Extracellular vesicles (EVs) are continually released from cancer cells into biofluids, carrying actionable molecular fingerprints of the underlying disease with considerable diagnostic and ...therapeutic potential. The scarcity, heterogeneity and intrinsic complexity of tumor EVs present a major technological challenge in real-time monitoring of complex cancers such as glioblastoma (GBM). Surface-enhanced Raman spectroscopy (SERS) outputs a label-free spectroscopic fingerprint for EV molecular profiling. However, it has not been exploited to detect known biomarkers at the single EV level. We developed a multiplex fluidic device with embedded arrayed nanocavity microchips (MoSERS microchip) that achieves 97% confinement of single EVs in a minute amount of fluid (<10 μL) and enables molecular profiling of single EVs with SERS. The nanocavity arrays combine two featuring characteristics: (1) An embedded MoS
monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS
edge sites and the lipid bilayer; and (2) A layered plasmonic cavity that enables sufficient electromagnetic field enhancement inside the cavities to obtain a single EV level signal resolution for stratifying the molecular alterations. We used the GBM paradigm to demonstrate the diagnostic potential of the SERS single EV molecular profiling approach. The MoSERS multiplexing fluidic achieves parallel signal acquisition of glioma molecular variants (EGFRvIII oncogenic mutation and MGMT expression) in GBM cells. The detection limit of 1.23% was found for stratifying these key molecular variants in the wild-type population. When interfaced with a convolutional neural network (CNN), MoSERS improved diagnostic accuracy (87%) with which GBM mutations were detected in 12 patient blood samples, on par with clinical pathology tests. Thus, MoSERS demonstrates the potential for molecular stratification of cancer patients using circulating EVs.
New treatments are needed to improve the prognosis of patients with recurrent high-grade glioma.
To compare overall survival for patients receiving tumor resection followed by vocimagene ...amiretrorepvec (Toca 511) with flucytosine (Toca FC) vs standard of care (SOC).
A randomized, open-label phase 2/3 trial (TOCA 5) in 58 centers in the US, Canada, Israel, and South Korea, comparing posttumor resection treatment with Toca 511 followed by Toca FC vs a defined single choice of approved (SOC) therapies was conducted from November 30, 2015, to December 20, 2019. Patients received tumor resection for first or second recurrence of glioblastoma or anaplastic astrocytoma.
Patients were randomized 1:1 to receive Toca 511/FC (n = 201) or SOC control (n = 202). For the Toca 511/FC group, patients received Toca 511 injected into the resection cavity wall at the time of surgery, followed by cycles of oral Toca FC 6 weeks after surgery. For the SOC control group, patients received investigators' choice of single therapy: lomustine, temozolomide, or bevacizumab.
The primary outcome was overall survival (OS) in time from randomization date to death due to any cause. Secondary outcomes reported in this study included safety, durable response rate (DRR), duration of DRR, durable clinical benefit rate, OS and DRR by IDH1 variant status, and 12-month OS.
All 403 randomized patients (median SD age: 56 11.46 years; 62.5% 252 men) were included in the efficacy analysis, and 400 patients were included in the safety analysis (3 patients on the SOC group did not receive resection). Final analysis included 271 deaths (141 deaths in the Toca 511/FC group and 130 deaths in the SOC control group). The median follow-up was 22.8 months. The median OS was 11.10 months for the Toca 511/FC group and 12.22 months for the control group (hazard ratio, 1.06; 95% CI 0.83, 1.35; P = .62). The secondary end points did not demonstrate statistically significant differences. The rates of adverse events were similar in the Toca 511/FC group and the SOC control group.
Among patients who underwent tumor resection for first or second recurrence of glioblastoma or anaplastic astrocytoma, administration of Toca 511 and Toca FC, compared with SOC, did not improve overall survival or other efficacy end points.
ClinicalTrials.gov Identifier: NCT02414165.
Abstract
BACKGROUND
Vascular anomalies, including thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of dysregulated cancer cell genome and epigenome. Up-regulation of podoplanin ...(PDPN) by cancer cells has recently been linked to an increased risk of venous thromboembolism in glioblastoma patients. Thus, regulation of this platelet activating protein by transforming events and release from cancer cells is of considerable interest.
AIMS
I. Investigate the pattern of PDPN expression and characterize PDPN-expressing cellular populations in GBM. II. Evaluate the contribution of oncogenic drivers to PDPN expression in GBM models. III. Investigate the potential involvement of extracellular vesicles (EVs) as a mechanism for systemic dissemination of PDPN and tissue factor (TF). IV. Examine the role of PDPN in intratumoral and systemic thrombosis.
METHODS
Bioinformatics (single-cell and bulk transcriptome data mining), GBM cell lines and stem cell lines, xenograft models in mice, ELISA assays for PDPN and TF, platelet (PF4) and clotting activation markers (D-dimer), EV electron microscopy, density gradient fractionation, and nano-flow cytometry.
RESULTS
PDPN is expressed by distinct glioblastoma cell subpopulations (mesenchymal) and downregulated by oncogenic mutations of EGFR and IDH1 genes, via changes in chromatin modifications (EZH2) and DNA methylation, respectively. GBM cells exteriorize PDPN and/or TF as cargo of exosome-like EVs shed both in vitro and in vivo. Injection of glioma PDPN-EVs activates platelets. Increase of platelet activation (PF4) or coagulation markers (D-dimer) occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Co-expression of PDPN and TF by GBM cells cooperatively increases tumor microthrombosis.
CONCLUSION
Distinct cellular subsets drive multiple facets of GBM-associated thrombosis and may represent targets for diagnosis and intervention. We suggest that the preponderance of PDPN expression as a risk factor in glioblastoma and the involvement of platelets may merit investigating anti-platelets for potential inclusion in thrombosis management in GBM.
Abstract
Recent breakthroughs in immunotherapy have revolutionized treatment for many types of cancer, but unfortunately trials of these therapies have failed to provide meaningful life-prolonging ...benefit for brain tumor patients, potentially due to abundant immunosuppressive myeloid cells in the tumor. Our ultimate goal is to reprogram immunosuppressive tumor associated myeloid cells to an antitumor state to enable effective immunotherapy. Towards this goal, we have deeply characterized the immune microenvironment of more than 50 primary high and low grade gliomas using high-throughput single-cell RNA-sequencing to reveal recurrent myeloid cell states and immunosuppressive programs across IDH1 wild-type and mutant tumors. We have also established a brain tumor organoid model from primary patient tissue that maintains all of the tumor microenvironment, including myeloid and other immune cells. We utilize the this model to functionally test data-driven reprogramming strategies and understand how they impact the states of tumor and immune cells in the ex vivo human tumor microenvironment.
Inhibition of carbonic anhydrase IX in glioblastoma multiforme Amiri, Abdolali; Le, Phuong Uyen; Moquin, Alexandre ...
European journal of pharmaceutics and biopharmaceutics,
December 2016, 2016-Dec, 2016-12-00, 20161201, Letnik:
109
Journal Article
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Carbonic anhydrase IX (CAIX) is a transmembrane enzyme upregulated in several types of tumors including glioblastoma multiforme (GBM). GBM is among the most aggressive tumors among ...gliomas. Temozolomide (TMZ) therapy combined with surgical or radiation approaches is the standard treatment but not effective in long term. In this study we tested the treatment with acetazolamide (ATZ), an inhibitor of CAIX, alone or combined with TMZ. The experiments were performed in 2D and 3D cultures (spheroids) using glioblastoma U251N and human brain tumor stem cells (BTSCs). Several proteins implicated in tumor cell death were also investigated. The key results from these studies suggest the following: (1) Cell death of human glioblastoma spheroids and BTSC is significantly increased with combined treatment after 7 days, and (2) the effectiveness of ATZ is significantly enhanced against BTSC and U251N when incorporated into nano-carriers. Collectively, these results point toward the usefulness of nano-delivery of CAIX inhibitors and their combination with chemotherapeutics for glioblastoma treatment.
Navigation‐guided brain biopsies are the standard of care for diagnosis of several brain pathologies. However, imprecise targeting and tissue heterogeneity often hinder obtaining high‐quality tissue ...samples, resulting in poor diagnostic yield. We report the development and first clinical testing of a navigation‐guided fiberoptic Raman probe that allows surgeons to interrogate brain tissue in situ at the tip of the biopsy needle prior to tissue removal. The 900 μm diameter probe can detect high spectral quality Raman signals in both the fingerprint and high wavenumber spectral regions with minimal disruption to the neurosurgical workflow. The probe was tested in three brain tumor patients, and the acquired spectra in both normal brain and tumor tissue demonstrated the expected spectral features, indicating the quality of the data. As a proof‐of‐concept, we also demonstrate the consistency of the acquired Raman signal with different systems and experimental settings. Additional clinical development is planned to further evaluate the performance of the system and develop a statistical model for real‐time tissue classification during the biopsy procedure.
Stereotactic brain biopsy is a critical procedure in neuro‐oncology but suffers from suboptimal diagnostic accuracy. Optical technologies have the potential to correct this setback by offering molecular information at the tip of the surgery needle, before a sample is extracted. This article presents the first in‐human use of a point‐probe Raman spectroscopy system that fits inside a biopsy needle. The results show high‐quality spectra, consistent with previous experiments in brain tissue.
Significance: Ensuring spectral quality is prerequisite to Raman spectroscopy applied to surgery. This is because the inclusion of poor-quality spectra in the training phase of Raman-based pathology ...detection models can compromise prediction robustness and generalizability to new data. Currently, there exists no quantitative spectral quality assessment technique that can be used to either reject low-quality data points in existing Raman datasets based on spectral morphology or, perhaps more importantly, to optimize the in vivo data acquisition process to ensure minimal spectral quality standards are met.
Aim: To develop a quantitative method evaluating Raman signal quality based on the variance associated with stochastic noise in important tissue bands, including C─C stretch, CH2 / CH3 deformation, and the amide bands.
Approach: A single-point hand-held Raman spectroscopy probe system was used to acquire 315 spectra from 44 brain cancer patients. All measurements were classified as either high or low quality based on visual assessment (qualitative) and using a quantitative quality factor (QF) metric. Receiver-operator-characteristic (ROC) analyses were performed to evaluate the performance of the quantitative metric to assess spectral quality and improve cancer detection accuracy.
Results: The method can separate high- and low-quality spectra with a sensitivity of 89% and a specificity of 90% which is shown to increase cancer detection sensitivity and specificity by up to 20% and 12%, respectively.
Conclusions: The QF threshold is effective in stratifying spectra in terms of spectral quality and the observed false negatives and false positives can be linked to limitations of qualitative spectral quality assessment.
Abstract
Background
Glioblastoma is a treatment-resistant brain cancer. Its hierarchical cellular nature and its tumor microenvironment (TME) before, during, and after treatments remain unresolved.
...Methods
Here, we used single-cell RNA sequencing to analyze new and recurrent glioblastoma and the nearby subventricular zone (SVZ).
Results
We found 4 glioblastoma neural lineages are present in new and recurrent glioblastoma with an enrichment of the cancer mesenchymal lineage, immune cells, and reactive astrocytes in early recurrences. Cancer lineages were hierarchically organized around cycling oligodendrocytic and astrocytic progenitors that are transcriptomically similar but distinct to SVZ neural stem cells (NSCs). Furthermore, NSCs from the SVZ of patients with glioblastoma harbored glioblastoma chromosomal anomalies. Lastly, mesenchymal cancer cells and TME reactive astrocytes shared similar gene signatures which were induced by radiotherapy in a myeloid-dependent fashion in vivo.
Conclusion
These data reveal the dynamic, immune-dependent nature of glioblastoma’s response to treatments and identify distant NSCs as likely cells of origin.
Abstract
Post-craniotomy recovery in a neuro-critical care unit (NCCU) has been routine for most patients. There is no evidence to support this practice. To address this, we assessed the development ...of post-operative adverse events in brain tumor resection patients surveilled in the NCCU or the post-anesthesia care unit (PACU).
METHODS: All brain tumor craniotomies performed between 2016-2021 (KP) were reviewed. Inclusion criteria were: age >18; craniotomy for biopsy or resection of intracerebral or extracerebral tumors. Cohorts were divided into two groups based on recovery destination: NCCU or PACU. Past medical history, pre- and post-operative neuroradiological imaging, surgical resection features, and neuropathology results were reviewed for both groups. RESULTS: There were 681 patients in NCCU group and 397 in the PACU group. Mean ages were 55.6 (NCCU) and 59.7 (PACU) (p <0.001). The American Society of Anesthesiologists (ASA) class was not significantly different between groups (p=0.89). Median recovery time in the NCCU group was 19 hours, and 4 hours in the PACU group. The incidence of adverse events within 24 hours post- surgery was 0.04 in NCCU group and 0.03 in PACU group (p=0.46). Post-operative hospital stay was 9.5 days in the NCCU group and 8.5 days in PACU group (p <0.001). CONCLUSION: The incidence of adverse events within 24 hours post brain tumor surgery is not different between patients being surveilled for a long period in a NCCU, or a short period in a PACU.
Morphological and emerging molecular studies have provided evidence for heterogeneity within the oligodendrocyte population. To address the regional and age‐related heterogeneity of human mature ...oligodendrocytes (MOLs) we applied single‐cell RNA sequencing to cells isolated from cortical/subcortical, subventricular zone brain tissue samples, and thoracolumbar spinal cord samples. Unsupervised clustering of cells identified transcriptionally distinct MOL subpopulations across regions. Spinal cord MOLs, but not microglia, exhibited cell‐type‐specific upregulation of immune‐related markers compared to the other adult regions. SVZ MOLs showed an upregulation of select number of development‐linked transcription factors compared to other regions; however, pseudotime trajectory analyses did not identify a global developmental difference. Age‐related analysis of cortical/subcortical samples indicated that pediatric MOLs, especially from under age 5, retain higher expression of genes linked to development and to immune activity with pseudotime analysis favoring a distinct developmental stage. Our regional and age‐related studies indicate heterogeneity of MOL populations in the human CNS that may reflect developmental and environmental influences.
Main Points
scRNA sequencing identified regionally distinct mature oligodendrocytes (OL) sub populations in the adult human CNS, including relative expression of immune markers.
Pediatric OLs retain higher expression of genes linked to development and to immune activity.
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