, H2O2 specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine γ-lyase (CTH), which converts cystathionine to the nonessential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.

OBJECTIVE: The 2017 World Health Organization classification of pituitary adenomas identified the plurihormonal PIT-1-positive (PP1) adenoma as a distinct subtype. The reported data suggest that PP1 adenomas encompass the former class of silent subtype 3 (SS3) adenomas and might have an aggressive phenotype. In the present study, we summarized the current clinical data on PP1 and SS3 adenomas and compared the reported data with the data from a single institutional cohort. METHODS: Medline and Google Scholar were searched from 1990 to 2020 for clinical series of PP1 and SS3 adenomas in accordance with the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. Studies were included if they had reported pituitary pathology as PP1 or SS3 adenomas and had reported the clinical outcomes after surgical intervention. To better define the PP1 phenotype compared with non-PP1 adenomas, we also reviewed the adenomas treated surgically at our institution from 2012 to 2019. RESULTS: Of all the tumors reported in the studies as PP1 or SS3, 99% were macroadenomas and 18% were giant adenomas (>4 cm). Of the reported patients, 31.8% had received radiotherapy, and 22.9% had undergone multiple surgeries for their pituitary tumor. In our single-center experience, 20 patients had an adenoma that met the criteria for a PP1 adenoma. Compared with the 1146 non-PP1 tumors, the PP1 tumors did not show statistically significant differences in the extent of resection, size, number of previous surgeries, future reoperations, rate of radiotherapy, p53 staining, or MIB-1 labeling index. CONCLUSIONS: The findings from the present large, single-center study comparing PP1 and non-PP1 adenomas do not suggest that PP1 tumors are more aggressive. Further work is warranted to identify the pathologic subtypes of pituitary adenomas that are consistently more clinically aggressive.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy. SIGNIFICANCE: Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1498/F1.large.jpg.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.