Single-nucleus and spatial landscape of the sub-ventricular zone in human glioblastoma.

The sub-ventricular zone (SVZ) is the most well-characterized neurogenic area in the mammalian brain. We previously showed that in 65% of patients with glioblastoma (GBM), the SVZ is a reservoir of cancer stem-like cells that contribute to treatment resistance and emergence of recurrence. Here, we built a single-nucleus RNA-sequencing-based microenvironment landscape of the tumor mass (T_Mass) and the SVZ (T_SVZ) of 15 GBM patients and 2 histologically normal SVZ (N_SVZ) samples as controls. We identified a mesenchymal signature in the T_SVZ of GBM patients: tumor cells from the T_SVZ relied on the ZEB1 regulatory network, whereas tumor cells in the T_Mass relied on the TEAD1 regulatory network. Moreover, the T_SVZ microenvironment was predominantly characterized by tumor-supportive microglia, which spatially co-exist and establish heterotypic interactions with tumor cells. Lastly, differential gene expression analyses, predictions of ligand-receptor and incoming/outgoing interactions, and functional assays revealed that the IL-1ß/IL-1RAcP and Wnt-5a/Frizzled-3 pathways are therapeutic targets in the T_SVZ microenvironment. Our data provide insights into the biology of the SVZ in GBM patients and identify specific targets of this microenvironment.

Evolutionary dynamics of residual disease in human glioblastoma.

BACKGROUND: Glioblastoma is the most common and aggressive adult brain malignancy against which conventional surgery and chemoradiation provide limited benefit. Even when a good treatment response is obtained, recurrence inevitably occurs either locally (∼80%) or distally (∼20%), driven by cancer clones that are often genomically distinct from those in the primary tumour. Glioblastoma cells display a characteristic infiltrative phenotype, invading the surrounding tissue and often spreading across the whole brain. Cancer cells responsible for relapse can reside in two compartments of residual disease that are left behind after treatment: the infiltrated normal brain parenchyma and the sub-ventricular zone. However, these two sources of residual disease in glioblastoma are understudied because of the difficulty in sampling these regions during surgery. PATIENT AND METHODS: Here, we present the results of whole-exome sequencing of 69 multi-region samples collected using fluorescence-guided resection from 11 patients, including the infiltrating tumour margin and the sub-ventricular zone for each patient, as well as matched blood. We used a phylogenomic approach to dissect the spatio-temporal evolution of each tumour and unveil the relation between residual disease and the main tumour mass. We also analysed two patients with paired primary-recurrence samples with matched residual disease. RESULTS: Our results suggest that infiltrative subclones can arise early during tumour growth in a subset of patients. After treatment, the infiltrative subclones may seed the growth of a recurrent tumour, thus representing the 'missing link' between the primary tumour and recurrent disease. CONCLUSIONS: These results are consistent with recognised clinical phenotypic behaviour and suggest that more specific therapeutic targeting of cells in the infiltrated brain parenchyma may improve patient's outcome.

Fluorescence-guided surgical sampling of glioblastoma identifies phenotypically distinct tumour-initiating cell populations in the tumour mass and margin.

BACKGROUND: Acquiring clinically annotated, spatially stratified tissue samples from human glioblastoma (GBM) is compromised by haemorrhage, brain shift and subjective identification of 'normal' brain. We tested the use of 5-aminolevulinic acid (5-ALA) fluorescence to objective tissue sampling and to derive tumour-initiating cells (TICs) from mass and margin. METHODS: The 5-ALA was administered to 30 GBM patients. Samples were taken from the non-fluorescent necrotic core, fluorescent tumour mass and non-fluorescent margin. We compared the efficiency of isolating TICs from these areas in 5-ALA versus control patients. HRMAS (1)H NMR was used to reveal metabolic alterations due to 5-ALA. We then characterised TICs for self-renewal in vitro and tumorigenicity in vivo. RESULTS: The derivation of TICs was not compromised by 5-ALA and the metabolic profile was similar between tumours from 5-ALA patients and controls. The TICs from the fluorescent mass were self-renewing in vitro and tumour-forming in vivo, whereas TICs from non-fluorescent margin did not self-renew in vitro but did form tumours in vivo. CONCLUSION: Our data show that 5-ALA does not compromise the derivation of TICs. It also reveals that the margin contains TICs, which are phenotypically different from those isolated from the corresponding mass.

Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution.

Glioblastomas (GBMs) contain transformed, self-maintaining, multipotent, tumour-initiating cancer stem cells, whose identification has radically changed our perspective on the physiology of these tumours. Currently, it is unknown whether multiple types of transformed precursors, which display alternative sets of the complement of properties of true cancer stem cells, can be found in a GBM. If different subsets of such cancer stem-like cells (CSCs) do exist, they might represent distinct cell targets, with a differential therapeutic importance, also depending on their characteristics and lineage relationship. Here, we report the presence of two types of CSCs within different regions of the same human GBM. Cytogenetic and molecular analysis shows that the two types of CSCs bear quite diverse tumorigenic potential and distinct genetic anomalies, and, yet, derive from common ancestor cells. This provides critical information to unravel the development of CSCs and the key molecular/genetic components underpinning tumorigenicity in human GBMs.

Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.

Bone morphogenetic proteins regulate tumorigenicity in human glioblastoma stem cells.

Human glioblastomas appear to be established and expanded by cancer stem cells, which are endowed with tumour-initiating and perpetuating ability. We report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, activate their cognate receptors (BMPRs) and trigger the Smad but not the MAP38 kinase signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation and increased expression of differentiated neural markers, without affecting cell viability. The concomitant reduction in the clonogenic ability, both in the size of the CD133+ side population and in the growth kinetics of GBM cells, indicates that BMP4 triggers a reduction in the in vitro cancer stem cell (CSC) pool. Accordingly, transient ex vivo exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most important, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality which occur in 100% of control mice in less than 12 weeks, following intracerebral grafting of human GBM cells. These findings show that the BMP-BMPR signalling system, which controls the activity of normal brain stem cells, may also act as a key inhibitory regulator of cancer-initiating, GBM stem-like cells and identifies BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.