Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma.

Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast-cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow-cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose-deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7-related metabolic pathways is a viable therapeutic strategy.

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma.

Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from submicrogram quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintain the structural integrity of epigenetic information and provide substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. In addition, considering each promoter individually, substantial epigenetic heterogeneity was observed across the sequenced molecules, indicating the presence of epigenetically distinct cellular subpopulations. At the divergent MLH1/EPM2AIP1 promoter, a locus with three well-defined, nucleosome-depleted regions (NDRs), a fraction of promoter copies with inaccessible chromatin was detected and enriched upon selection of temozolomide-tolerant GBM cells. These results illustrate the biological relevance of epigenetically distinct subpopulations that in part underlie the phenotypic heterogeneity of tumor cell populations. Furthermore, these findings show that alterations in chromatin accessibility without accompanying changes in DNA methylation may constitute a novel class of epigenetic biomarker.

Tissue Response to Deep Brain Stimulation and Microlesion: A Comparative Study.

OBJECTIVES: Deep brain stimulation (DBS) is used for a variety of movement disorders, including Parkinson's disease. There are several theories regarding the biology and mechanisms of action of DBS. Previously, we observed an up-regulation of neural progenitor cell proliferation in post-mortem tissue suggesting that DBS can influence cellular plasticity in regions beyond the site of stimulation. We wanted to support these observations and investigate the relationship if any, between DBS, neural progenitor cells, and microglia. METHODS: We used naïve rats in this study for DBS electrode implantation, stimulation, and microlesions. We used immunohistochemistry techniques for labeling microglial and progenitor cells, and fluorescence microscopy for viewing and quantification of labeled cells. RESULTS: We present data that demonstrates a reciprocal relationship of microglia and neural precursor cells in the presence of acute high frequency stimulation. In our hands, stimulated animals demonstrate significantly lower numbers of activated microglia (p = 0.026) when compared to microlesion and sham animals. The subthalamic region surrounding the DBS stimulating electrode reveals a significant increase in the number of neural precursor cells expressing cell cycle markers, plasticity and precursor cell markers (Ki67; p = 0.0013, MCM2; p = 0.0002). INTERPRETATION: We conclude that in this animal model, acute DBS results in modest local progenitor cell proliferation and influenced the total number of activated microglia. This could be of clinical significance in patients with PD, as it is thought to progress via neuroinflammatory processes involving microglia, cytokines, and the complement system. Further studies are required to comprehend the behavior of microglia in different activation states and their ability to regulate adult neurogenesis under physiologic and pathologic conditions.

Brain tumour stem cells.

The dogma that the genesis of new cells is a negligible event in the adult mammalian brain has long influenced our perception and understanding of the origin and development of CNS tumours. The discovery that new neurons and glia are produced throughout life from neural stem cells provides new possibilities for the candidate cells of origin of CNS neoplasias. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis, thereby allowing the identification of new therapeutic strategies.

Chondroitin sulfate proteoglycans potently inhibit invasion and serve as a central organizer of the brain tumor microenvironment.

Glioblastoma (GBM) remains the most pervasive and lethal of all brain malignancies. One factor that contributes to this poor prognosis is the highly invasive character of the tumor. GBM is characterized by microscopic infiltration of tumor cells throughout the brain, whereas non-neural metastases, as well as select lower grade gliomas, develop as self-contained and clearly delineated lesions. Illustrated by rodent xenograft tumor models as well as pathological human patient specimens, we present evidence that one fundamental switch between these two distinct pathologies--invasion and noninvasion--is mediated through the tumor extracellular matrix. Specifically, noninvasive lesions are associated with a rich matrix containing substantial amounts of glycosylated chondroitin sulfate proteoglycans (CSPGs), whereas glycosylated CSPGs are essentially absent from diffusely infiltrating tumors. CSPGs, acting as central organizers of the tumor microenvironment, dramatically influence resident reactive astrocytes, inducing their exodus from the tumor mass and the resultant encapsulation of noninvasive lesions. Additionally, CSPGs induce activation of tumor-associated microglia. We demonstrate that the astrogliotic capsule can directly inhibit tumor invasion, and its absence from GBM presents an environment favorable to diffuse infiltration. We also identify the leukocyte common antigen-related phosphatase receptor (PTPRF) as a putative intermediary between extracellular glycosylated CSPGs and noninvasive tumor cells. In all, we present CSPGs as critical regulators of brain tumor histopathology and help to clarify the role of the tumor microenvironment in brain tumor invasion.

Detection of primary cilia in human glioblastoma.

Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies.

Controlling tumor invasion: bevacizumab and BMP4 for glioblastoma.

AIM: Bevacizumab has been reported to result in increased tumor invasion when used to treat malignant glioma. We hypothesized that BMP4 would prevent diffuse tumor infiltration induced by bevacizumab for malignant glioma in a xenograft model. METHODS: Human glioblastoma (GBM) tumor cells were implanted in the striatum of immunocompromised mice. The animals were treated with bevacizumab and BMP4. Tumor growth and invasion were measured. RESULTS: The bevacizumab-treated mice had increased survival compared with control animals (p = 0.02). BMP4 alone did not result in improved survival (p = 1.0). The bevacizumab (p = 0.006) and bevacizumab plus BMP4 (p = 0.006) groups demonstrated significantly decreased total tumor size compared with control. Tumor invasion was significantly decreased in the bevacizumab (p = 0.005), BMP4 (p = 0.04) alone and bevacizumab plus BMP4 (p = 0.002) groups compared with control. No synergistic effect between bevacizumab and BMP4 was observed. CONCLUSION: Bevacizumab treatment did not result in diffuse infiltration of human GBM in a mouse xenograft model. BMP4 did have an independent favorable effect on GBM that was not synergistic with bevacizumab treatment.

Evidence for label-retaining tumour-initiating cells in human glioblastoma.

Individual tumour cells display diverse functional behaviours in terms of proliferation rate, cell-cell interactions, metastatic potential and sensitivity to therapy. Moreover, sequencing studies have demonstrated surprising levels of genetic diversity between individual patient tumours of the same type. Tumour heterogeneity presents a significant therapeutic challenge as diverse cell types within a tumour can respond differently to therapies, and inter-patient heterogeneity may prevent the development of general treatments for cancer. One strategy that may help overcome tumour heterogeneity is the identification of tumour sub-populations that drive specific disease pathologies for the development of therapies targeting these clinically relevant sub-populations. Here, we have identified a dye-retaining brain tumour population that displays all the hallmarks of a tumour-initiating sub-population. Using a limiting dilution transplantation assay in immunocompromised mice, label-retaining brain tumour cells display elevated tumour-initiation properties relative to the bulk population. Importantly, tumours generated from these label-retaining cells exhibit all the pathological features of the primary disease. Together, these findings confirm dye-retaining brain tumour cells exhibit tumour-initiation ability and are therefore viable targets for the development of therapeutics targeting this sub-population.

Method for Isolating Extracellular Vesicles from Human Neural Stem Cells Expanded Under Neurosphere Culture.

Neural stem cells (NSCs) transplantation enhances plasticity and restores functions in neurological diseases. Therapeutic benefits of NSCs are due to their ability to replace the lost neurons and glial cells and also secreting a wide array of free and membrane-bound bioactive molecules that can reduce the hostility of diseased microenvironment, resolve inflammation, and rescue damaged neural cells. Membrane-encircled spherical nanostructures that are collectively known as extracellular vesicles (EVs) contain mRNA, miRNA, lipids, and specific proteins that affect different biological processes in cells located nearby or at far distances. Using EVs as an alternative non-cell-based therapy has gained huge attention, and developing methods for large-scale production of EVs is of great clinical importance. Here, we describe an efficient method to yield significant quantity of EVs from human NSCs that are expanded under free floating neurosphere assay culture system. Using the neurosphere assay in bioreactors under GMP-compliant conditions can result in scalable NSC-EVs required for human trials.

Slow-Cycling Cells in Glioblastoma: A Specific Population in the Cellular Mosaic of Cancer Stem Cells.

Glioblastoma (GBM) exhibits populations of cells that drive tumorigenesis, treatment resistance, and disease progression. Cells with such properties have been described to express specific surface and intracellular markers or exhibit specific functional states, including being slow-cycling or quiescent with the ability to generate proliferative progenies. In GBM, each of these cellular fractions was shown to harbor cardinal features of cancer stem cells (CSCs). In this study, we focus on the comparison of these cells and present evidence of great phenotypic and functional heterogeneity in brain cancer cell populations with stemness properties, especially between slow-cycling cells (SCCs) and cells phenotypically defined based on the expression of markers commonly used to enrich for CSCs. Here, we present an integrative analysis of the heterogeneity present in GBM cancer stem cell populations using a combination of approaches including flow cytometry, bulk RNA sequencing, and single cell transcriptomics completed with functional assays. We demonstrated that SCCs exhibit a diverse range of expression levels of canonical CSC markers. Importantly, the property of being slow-cycling and the expression of these markers were not mutually inclusive. We interrogated a single-cell RNA sequencing dataset and defined a group of cells as SCCs based on the highest score of a specific metabolic signature. Multiple CSC groups were determined based on the highest expression level of CD133, SOX2, PTPRZ1, ITGB8, or CD44. Each group, composed of 22 cells, showed limited cellular overlap, with SCCs representing a unique population with none of the 22 cells being included in the other groups. We also found transcriptomic distinctions between populations, which correlated with clinicopathological features of GBM. Patients with strong SCC signature score were associated with shorter survival and clustered within the mesenchymal molecular subtype. Cellular diversity amongst these populations was also demonstrated functionally, as illustrated by the heterogenous response to the chemotherapeutic agent temozolomide. In conclusion, our study supports the cancer stem cell mosaicism model, with slow-cycling cells representing critical elements harboring key features of disseminating cells.

Chemokine receptor CXCR3 promotes growth of glioma.

Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3(+), which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this receptor may be a potential therapeutic target for treating human GBM.

Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells.

Thymidine analogs (TAs) are synthetic nucleosides that incorporate into newly synthesized DNA. Halogenated pyrimidines (HPs), such as bromodeoxyuridine (BrdU), are a class of TAs that can be detected with antibodies and are commonly used for birthdating individual cells and for assessing the proliferative index of cell populations. It is well established that HPs can act as radiosensitizers when incorporated into DNA chains, but they are generally believed not to impair normal cell function in the absence of secondary stressors. However, we and others have shown that HP incorporation leads to a sustained suppression of cell cycle progression in mammalian cells, resulting in cellular senescence in somatic cells. In addition, we have shown that HP incorporation results in delayed tumor progression in a syngeneic rat model of glioma. Here we examine ethynyldeoxyuridine (EdU), a newly developed and alkylated TA, for its anti-cancer activity, both in vitro and in vivo. We show that EdU, like HPs, leads to a severe reduction in the proliferation rate of normal and transformed cells in vitro. Unlike HPs, however, EdU incorporation also causes DNA damage resulting in the death of a substantial subset of treated cells. When administered over an extended time as a monotherapy to mice bearing subcutaneous xenografts of human glioblastoma multiforme tumors, EdU significantly reduces tumor volume and increases survival without apparent significant toxicity. These results, combined with the fact that EdU readily crosses the blood-brain barrier, support the continued investigation of EdU as a potential therapy for malignant brain tumors.

Stem cell transplantation for ischemic stroke.

BACKGROUND: Studies in animal models of ischemic stroke have shown that stem cells transplanted into the brain can lead to functional improvement. However, to date, evidence for the benefits of stem cell transplantation in ischemic stroke patients is lacking. OBJECTIVES: To assess the efficacy and safety of stem cell transplantation compared with conventional treatments in patients with ischemic stroke. SEARCH STRATEGY: We searched the Cochrane Stroke Group Trials Register (last searched February 2010), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2009, Issue 3), MEDLINE (1966 to August 2008), EMBASE (1980 to August 2008), Science Citation Index (1900 to August 2008), and BIOSIS (1926 to August 2008). We handsearched potentially relevant conference proceedings, screened reference lists, and searched ongoing trials and research registers (last searched November 2008). We also contacted individuals active in the field and stem cell manufacturers (last contacted December 2008). SELECTION CRITERIA: We included randomized controlled trials (RCTs) recruiting patients with ischemic stroke, in any phase of the disease, and an ischemic lesion confirmed by computerized tomography or magnetic resonance imaging scan. We included all types of stem cell transplantation regardless of cell source (autograft, allograft, or xenograft; embryonic, fetal, or adult; from brain or other tissues), route of cell administration (systemic or local), and dosage. The primary outcome was efficacy (assessed as combined functional outcome or disability and dependency) at longer follow-up (minimum six months). Secondary outcomes included post-procedure safety outcomes (death, worsening of neurological deficit, infections and neoplastic transformation). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed trial quality. We contacted study authors for additional information. MAIN RESULTS: We identified three very small RCTs. Two are still awaiting classification because only subgroups of patients could be included in this meta-analysis and additional unpublished data are needed. The third trial randomized 30 patients to intravenous transplantation of autologous mesenchymal stem cell (10 participants) or reference group (20 participants) (five participants, initially randomized to the intervention group, refused the treatment and were allocated to the reference group) and found a statistically non-significant functional improvement in treated patients at longer follow-up. No adverse cell-related events were reported. AUTHORS' CONCLUSIONS: No large trials of stem cell transplantation have been performed in ischemic stroke patients and it is too early to know whether this intervention can improve functional outcome. Large, well-designed trials are needed.

Eco-oncology: Applying ecological principles to understand and manage cancer.

Cancer is a disease of single cells that expresses itself at the population level. The striking similarities between initiation and growth of tumors and dynamics of biological populations, and between metastasis and ecological invasion and community dynamics suggest that oncology can benefit from an ecological perspective to improve our understanding of cancer biology. Tumors can be viewed as complex, adaptive, and evolving systems as they are spatially and temporally heterogeneous, continually interacting with each other and with the microenvironment and evolving to increase the fitness of the cancer cells. We argue that an eco-evolutionary perspective is essential to understand cancer biology better. Furthermore, we suggest that ecologically informed therapeutic approaches that combine standard of care treatments with strategies aimed at decreasing the evolutionary potential and fitness of neoplastic cells, such as disrupting cell-to-cell communication and cooperation, and preventing successful colonization of distant organs by migrating cancer cells, may be effective in managing cancer as a chronic condition.

Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use.

Bromodeoxyuridine induces senescence in neural stem and progenitor cells.

Bromodeoxyuridine (BrdU) is a halogenated pyrimidine that incorporates into newly synthesized DNA during the S phase. BrdU is used ubiquitously in cell birthdating studies and as a means of measuring the proliferative index of various cell populations. In the absence of secondary stressors, BrdU is thought to incorporate relatively benignly into replicating DNA chains. However, we report here that a single, low-dose pulse of BrdU exerts a profound and sustained antiproliferative effect in cultured murine stem and progenitor cells. This is accompanied by altered terminal differentiation, cell morphology, and protein expression consistent with the induction of senescence. There is no evidence of a significant increase in spontaneous cell death; however, cells are rendered resistant to chemically induced apoptosis. Finally, we show that a brief in vivo BrdU regimen reduces the proliferative potential of subsequently isolated subependymal zone neurosphere-forming cells. We conclude, therefore, that BrdU treatment induces a senescence pathway that causes a progressive decline in the replication of rapidly dividing stem/progenitor cells, suggesting a novel and uncharacterized effect of BrdU. This finding is significant in that BrdU-incorporating neural stem/progenitor cells and their progeny should not be expected to behave normally with respect to proliferative potential and downstream functional parameters. This effect highlights the need for caution when results based on long-term BrdU tracking over multiple rounds of replication are interpreted. Conversely, the reliable induction of senescence in stem/progenitor cells in vitro and in vivo may yield a novel platform for molecular studies designed to address multiple aspects of aging and neurogenesis.

Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 +/- 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques.

Isolation, expansion, and differentiation of adult Mammalian neural stem and progenitor cells using the neurosphere assay.

During development and continuing into adulthood, stem cells function as a reservoir of undifferentiated cell types, whose role is to support cell genesis in several tissues and organs. In the adult, they play an essential homeostatic role by replacing differentiated cells that are lost due to physiological turnover, injury, or disease. The discovery of such cells in the adult mammalian central nervous system (CNS), an organ traditionally thought to have little or no regenerative capacity, has opened the door to the possibility of designing innovative regenerative therapeutics, an unexpected concept in neurobiology 15 years ago. In 1992, to detect precursor cells in the adult brain, we employed a serum-free culture system whereby the majority of primary differentiated CNS cells did not survive but a small population of EGF-responsive cells were maintained in an undifferentiated state and proliferated to form clusters, called neurospheres (Reynolds and Weiss, Science 255:1707-1710, 1992). These neurospheres could be (a) dissociated to form numerous secondary spheres or (b) induced to differentiate, generating the three major cell types of the CNS. This chapter outlines the adult mammalian NSC culture methodology and provides technical details of the neurosphere assay to achieve reproducible cultures.

Identification of CD24 as a marker of Patched1 deleted medulloblastoma-initiating neural progenitor cells.

High morbidity and mortality are common traits of malignant tumours and identification of the cells responsible is a focus of on-going research. Many studies are now reporting the use of antibodies specific to Clusters of Differentiation (CD) cell surface antigens to identify tumour-initiating cell (TIC) populations in neural tumours. Medulloblastoma is one of the most common malignant brain tumours in children and despite a considerable amount of research investigating this tumour, the identity of the TICs, and the means by which such cells can be targeted remain largely unknown. Current prognostication and stratification of medulloblastoma using clinical factors, histology and genetic profiling have classified this tumour into four main subgroups: WNT, Sonic hedgehog (SHH), Group 3 and Group 4. Of these subgroups, SHH remains one of the most studied tumour groups due to the ability to model medulloblastoma formation through targeted deletion of the Shh pathway inhibitor Patched1 (Ptch1). Here we sought to utilise CD antibody expression to identify and isolate TIC populations in Ptch1 deleted medulloblastoma, and determine if these antibodies can help classify the identity of human medulloblastoma subgroups. Using a fluorescence-activated cell sorted (FACS) CD antibody panel, we identified CD24 as a marker of TICs in Ptch1 deleted medulloblastoma. CD24 expression was not correlated with markers of astrocytes or oligodendrocytes, but co-labelled with markers of neural progenitor cells. In conjunction with CD15, proliferating CD24+/CD15+ granule cell precursors (GCPs) were identified as a TIC population in Ptch1 deleted medulloblastoma. On human medulloblastoma, CD24 was found to be highly expressed on Group 3, Group 4 and SHH subgroups compared with the WNT subgroup, which was predominantly positive for CD15, suggesting CD24 is an important marker of non-WNT medulloblastoma initiating cells and a potential therapeutic target in human medulloblastoma. This study reports the use of CD24 and CD15 to isolate a GCP-like TIC population in Ptch1 deleted medulloblastoma, and suggests CD24 expression as a marker to help stratify human WNT tumours from other medulloblastoma subgroups.