Single-cell spatial immune landscapes of primary and metastatic brain tumours.

Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell-cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

MAFG-driven astrocytes promote CNS inflammation.

Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

A clinical Raman spectroscopy imaging system and safety requirements for in situ intraoperative tissue characterization.

Raman spectroscopy imaging is a technique that can be adapted for intraoperative tissue characterization to be used for surgical guidance. Here we present a macroscopic line scanning Raman imaging system that has been modified to ensure suitability for intraoperative use. The imaging system has a field of view of 1 × 1 cm2 and acquires Raman fingerprint images of 40 × 42 pixels, typically in less than 5 minutes. The system is mounted on a mobile cart, it is equiped with a passive support arm and possesses a removable and sterilizable probe muzzle. The results of a proof of concept study are presented in porcine adipose and muscle tissue. Supervised machine learning models (support vector machines and random forests) were trained and they were tested on a holdout dataset consisting of 7 Raman images (10 080 spectra) acquired in different animal tissues. This led to a detection accuracy >96% and prediction confidence maps providing a quantitative detection assessment for tissue border visualization. Further testing was accomplished on a dataset acquired with the imaging probe's contact muzzle and tailored classification models showed robust classifications capabilities with specificity, sensitivity and accuracy all surpassing 95% with a support vector machine classifier. Finally, laser safety, biosafety and sterilization of the system was assest. The safety assessment showed that the system's laser can be operated safetly according to the American National Standards Institute's standard for maximum permissible exposures for eyes and skin. It was further shown that during tissue interrogation, the temperature-history in cumulative equivalent minutes at 43 °C (CEM43 °C) never exceeded a safe threshold of 5 min.

Regional and age-related diversity of human mature oligodendrocytes.

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.

Developmental trajectory of oligodendrocyte progenitor cells in the human brain revealed by single cell RNA sequencing.

Characterizing the developmental trajectory of oligodendrocyte progenitor cells (OPC) is of great interest given the importance of these cells in the remyelination process. However, studies of human OPC development remain limited by the availability of whole cell samples and material that encompasses a wide age range, including time of peak myelination. In this study, we apply single cell RNA sequencing to viable whole cells across the age span and link transcriptomic signatures of oligodendrocyte-lineage cells with stage-specific functional properties. Cells were isolated from surgical tissue samples of second-trimester fetal, 2-year-old pediatric, 13-year-old adolescent, and adult donors by mechanical and enzymatic digestion, followed by percoll gradient centrifugation. Gene expression was analyzed using droplet-based RNA sequencing (10X Chromium). Louvain clustering analysis identified three distinct cellular subpopulations based on 5,613 genes, comprised of an early OPC (e-OPC) group, a late OPC group (l-OPC), and a mature OL (MOL) group. Gene ontology terms enriched for e-OPCs included cell cycle and development, for l-OPCs included extracellular matrix and cell adhesion, and for MOLs included myelination and cytoskeleton. The e-OPCs were mostly confined to the premyelinating fetal group, and the l-OPCs were most highly represented in the pediatric age group, corresponding to the peak age of myelination. Cells expressing a signature characteristic of l-OPCs were identified in the adult brain in situ using RNAScope. These findings highlight the transcriptomic variability in OL-lineage cells before, during, and after peak myelination and contribute to identifying novel pathways required to achieve remyelination.

Feature engineering applied to intraoperative in vivo Raman spectroscopy sheds light on molecular processes in brain cancer: a retrospective study of 65 patients.

Raman spectroscopy is a promising tool for neurosurgical guidance and cancer research. Quantitative analysis of the Raman signal from living tissues is, however, limited. Their molecular composition is convoluted and influenced by clinical factors, and access to data is limited. To ensure acceptance of this technology by clinicians and cancer scientists, we need to adapt the analytical methods to more closely model the Raman-generating process. Our objective is to use feature engineering to develop a new representation for spectral data specifically tailored for brain diagnosis that improves interpretability of the Raman signal while retaining enough information to accurately predict tissue content. The method consists of band fitting of Raman bands which consistently appear in the brain Raman literature, and the generation of new features representing the pairwise interaction between bands and the interaction between bands and patient age. Our technique was applied to a dataset of 547 in situ Raman spectra from 65 patients undergoing glioma resection. It showed superior predictive capacities to a principal component analysis dimensionality reduction. After analysis through a Bayesian framework, we were able to identify the oncogenic processes that characterize glioma: increased nucleic acid content, overexpression of type IV collagen and shift in the primary metabolic engine. Our results demonstrate how this mathematical transformation of the Raman signal allows the first biological, statistically robust analysis of in vivo Raman spectra from brain tissue.

DZ-2384 has a superior preclinical profile to taxanes for the treatment of triple-negative breast cancer and is synergistic with anti-CTLA-4 immunotherapy.

Triple-negative breast cancer (TNBC) is typically aggressive, difficult to treat, and commonly metastasizes to the visceral organs and soft tissues, including the lungs and the brain. Taxanes represent the most effective and widely used therapeutic class in metastatic TNBC but possess limiting adverse effects that often result in a delay, reduction, or cessation of their use. DZ-2384 is a candidate microtubule-targeting agent with a distinct mechanism of action and strong activity in several preclinical cancer models, with reduced toxicities. DZ-2384 is highly effective in patient-derived taxane-sensitive and taxane-resistant xenograft models of TNBC at lower doses and over a wider range relative to paclitaxel. When comparing compound exposure at minimum effective doses relative to safe exposure levels, the therapeutic window for DZ-2384 is 14-32 compared with 2.0 and less than 2.8 for paclitaxel and docetaxel, respectively. DZ-2384 is effective at reducing brain metastatic lesions when used at maximum tolerated doses and is equivalent to paclitaxel. Drug distribution experiments indicate that DZ-2384 is taken up more efficiently by tumor tissue but at equivalent levels in the brain compared with paclitaxel. Selective DZ-2384 uptake by tumor tissue may in part account for its wider therapeutic window compared with taxanes. In view of the current clinical efforts to combine chemotherapy with immune checkpoint inhibitors, we demonstrate that DZ-2384 acts synergistically with anti-CTLA-4 immunotherapy in a syngeneic murine model. These results demonstrate that DZ-2384 has a superior pharmacologic profile over currently used taxanes and is a promising therapeutic agent for the treatment of metastatic TNBC.

Raman spectroscopy in microsurgery: impact of operating microscope illumination sources on data quality and tissue classification.

Ambient light artifacts can confound Raman spectroscopy measurements performed in a clinical setting such as during open surgery. However, requiring light sources to be turned off during intraoperative spectral acquisition can be impractical because it can slow down the procedure by requiring surgeons to acquire data under light conditions different from the routine clinical practice. Here a filter system is introduced allowing in vivo Raman spectroscopy measurements to be performed with the light source of a neurosurgical microscope turned on, without interfering with the standard procedure. Ex vivo and in vivo results on calf and human brain, respectively, show that when the new filter system is used there is no significant difference between Raman spectra acquired under pitch dark conditions or with the microscope light source turned on. This is important for the clinical translation of Raman spectroscopy because of the resulting decrease in total imaging time for each measurement and because the surgeon can now acquire spectroscopic data with no disruption of the surgical workflow.

Decompressive Craniectomy for Ischemic Stroke: Effect of Hemorrhagic Transformation on Outcome.

BACKGROUND: Decompressive hemicraniectomy (DhC) is a life-saving surgical procedure being increasingly employed for malignant middle cerebral artery strokes. We examined the incidence of hemorrhagic transformation following DhC. METHODS: We retrospectively reviewed the charts and radiological images of patients who underwent DhC for malignant middle cerebral artery strokes. We classified the hemorrhagic events and assessed the short-term 30-day outcome associated with these events. RESULTS: A total of 23 DhCs were performed for supratentorial ischemic strokes in 22 patients. There were 16 males and 6 females with an average age of 47 years (21-69 years). Of the 22 patients, 13 (59%) developed a new hemorrhage following DhC. There were 3 mortalities (14%). Of the survivors, 6 (27%) were discharged home with a modified Rankin Scale (mRS) score of 2. The remaining 13 patients (59%) recovered to a degree wherein they were discharged to a rehabilitation center (mRS score 3-4). No patient persisted in a vegetative or semivegetative state (mRS score 5). CONCLUSIONS: In this study, the rate of hemorrhagic transformation following DhC for ischemic stroke was 59%. This is much higher than that reported in the stroke thrombolysis literature. The presence of any type of new hemorrhagic transformation in this patient population does not appear to alter the natural history of their ischemic strokes in terms of Glasgow outcome scores or destination of disposition.

Sub-diffuse interstitial optical tomography to improve the safety of brain needle biopsies: a proof-of-concept study.

The extraction of tissue samples during brain needle biopsy can cause life-threatening hemorrhage because of significant blood vessel injury during the procedure. Vessel rupture can have significant consequences for patient health, ranging from transient neurological deficits to death. Here, we present a sub-diffuse optical tomography technique that can be integrated into neurosurgical workflow to detect the presence of blood vessels. A proof-of-concept study performed on a realistic brain tissue phantom is presented and demonstrates that interstitial optical tomography (iOT) can detect several 1 mm diameter high-contrast absorbing objects located <2 mm from the needle.

Patterns of care at end of life for people with primary intracranial tumors: lessons learned.

To determine the variability in processes of care in the last 6 months of life experienced by patients dying of primary intracranial tumors and potential predictors of place of death, a death-backwards cohort was assembled using historical data and 1,623 decedents were identified. 90 % of people had ≥ 1 admission to an acute care hospital and 23 % spent ≥ 3 months of their last 6 months of life in acute care. 44 % had ≥ 1 ER visits and 30 % were admitted ≥ 1 times to ICU. Only 18 % had a home visit by a physician. 10 % died at home but 49 % died in hospital, while 40 % died in a palliative care facility. Age, comorbidities, and being diagnosed with grade 4 astrocytoma were associated with greater burden of care. Level of care burden and age were associated with higher odds of dying in a treatment intensive place of death, being diagnosed with grade 4 astrocytoma had opposite effect. Despite valuable research efforts to improve the treatment of primary intracranial tumors that focus on biology, refinements to surgery, radiation, and chemotherapy, there is also room to improve aspects of care at the end of life situation. An integrative approach for this patients' population, from diagnosis to death, could potentially reduce the care burden in the final period on the health care system, patient's family and improve access to a better place of death.

Identity and nature of neural stem cells in the adult human subventricular zone.

The existence of neural stem cells (NSCs) in adult human brain neurogenic regions remains unresolved. To address this, we created a cell atlas of the adult human subventricular zone (SVZ) derived from fresh neurosurgical samples using single-cell transcriptomics. We discovered 2 adult radial glia (RG)-like populations, aRG1 and aRG2. aRG1 shared features with fetal early RG (eRG) and aRG2 were transcriptomically similar to fetal outer RG (oRG). We also captured early neuronal and oligodendrocytic NSC states. We found that the biological programs driven by their transcriptomes support their roles as early lineage NSCs. Finally, we show that these NSCs have the potential to transition between states and along lineage trajectories. These data reveal that multipotent NSCs reside in the adult human SVZ.

In situ brain tumor detection using a Raman spectroscopy system-results of a multicenter study.

Safe and effective brain tumor surgery aims to remove tumor tissue, not non-tumoral brain. This is a challenge since tumor cells are often not visually distinguishable from peritumoral brain during surgery. To address this, we conducted a multicenter study testing whether the Sentry System could distinguish the three most common types of brain tumors from brain tissue in a label-free manner. The Sentry System is a new real time, in situ brain tumor detection device that merges Raman spectroscopy with machine learning tissue classifiers. Nine hundred and seventy-six in situ spectroscopy measurements and colocalized tissue specimens were acquired from 67 patients undergoing surgery for glioblastoma, brain metastases, or meningioma to assess tumor classification. The device achieved diagnostic accuracies of 91% for glioblastoma, 97% for brain metastases, and 96% for meningiomas. These data show that the Sentry System discriminated tumor containing tissue from non-tumoral brain in real time and prior to resection.

Invasive growth of brain metastases is linked to CHI3L1 release from pSTAT3-positive astrocytes.

BACKGROUND: Compared to minimally invasive brain metastases (MI BrM), highly invasive (HI) lesions form abundant contacts with cells in the peritumoral brain parenchyma and are associated with poor prognosis. Reactive astrocytes (RAs) labeled by phosphorylated STAT3 (pSTAT3) have recently emerged as a promising therapeutic target for BrM. Here, we explore whether the BrM invasion pattern is influenced by pSTAT3+ RAs and may serve as a predictive biomarker for STAT3 inhibition. METHODS: We used immunohistochemistry to identify pSTAT3+ RAs in HI and MI human and patient-derived xenograft (PDX) BrM. Using PDX, syngeneic, and transgenic mouse models of HI and MI BrM, we assessed how pharmacological STAT3 inhibition or RA-specific STAT3 genetic ablation affected BrM growth in vivo. Cancer cell invasion was modeled in vitro using a brain slice-tumor co-culture assay. We performed single-cell RNA sequencing of human BrM and adjacent brain tissue. RESULTS: RAs expressing pSTAT3 are situated at the brain-tumor interface and drive BrM invasive growth. HI BrM invasion pattern was associated with delayed growth in the context of STAT3 inhibition or genetic ablation. We demonstrate that pSTAT3+ RAs secrete Chitinase 3-like-1 (CHI3L1), which is a known STAT3 transcriptional target. Furthermore, single-cell RNA sequencing identified CHI3L1-expressing RAs in human HI BrM. STAT3 activation, or recombinant CHI3L1 alone, induced cancer cell invasion into the brain parenchyma using a brain slice-tumor plug co-culture assay. CONCLUSIONS: Together, these data reveal that pSTAT3+ RA-derived CHI3L1 is associated with BrM invasion, implicating STAT3 and CHI3L1 as clinically relevant therapeutic targets for the treatment of HI BrM.

Failure pattern following complete resection plus radiotherapy and temozolomide is at the resection margin in patients with glioblastoma.

Glioblastomas (GBM) are highly motile cancers that invade through normal brain. In the absence of curative chemotherapy this invasion, beyond surgical and radiotherapy margins, to distant brain sites is thought to be an important cause of treatment failure. Paradoxically, studies analyzing failure patterns have consistently shown that the large majority of failures occur at the original tumor site. This conflict may be explained by the fact these cancers are often only sub-totally resected and radiotherapy and chemotherapies fail to control this significant local cancer burden. We analyzed the failure pattern in 20 consecutive patients with complete resection of the gadolinium-enhancing portion of GBM demonstrated on the immediate post-operative magnetic resonance study, and who underwent a radical course of radiotherapy and chemotherapy. We found that recurrences occurred only at the resection margin in 17 of 20 patients. Recurrences were exclusively distant in 2 of 20 patients and occurred at both the resection margin and a distant site in 1 of 20 cases. We found that even in cases of complete resection of the gadolinium-enhancing portion of GBM 85 % of recurrences are localized to the resection margin.

Bevacizumab use for recurrent high-grade glioma at McGill University Hospital.

BACKGROUND: Bevacizumab, a humanized recombinant anti-vascular endothelial growth factor antibody, was approved in Canada in 2010 for the treatment of high-grade glioma. We report the effectiveness and safety of bevacizumab in the treatment of patients with recurrent high-grade gliomas at a single institution. METHODS: Twenty-seven consecutive patients with high-grade glioma (anaplastic glioma and glioblastoma) at first or subsequent relapse were treated with bevacizumab alone or in combination with chemotherapy. The primary endpoint was progression-free survival (PFS) and secondary endpoints were objective response rate, six month PFS, overall survival (OS), and safety profile. RESULTS: The clinical benefit rate (complete and partial responses plus stable disease) was 59%. Median PFS was 4.3 (95% CI, 3.0-10.9) months, with a six month PFS rate of 43%. Median OS after current relapse was 8.9 (95% CI, 5.8-not reached) months. Ten episodes of grade 3/4 adverse events were observed in nine patients, including fatigue (n = 3), thrombocytopenia (n = 4), and myelotoxicity, febrile neutropenia, and pulmonary embolism (each n = 1). CONCLUSIONS: We consider the efficacy and safety profile of bevacizumab is comparable to other cohorts of patients treated for recurrent high-grade glioma at other international institutions.

Open-sourced Raman spectroscopy data processing package implementing a baseline removal algorithm validated from multiple datasets acquired in human tissue and biofluids.

Significance: Standardized data processing approaches are required in the field of bio-Raman spectroscopy to ensure information associated with spectral data acquired by different research groups, and with different systems, can be compared on an equal footing. Aim: An open-sourced data processing software package was developed, implementing algorithms associated with all steps required to isolate the inelastic scattering component from signals acquired using Raman spectroscopy devices. The package includes a novel morphological baseline removal technique (BubbleFill) that provides increased adaptability to complex baseline shapes compared to current gold standard techniques. Also incorporated in the package is a versatile tool simulating spectroscopic data with varying levels of Raman signal-to-background ratios, baselines with different morphologies, and varying levels of stochastic noise. Results: Application of the BubbleFill technique to simulated data demonstrated superior baseline removal performance compared to standard algorithms, including iModPoly and MorphBR. The data processing workflow of the open-sourced package was validated in four independent in-human datasets, demonstrating it leads to inter-systems data compatibility. Conclusions: A new open-sourced spectroscopic data pre-processing package was validated on simulated and real-world in-human data and is now available to researchers and clinicians for the development of new clinical applications using Raman spectroscopy.

Phase II trial of concurrent sunitinib, temozolomide, and radiotherapy with adjuvant temozolomide for newly diagnosed MGMT unmethylated glioblastoma.

Background: The overall prognosis of glioblastoma (GBM) remains dismal, particularly for patients with unmethylated O6-methylguanine-DNA-methyltransferase (MGMT) promoter. In this phase II trial, we tested the combination of the antiangiogenic agent sunitinib with radiotherapy and temozolomide (TMZ) for newly diagnosed unmethylated MGMT GBM patients. Methods: We enrolled 37 patients with unmethylated MGMT promoter GBM, age 18-70, and KPS ≥70. Patients received 12.5 mg of daily sunitinib for 7 days, followed by concurrent chemoradiation plus 12.5 mg sunitinib, then adjuvant TMZ. The primary endpoint was progression-free survival (PFS), and secondary endpoints were overall survival (OS), safety, and neutrophil-to-lymphocyte ratio (NLR) biomarker. Results: At a median follow-up time of 15.3 months (range: 3.1-71.3 months), the median PFS was 7.15 months (95% CI: 5.4-10.5) and the 6-month PFS was 54.0%. Median OS was 15.0 months (95% CI: 13.8-19.4) and 2-year OS rate was 17.1%. Patients receiving >3 cycles of adjuvant TMZ, undergoing surgery at progression, and presenting a post-concurrent NLR ≤6 experienced a significant improved OS with hazard ratios of 0.197 (P = .001), 0.46 (P = .049), and 0.38 (P = .021), respectively, on multivariable analysis. Age >65 years predicted for worse OS with hazard ratio of 3.92 (P = .037). Grade ≥3 thrombocytopenia occurred in 22.9%, grade ≥3 neutropenia in 20%, and grade ≥3 thromboembolic events in 14.3% of patients. There were no grade 5 events. Conclusion: Our findings suggest a potential benefit of combining sunitinib with chemoradiation in newly diagnosed GBM patients with unmethylated MGMT status and provide a strong rationale to test this combination in future studies.

MoS2-Plasmonic Nanocavities for Raman Spectra of Single Extracellular Vesicles Reveal Molecular Progression in Glioblastoma.

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 MoS2 monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS2 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.