Sara Piccirillo

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.

Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics

Glioblastoma (GB) is the most common and aggressive primary brain malignancy, with poor prognosis and a lack of effective therapeutic options. Accumulating evidence suggests that intratumor heterogeneity likely is the key to understanding treatment failure. However, the extent of intratumor heterogeneity as a result of tumor evolution is still poorly understood. To address this, we developed a unique surgical multisampling scheme to collect spatially distinct tumor fragments from 11 GB patients. We present an integrated genomic analysis that uncovers extensive intratumor heterogeneity, with most patients displaying different GB subtypes within the same tumor. Moreover, we reconstructed the phylogeny of the fragments for each patient, identifying copy number alterations in EGFR and CDKN2A/B/p14ARF as early events, and aberrations in PDGFRA and PTEN as later events during cancer progression. We also characterized the clonal organization of each tumor fragment at the single-molecule level, detecting multiple coexisting cell lineages. Our results reveal the genome-wide architecture of intratumor variability in GB across multiple spatial scales and patient-specific patterns of cancer evolution, with consequences for treatment design.

Cyclopamine-Mediated Hedgehog Pathway Inhibition Depletes Stem-Like Cancer Cells in Glioblastoma

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non‐neoplastic stem cells, we investigated whether Hedgehog blockade could target the stem‐like population in glioblastoma multiforme (GBM). We found that Gli1, a key Hedgehog pathway target, was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors, and in GBM‐derived neurospheres, suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%–60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM‐derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis, the stem‐like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment of our GBM neurospheres increased the percentage of these stem‐like cells, suggesting that this standard therapy preferentially targets better‐differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed.

NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long‐term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem‐like cells with higher NOTCH activity. We therefore used low‐passage and established GBM‐derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by γ‐secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug‐impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem‐like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem‐like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas. STEM CELLS 2010;28:5–16

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.

NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor and a highly malignant and heterogeneous cancer. Current conventional therapies fail to eradicate or curb GBM cell growth. Hence, exploring the cellular and molecular basis of GBM cell growth is vital to develop novel therapeutic approaches. Neuroglia (NG)-2 is a transmembrane proteoglycan expressed by NG2+ progenitors and is strongly linked to cell proliferation in the normal brain. By using NG2 as a biomarker we identify a GBM cell population (GBM NG2+ cells) with robust proliferative, clonogenic, and tumorigenic capacity. We show that a significant proportion (mean 83%) of cells proliferating in the tumor mass express NG2 and that over 50% of GBM NG2+ cells are proliferating. Compared with the GBM NG2- cells from the same tumor, the GBM of NG2+ cells overexpress genes associated with aggressive tumorigenicity, including overexpression of Mitosis and Cell Cycling Module genes (e.g., MELK, CDC, MCM, E2F), which have been previously shown to correlate with poor survival in GBM. We also show that the coexpression pattern of NG2 with other glial progenitor markers in GBM does not recapitulate that described in the normal brain. The expression of NG2 by such an aggressive and actively cycling GBM population combined with its location on the cell surface identifies this cell population as a potential therapeutic target in a subset of patients with GBM.

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 1H 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.

Brain cancer stem cells

Cancers comprise heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. In the last decade, several groups have demonstrated the existence of cancer stem cells in both nonsolid solid tumors, including some of the brain: glioblastoma multiforme (GBM), medulloblastoma, and ependymoma. These cells, like their normal counterpart in homologous tissues, are multipotent, undifferentiated, self-sustaining, yet transformed cells. In particular, glioblastoma-stem like cells (GBSCs) self-renew under clonal conditions and differentiate into neuron- and glia-like cells, with aberrant, mixed neuronal/astroglial phenotypes. Remarkably, upon subcutaneous and intracerebral transplantation in immunosuppressed mice, GBSCs are able to form secondary tumors that closely resemble the human pathology, a property retained also throughout serial transplantation. The search is up for the identification of the markers and the molecular mechanisms that underpin the tumorigenic potential of these cells. This is critical if we aim at defining new therapeutic approaches for the treatment of malignant brain tumors. Lately, it has been shown that some key regulatory system that plays pivotal roles in neural stem cell physiology can also regulate the tumorigenic ability of cancer stem cells in GBMs. This suggests that the study of cancer stem cells in brain tumors might help to identify new and more specific therapeutic molecular effectors, with the cancer stem cells themselves representing one of the main targets, in fact the Holy Grail, in cancer cell therapy. This review includes a summary review on brain cancer cells and their usefulness as emerging targets in cancer cell therapy.

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.

Glioblastoma cell growth is suppressed by disruption of fibroblast growth factor pathway signaling

The Fibroblast Growth Factor (FGF) signaling pathway is reported to stimulate glioblastoma (GBM) growth. In this work we evaluated the effect of FGF2, FGF receptor (FGFR), and small molecule inhibition on GBM cells grown in traditional media, or cultured directly in stem-cell media. These lines each expressed the FGFR1, FGFR3 and FGFR4 receptors. Addition of FGF2 ligand showed significant growth stimulation in 8 of 10 cell lines. Disruption of FGF signaling by a neutralizing FGF2 monoclonal antibody and FGFR1 suppression by RNA interference both partially inhibited cell proliferation. Growth inhibition was temporally correlated with a reduction in MAPK signaling. A receptor tyrosine kinase inhibitor with known FGFR/VEGFR activity, PD173074, showed reproducible growth inhibition. Possible mechanisms of growth suppression by PD173074 were implicated by reduced phosphorylation of AKT and MAPK, known oncogenic signal transducers. Subsequent reduction in the cyclin D1, cyclin D2 and CDK4 cell cycle regulators was also observed. Our results indicate that FGF signaling pathway inhibition as a monotherapy will slow, but not arrest growth of glioblastoma cells.