Evaluation of Microvascular Density in Glioblastomas in Relation to p53 and Ki67 Immunoexpression.

Glioblastoma is the most aggressive tumor in the central nervous system, with a survival rate of less than 15 months despite multimodal therapy. Tumor recurrence frequently occurs after removal. Tumoral angiogenesis, the formation of neovessels, has a positive impact on tumor progression and invasion, although there are controversial results in the specialized literature regarding its impact on survival. This study aims to correlate the immunoexpression of angiogenesis markers (CD34, CD105) with the proliferation index Ki67 and p53 in primary and secondary glioblastomas. This retrospective study included 54 patients diagnosed with glioblastoma at the Pathology Department of County Emergency Clinical Hospital Târgu Mureș. Microvascular density was determined using CD34 and CD105 antibodies, and the results were correlated with the immunoexpression of p53, IDH1, ATRX and Ki67. The number of neoformed blood vessels varied among cases, characterized by different shapes and calibers, with endothelial cells showing modified morphology and moderate to marked pleomorphism. Neovessels with a glomeruloid aspect, associated with intense positivity for CD34 or CD105 in endothelial cells, were observed, characteristic of glioblastomas. Mean microvascular density values were higher for the CD34 marker in all cases, though there were no statistically significant differences compared to CD105. Mutant IDH1 and ATRX glioblastomas, wild-type p53 glioblastomas, and those with a Ki67 index above 20% showed a more abundant microvascular density, with statistical correlations not reaching significance. This study highlighted a variety of percentage intervals of microvascular density in primary and secondary glioblastomas using immunohistochemical markers CD34 and CD105, respectively, with no statistically significant correlation between evaluated microvascular density and p53 or Ki67.

HNRNPA2B1 stabilizes NFATC3 levels to potentiate its combined actions with FOSL1 to mediate vasculogenic mimicry in GBM cells.

BACKGROUND: Vasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM. AIMS: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM. METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo. RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo. CONCLUSION: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.

Protocol to quantify immune cell distribution from the vasculature to the glioma microenvironment on sequential immunofluorescence multiplex images.

Although myeloid-derived immune cells can be dispersed throughout the tumor microenvironment (TME), anti-tumor effector cells are confined to the perivascular space. Here, we present a protocol to quantify immune cell distribution from tumor vasculature to its glioma microenvironment on sequential immunofluorescence multiplex images. We describe steps for sequential immunofluorescence multiplex staining, image generation, and storage. We then detail the procedures for tissue, vessel, and nuclei segmentation; cell phenotyping; data extraction; and training using RStudio and Spyder.

PDZK1 is correlated with DCE-MRI perfusion parameters in high-grade glioma.

OBJECTIVE: This study investigated the relationship between PDZK1 expression and Dynamic Contrast-Enhanced MRI (DCE-MRI) perfusion parameters in High-Grade Glioma (HGG). METHODS: Preoperative DCE-MRI scanning was performed on 80 patients with HGG to obtain DCE perfusion transfer coefficient (Ktrans), vascular plasma volume fraction (vp), extracellular volume fraction (ve), and reverse transfer constant (kep). PDZK1 in HGG patients was detected, and its correlation with DCE-MRI perfusion parameters was assessed by the Pearson method. An analysis of Cox regression was performed to determine the risk factors affecting survival, while Kaplan-Meier and log-rank tests to evaluate PDZK1's prognostic significance, and ROC curve analysis to assess its diagnostic value. RESULTS: PDZK1 was upregulated in HGG patients and predicted poor overall survival and progression-free survival. Moreover, PDZK1 expression distinguished grade III from grade IV HGG. PDZK1 expression was positively correlated with Ktrans 90, and ve_90, and negatively correlated with kep_max, and kep_90. CONCLUSION: PDZK1 is upregulated in HGG, predicts poor survival, and differentiates tumor grading in HGG patients. PDZK1 expression is correlated with DCE-MRI perfusion parameters.

Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization.

Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.

Microbeam Radiation Therapy Opens a Several Days' Vessel Permeability Window for Small Molecules in Brain Tumor Vessels.

PURPOSE: Synchrotron microbeam radiation therapy (MRT), based on an inhomogeneous geometric and microscopic irradiation pattern of the tissues with high-dose and high-dose-rate x-rays, enhances the permeability of brain tumor vessels. This study attempted to determine the time and size range of the permeability window induced by MRT in the blood-brain (tumor) barrier. METHODS AND MATERIALS: Rats-bearing 9L gliomas were exposed to MRT, either unidirectional (tumor dose, 406 Gy) or bidirectional (crossfired) (2 × 203 Gy). We measured vessel permeability to molecules of 3 sizes (Gd-DOTA, Dotarem, 0.56 kDa; gadolinium-labeled albumin, ∼74 kDa; and gadolinium-labeled IgG, 160 kDa) by daily in vivo magnetic resonance imaging, from 1 day before to 10 days after irradiation. RESULTS: An equivalent tumor dose of bidirectional MRT delivered from 2 orthogonal directions increased tumor vessel permeability for the smallest molecule tested more effectively than unidirectional MRT. Bidirectional MRT also affected the permeability of normal contralateral vessels to a different extent than unidirectional MRT. Conversely, bidirectional MRT did not modify the permeability of normal or tumor vessels for both larger molecules (74 and 160 kDa). CONCLUSIONS: High-dose bidirectional (cross-fired) MRT induced a significant increase in tumor vessel permeability for small molecules between the first and the seventh day after irradiation, whereas permeability of vessels in normal brain tissue remained stable. Such a permeability window could facilitate an efficient and safe delivery of intravenous small molecules (≤0.56 kDa) to tumoral tissues. A permeability window was not achieved by molecules larger than gado-grafted albumin (74 kDa). Vascular permeability for molecules between these 2 sizes has not been determined.

The utility of arterial spin labeling imaging for predicting prognosis after a recurrence of high-grade glioma in patients under bevacizumab treatment.

BACKGROUND AND PURPOSE: Currently, the antiangiogenic agent bevacizumab (BVZ) is used as a treatment option for high-grade glioma (HGG) patients. However, BVZ restores disruptions of the blood-brain barrier, which leads to the disappearance of contrast enhancement during radiological examinations and therefore complicates evaluations of treatment efficacy. This study aimed to investigate the radio-morphological features of recurrent lesions that newly appeared under BVZ therapy, as well as the utility of arterial spin labeling (ASL) perfusion imaging for evaluating treatment response and prognosis in HGG patients receiving BVZ. METHODS: Thirty-two patients (20 males, 12 females; age range, 35-84 years) with HGG who experienced a recurrence under BVZ therapy were enrolled. We measured the relative cerebral blood flow (rCBF) values of each recurrent lesion using ASL, and retrospectively investigated the correlation between rCBF values and prognosis. RESULTS: The optimal rCBF cut-off value for predicting prognosis was defined as 1.67 using receiver operating characteristic curve analysis. The patients in the rCBF < 1.67 group had significantly longer overall survival (OS) and post-progression survival (PPS) than those in the rCBF ≥ 1.67 group (OS: 34.0 months vs. 13.0 months, p = 0.03 and PPS: 13.0 months vs. 6.0 months, p < 0.001, respectively). CONCLUSION: The ASL-derived rCBF values of recurrent lesions may serve as an effective imaging biomarker for prognosis in HGG patients undergoing BVZ therapy. Low rCBF values may indicate that BVZ efficacy is sustainable, which will influence BVZ treatment strategies in HGG patients.

Reproducibility of rCBV in glioblastomas using T2*-weighted perfusion MRI: an evaluation of sampling, normalization, and experience.

PURPOSE: The reproducibility of relative cerebral blood volume (rCBV) measurements among readers with different levels of experience is a concern. This study aimed to investigate the inter-reader reproducibility of rCBV measurement of glioblastomas using the hotspot method in dynamic susceptibility contrast perfusion magnetic resonance imaging (DSC-MRI) with various strategies. METHODS: In this institutional review board-approved single-center study, 30 patients with glioblastoma were retrospectively evaluated with DSC-MRI at a 3.0 Tesla scanner. Three groups of reviewers, including neuroradiologists, general radiologists, and radiology residents, calculated the rCBV based on the number of regions of interest (ROIs) and reference areas. For statistical analysis of feature reproducibility, the intraclass correlation coefficient (ICC) and Bland-Altman plots were used. Analyses were made among individuals, reader groups, reader-group pooling, and a population that contained all of them. RESULTS: For individuals, the highest inter-reader reproducibility was observed between neuroradiologists [ICC: 0.527; 95% confidence interval (CI): 0.21-0.74] and between residents (ICC: 0.513; 95% CI: 0.20-0.73). There was poor reproducibility in the analyses of individuals with different levels of experience (ICC range: 0.296-0.335) and in reader-wise and group-wise pooling (ICC range: 0.296-0.335 and 0.397-0.427, respectively). However, an increase in ICC values was observed when five ROIs were used. In an analysis of all strategies, the ICC for the centrum semiovale was significantly higher than that for contralateral white matter (P < 0.001). CONCLUSION: The inter-reader reproducibility of rCBV measurement was poor to moderate regardless of whether it was calculated by neuroradiologists, general radiologists, or residents, which may indicate the need for automated methods. Choosing five ROIs and using the centrum semiovale as a reference area may increase reliability for all users.

Tailoring vascular phenotype through AAV therapy promotes anti-tumor immunity in glioma.

Glioblastomas are aggressive brain tumors that are largely immunotherapy resistant. This is associated with immunosuppression and a dysfunctional tumor vasculature, which hinder T cell infiltration. LIGHT/TNFSF14 can induce high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), suggesting that its therapeutic expression could promote T cell recruitment. Here, we use a brain endothelial cell-targeted adeno-associated viral (AAV) vector to express LIGHT in the glioma vasculature (AAV-LIGHT). We found that systemic AAV-LIGHT treatment induces tumor-associated HEVs and T cell-rich TLS, prolonging survival in αPD-1-resistant murine glioma. AAV-LIGHT treatment reduces T cell exhaustion and promotes TCF1+CD8+ stem-like T cells, which reside in TLS and intratumoral antigen-presenting niches. Tumor regression upon AAV-LIGHT therapy correlates with tumor-specific cytotoxic/memory T cell responses. Our work reveals that altering vascular phenotype through vessel-targeted expression of LIGHT promotes efficient anti-tumor T cell responses and prolongs survival in glioma. These findings have broader implications for treatment of other immunotherapy-resistant cancers.

Arterial spin labeling clinical applications for brain tumors and tumor treatment complications: A comprehensive case-based review.

Arterial spin labeling (ASL) is a noninvasive neuroimaging technique that allows for quantifying cerebral blood flow without intravenous contrast. Various neurovascular disorders and tumors have cerebral blood flow alterations. Identifying these perfusion changes through ASL can aid in the diagnosis, especially in entities with normal structural imaging. In addition, complications of tumor treatment and tumor progression can also be monitored using ASL. In this case-based review, we demonstrate the clinical applications of ASL in diagnosing and monitoring brain tumors and treatment complications.

Vessel size and perfusion-derived vascular habitat refines prediction of treatment failure to bevacizumab in recurrent glioblastomas: validation in a prospective cohort.

OBJECTIVES: Anti-angiogenic therapy may not benefit all patients with recurrent glioblastomas, and imaging biomarker predicting treatment response to anti-angiogenic therapy is currently limited. We aimed to develop and validate vascular habitats based on perfusion and vessel size to predict time to progression (TTP) in patients with recurrent glioblastomas treated with bevacizumab. METHODS: Sixty-nine patients with recurrent glioblastomas treated with bevacizumab who underwent pretreatment MRI with dynamic susceptibility contrast imaging and vessel architectural imaging were enrolled. Vascular habitats were constructed using vessel size index (VSI) and relative cerebral blood volume (rCBV). Associations with vascular habitats and TTP were analyzed using Cox proportional hazard regression analysis. In a prospectively enrolled validation cohort consisting of 15 patients ( ClinicalTrials.gov identifier; NCT04143425), stratification of TTP was demonstrated by the Kaplan-Meier method (log-rank test) using vascular habitats. RESULTS: Three vascular habitats consisting of high, intermediate, and low angiogenic habitats were identified with rCBV and VSI. Both high angiogenic and intermediate angiogenic habitats were significantly associated with a shorter TTP (hazard ratio [HR], 2.78 and 1.82, respectively; largest p = .003) and so was rCBV (HR, 2.15; p = .02). Concordance probability index of vascular habitat combining high and intermediate angiogenic habitats was 0.74. Vascular habitats stratified patients as good or poor responder in a prospective cohort (p = .059). CONCLUSIONS: Perfusion- and vessel size-derived vascular habitats predicted TTP in recurrent glioblastomas treated with anti-angiogenic therapy and aided patient stratification in a prospective validation cohort. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04143425 KEY POINTS: • High and intermediate angiogenic habitats predicted TTP in recurrent glioblastomas treated with anti-angiogenic therapy. • Vascular habitats combining high and intermediate angiogenic habitats aided patient stratification for anti-angiogenic therapy in recurrent glioblastomas.

A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier-penetrant nanoparticles.

The blood­brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood­brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood­brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood­brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

3D pCASL-perfusion in preoperative assessment of brain gliomas in large cohort of patients.

The aim of the study was to evaluate the role of pseudocontinuous arterial spin labeling perfusion (pCASL-perfusion) in preoperative assessment of cerebral glioma grades. The study group consisted of 253 patients, aged 7-78 years with supratentorial gliomas (65 low-grade gliomas (LGG), 188 high-grade gliomas (HGG)). We used 3D pCASL-perfusion for each patient in order to calculate the tumor blood flow (TBF). We obtained maximal tumor blood flow (maxTBF) in small regions of interest (30 ± 10 mm2) and then normalized absolute maximum tumor blood flow (nTBF) to that of the contralateral normal-appearing white matter of the centrum semiovale. MaxTBF and nTBF values significantly differed between HGG and LGG groups (p < 0.001), as well as between patient groups separated by the grades (grade II vs. grade III) (p < 0.001). Moreover, we performed ROC-analysis which demonstrated high sensitivity and specificity in differentiating between HGG and LGG. We found significant differences for maxTBF and nTBF between grade III and IV gliomas, however, ROC-analysis showed low sensitivity and specificity. We did not observe a significant difference in TBF for astrocytomas and oligodendrogliomas. Our study demonstrates that 3D pCASL-perfusion as an effective diagnostic tool for preoperative differentiation of glioma grades.

FRAT1 promotes the angiogenic properties of human glioblastoma cells via VEGFA.

Glioblastoma is a common central nervous system tumor and despite considerable advancements in treatment patient prognosis remains poor. Angiogenesis is a significant prognostic factor in glioblastoma, anti­angiogenic treatments represent a promising therapeutic approach. Vascular endothelial growth factor A (VEGFA) is a predominant regulator of angiogenesis and mounting evidence suggests that the Wnt signaling pathway serves a significant role in tumor angiogenesis. As a positive regulator of the Wnt/ß­catenin signaling pathway, frequently rearranged in advanced T­cell lymphomas­1 (FRAT1) is highly expressed in human glioblastoma and is significantly associated with glioblastoma growth, invasion and migration, as well as poor patient prognosis. Bioinformatics analysis demonstrated that both VEGFA and FRAT1 were highly expressed in most tumor tissues and associated with prognosis. However, whether and how FRAT1 is involved in angiogenesis remains to be elucidated. In the present study, the relationship between FRAT1 and VEGFA in angiogenesis was investigated using the human glioblastoma U251 cell line. Small interfering RNAs (siRNAs) were used to silence FRAT1 expression in U251 cells, and the mRNA and protein expression levels of VEGFA, as well as the concentration of VEGFA in U251 cell supernatants, were determined using reverse transcription­quantitative PCR, western blotting and ELISA. A tube formation assay was conducted to assess angiogenesis. The results demonstrated that siRNA knockdown significantly decreased the protein expression levels of FRAT1 in U251 cells and markedly decreased the mRNA and protein expression levels of VEGFA. Furthermore, the concentration of VEGFA in the cell supernatant was significantly reduced and angiogenesis was suppressed. These results suggested that FRAT1 may promote VEGFA secretion and angiogenesis in human glioblastoma cells via the Wnt/ß­catenin signaling pathway, supporting the potential use of FRAT1 as a promising therapeutic target in human glioblastoma.

The Study of Cerebral Blood Flow Variations during Brain Metastases Radiotherapy.

PURPOSE: The aim of this study was to investigate the cerebral blood flow (CBF) variations during brain metastases (BMs) radiotherapy (RT) applying with magnetic resonance (MR) 3D-arterial spin labeling (ASL). MATERIALS AND METHODS: A total of 26 BM patients with 54 tumors were retrospectively enrolled. MR examinations were performed before and during RT (30-50 Gy) with a total dose of 36-60 Gy (12-30 fractions) including contrast-enhanced T1-weighted, T2 Flair, and 3D-ASL images. The relationship between CBF changes and the largest cross-sectional area changes in BMs was investigated. And CBF changes in BMs, normal brain tissue, and peritumoral edema areas were analyzed under different dose gradients that were divided into 10 Gy intervals. RESULTS: The largest cross-sectional areas and CBF of 54 BMs decreased by 26.46% and 29.64%, respectively, during RT (p < 0.05), but there was no correlation between the 2 changes (p > 0.05). The rates of CBF decrease in BMs were 33.75%, 24.61%, and 27.55% at 30-40, 40-50, and >50 Gy, respectively (p < 0.05). In normal brain tissue with dose gradients of 0-10, 10-20, 20-30, 30-40, 40-50, and >50 Gy, the CBF decreased by 7.65%, 11.12%, 18.42%, 20.23%, 19.79%, and 17.89%, respectively (p < 0.05). The CBF decreases reached a maximum at 30-40 Gy in normal brain tissue as well as BMs. In contrast, the CBF decreases of peritumoral edema areas increased as the dose gradients increased. Moreover, the CBF changes of BMs were more notable than those in normal brain tissue and peritumoral edema areas. CONCLUSION: CBF changes can be feasibly assessed in different brain regions during RT based on 3D-ASL. The changes should be considered as a critical factor to determine the personal radiation dose for BMs, normal brain tissue, and peritumoral edema areas.

Canonical and Non-Canonical Roles of PFKFB3 in Brain Tumors.

PFKFB3 is a bifunctional enzyme that modulates and maintains the intracellular concentrations of fructose-2,6-bisphosphate (F2,6-P2), essentially controlling the rate of glycolysis. PFKFB3 is a known activator of glycolytic rewiring in neoplastic cells, including central nervous system (CNS) neoplastic cells. The pathologic regulation of PFKFB3 is invoked via various microenvironmental stimuli and oncogenic signals. Hypoxia is a primary inducer of PFKFB3 transcription via HIF-1alpha. In addition, translational modifications of PFKFB3 are driven by various intracellular signaling pathways that allow PFKFB3 to respond to varying stimuli. PFKFB3 synthesizes F2,6P2 through the phosphorylation of F6P with a donated PO4 group from ATP and has the highest kinase activity of all PFKFB isoenzymes. The intracellular concentration of F2,6P2 in cancers is maintained primarily by PFKFB3 allowing cancer cells to evade glycolytic suppression. PFKFB3 is a primary enzyme responsible for glycolytic tumor metabolic reprogramming. PFKFB3 protein levels are significantly higher in high-grade glioma than in non-pathologic brain tissue or lower grade gliomas, but without relative upregulation of transcript levels. High PFKFB3 expression is linked to poor survival in brain tumors. Solitary or concomitant PFKFB3 inhibition has additionally shown great potential in restoring chemosensitivity and radiosensitivity in treatment-resistant brain tumors. An improved understanding of canonical and non-canonical functions of PFKFB3 could allow for the development of effective combinatorial targeted therapies for brain tumors.

Evaluation of single bolus, dual-echo dynamic susceptibility contrast MRI protocols in brain tumor patients.

Relative cerebral blood volume (rCBV) obtained from dynamic susceptibility contrast (DSC) MRI is adversely impacted by contrast agent leakage in brain tumors. Using simulations, we previously demonstrated that multi-echo DSC-MRI protocols provide improvements in contrast agent dosing, pulse sequence flexibility, and rCBV accuracy. The purpose of this study is to assess the in-vivo performance of dual-echo acquisitions in patients with brain tumors (n = 59). To verify pulse sequence flexibility, four single-dose dual-echo acquisitions were tested with variations in contrast agent dose, flip angle, and repetition time, and the resulting dual-echo rCBV was compared to standard single-echo rCBV obtained with preload (double-dose). Dual-echo rCBV was comparable to standard double-dose single-echo protocols (mean (standard deviation) tumor rCBV 2.17 (1.28) vs. 2.06 (1.20), respectively). High rCBV similarity was observed (CCC = 0.96), which was maintained across both flip angle (CCC = 0.98) and repetition time (CCC = 0.96) permutations, demonstrating that dual-echo acquisitions provide flexibility in acquisition parameters. Furthermore, a single dual-echo acquisition was shown to enable quantification of both perfusion and permeability metrics. In conclusion, single-dose dual-echo acquisitions provide similar rCBV to standard double-dose single-echo acquisitions, suggesting contrast agent dose can be reduced while providing significant pulse sequence flexibility and complementary tumor perfusion and permeability metrics.

Exploring the longitudinal glioma microenvironment landscape uncovers reprogrammed pro-tumorigenic neutrophils in the bone marrow.

Recent multi-omics studies show different immune tumor microenvironment (TME) compositions in glioblastoma (GBM). However, temporal comprehensive knowledge of the TME from initiation of the disease remains sparse. We use Cre recombinase (Cre)-inducible lentiviral murine GBM models to compare the cellular evolution of the immune TME in tumors initiated from different oncogenic drivers. We show that neutrophils infiltrate early during tumor progression primarily in the mesenchymal GBM model. Depleting neutrophils in vivo at the onset of disease accelerates tumor growth and reduces the median overall survival time of mice. We show that, as a tumor progresses, bone marrow-derived neutrophils are skewed toward a phenotype associated with pro-tumorigenic processes. Our findings suggest that GBM can remotely regulate systemic myeloid differentiation in the bone marrow to generate neutrophils pre-committed to a tumor-supportive phenotype. This work reveals plasticity in the systemic immune host microenvironment, suggesting an additional point of intervention in GBM treatment.

Optical tissue clearing and machine learning can precisely characterize extravasation and blood vessel architecture in brain tumors.

Precise methods for quantifying drug accumulation in brain tissue are currently very limited, challenging the development of new therapeutics for brain disorders. Transcardial perfusion is instrumental for removing the intravascular fraction of an injected compound, thereby allowing for ex vivo assessment of extravasation into the brain. However, pathological remodeling of tissue microenvironment can affect the efficiency of transcardial perfusion, which has been largely overlooked. We show that, in contrast to healthy vasculature, transcardial perfusion cannot remove an injected compound from the tumor vasculature to a sufficient extent leading to considerable overestimation of compound extravasation. We demonstrate that 3D deep imaging of optically cleared tumor samples overcomes this limitation. We developed two machine learning-based semi-automated image analysis workflows, which provide detailed quantitative characterization of compound extravasation patterns as well as tumor angioarchitecture in large three-dimensional datasets from optically cleared samples. This methodology provides a precise and comprehensive analysis of extravasation in brain tumors and allows for correlation of extravasation patterns with specific features of the heterogeneous brain tumor vasculature.

Long non-coding RNA HULC stimulates the epithelial-mesenchymal transition process and vasculogenic mimicry in human glioblastoma.

BACKGROUND: Long non-coding RNA (lncRNA) HULC (highly upregulated in liver cancer) is considered as an oncogenic factor for various malignant tumors. This study aimed to reveal the role of lncRNA HULC in the malignant behavior of glioblastoma (GBM) by exploring its effects on the epithelial-mesenchymal transition (EMT) and vasculogenic mimicry (VM) of human GBM. MATERIALS AND METHODS: The contents of VM in 27 GBM samples were assessed by immunohistochemistry-histology and their association with progress-free survival (PFS) was analyzed. Human GBM SHG44 and U87 cells were manipulated to establish stable lncRNA HULC overexpressing and silencing cells by lentivirus-based technology. The effects of altered lncRNA HULC on vasculogenic tubular formation, invasion, and EMT process in GBM cells were tested in vitro and the growth of implanted GBM tumors and their EMT process were examined in vivo. RESULTS: The numbers of VM were positively associated with disease progression, but negatively with PFS periods of GBM patients. Compared with the control vec cells, lncRNA HULC overexpression significantly increased the tubular formation, invasion, and EMT process of both SHG44 and U87 cells, accompanied by promoting the growth of implanted GBM tumors and EMT process in mice. LncRNA HULC silencing had opposite effects on the tubular formation, invasion, and EMT process as well as tumor growth of GBM cells. CONCLUSION: LncRNA HULC stimulates the EMT process and VM in human GBM, and may be a therapeutic target for intervention of GBM.