Brain Tumor-initiating Cells Research Articles

Expression and prognostic value of JAM-A in gliomas

Gliomas are among the most lethal cancers, being highly resistant to both chemo- and radiotherapy. The expression of junctional adhesion molecule-A (JAM-A) was recently identified on the surface of stem cell-like brain tumor-initiating cells and suggested to function as a unique glioblastoma niche adhesion factor influencing the tumorigenic potential of brain tumor-initiating cells. We have recently identified high JAM-A expression to be associated with poor outcome in glioblastomas, and our aim was to further investigate the expression of JAM-A in gliomas focusing especially on the prognostic value in WHO grade II and III gliomas. JAM-A protein expression was evaluated by immunohistochemistry and advanced quantitative image analysis with continuous estimates of staining intensity. The JAM-A antibody stained tumor cell membranes and cytoplasm to various extent in different glioma subtypes, and the intensity was higher in glioblastomas than low-grade gliomas. We could not detect an association with overall survival in patients with grade II and III tumors. Double-immunofluorescence stainings in glioblastomas revealed co-expression of JAM-A with CD133, SOX2, nestin, and GFAP in tumor cells as well as some co-expression with the microglial/macrophage marker IBA-1. In conclusion, JAM-A expression was higher in glioblastomas compared to low-grade gliomas and co-localized with recognized stem cell markers suggesting an association of JAM-A with glioma aggressiveness. No significant association between JAM-A expression and overall survival was found in grade II and III gliomas. Further research is needed to determine the function and clinical impact of JAM-A in gliomas.

Abstract 3844: Identification and validation of novel therapeutic targets driving clonal heterogeneity in treatment-refractory GBM

Abstract Glioblastoma (GBM) is the most common primary adult brain tumor, characterized by extensive cellular and genetic heterogeneity. Even with surgery, standard chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth (or recurrence) and patient relapse are inevitable. Patients face a median survival of <15 months, with uniformly fatal outcomes upon disease progression post-therapy. Recent profiling of GBM-initiating genes has shown that evolution of cancer-driving clones or cell populations within a solid tumor may progress through (and possibly be driven by) cancer treatment, such that GBM recurrence may no longer resemble the genetic landscape of the original primary tumor. Understanding and mapping clonal evolution of the primary GBM through therapy and at recurrence will allow for the discovery of novel targets specific to treatment-refractory GBM. Here, we have developed early passage patient-derived brain tumor initiating cell (BTIC) lines that have been annotated by genomic deep-sequencing technologies to systematically characterize and describe the extent of intratumoral heterogeneity. Tagged with a red florescent protein, these BTIC lines were engrafted into immunocompromised NOD SCID mice. Following half-maximal tumor engraftment, tumor bearing mice underwent a clinically relevant chemoradiotherapy regimen, with 2 Gy gamma-irradiation on the first day and 66 mg/kg temozolomide for five consecutive days. Following therapy, mice were kept alive until tumor recurrence. Engrafted BTICs were harvested at initial tumor formation, minimal residual disease after chemoradiotherapy, and tumor recurrence. Samples were analyzed by RNA and genomic deep-sequencing technologies to map cancer progression and identify novel therapeutic targets in treatment-refractory GBM. Potential therapeutic targets were validated by their effect on self-renewal and proliferation of patient-derived BTIC lines of human GBM in vitro and in vivo. Using CRISPR Cas9, potential targets were knocked out in patient-derived BTIC lines of human GBM in order to characterize the effect on sphere formation and proliferation in vitro, and tumor formation in vivo. Following validation of new therapeutic targets of treatment-refractor GBM, we aim to build novel biotherapeutics against highly validated cell surface targets, and establish preclinical testing protocols using our novel patient-derived and therapy-adapted xenograft model of treatment-resistant GBM. Citation Format: Chirayu Chokshi, Nick Yelle, Parvez Vora, Chitra Venugopal, Maleeha Qazi, Mohini Singh, Minomi Subapanditha, Avrilynn Ding, Sheila K. Singh. Identification and validation of novel therapeutic targets driving clonal heterogeneity in treatment-refractory GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3844. doi:10.1158/1538-7445.AM2017-3844

Abstract 5462: GTP cyclohydrolase in brain tumor stem cells is implicated in glioblastoma growth

Abstract Glioblastomas (GBMs) are the most common primary brain tumors in adults and one of the most aggressive cancers with high rates of recurrence and therapeutic resistance. In GBMs, subpopulations of highly tumorigenic cells called brain tumor initiating cells (BTICs) have been characterized by their unique capacity to promote tumor maintenance, therapeutic resistance, and angiogenesis. BTIC maintenance is known to be regulated by reactive oxygen and nitrogen species. GTP cyclohydrolase, or GCH1, is a critical molecule regulating reactive species levels. We found that expression of GCH1 RNA and protein were upregulated in BTICs in comparison to non-BTICs. Overexpression of GCH1 in glioma cells increased cell growth in vitro and increased tumor formation and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 depletion with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal orthotopic models. Furthermore, genetic modulation of GCH1 led to altered ROS levels in GBM xenolines. In silico analyses demonstrate that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in glioblastoma. Citation Format: Anh N. Tran, Kiera Walker, David Harisson, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee S. Sedaka, Jennifer S. Pollock, Sara J. Cooper, George Y. Gillespie, Anita B. Hjelmeland. GTP cyclohydrolase in brain tumor stem cells is implicated in glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2017-5462

Abstract 3870: Clonal evolution of medulloblastoma BTICs in response to therapy

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumour. Global gene expression arrays performed on human MBs have divided this tumour entity into 4 distinct molecular subgroups. Out of all the subgroups, Group 3 patients face the highest incidence of leptomeningeal spread and overall patient survival of less than 50%. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumours, provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumors. By adapting the existing COG (Children’s Oncology Group) Protocol for children with newly diagnosed high-risk MB for treatment of immuno-deficient mice intracranially engrafted with human MB brain tumour initiating cells (BTICs), we aim to identify and characterize the rare treatment-refractory cell population in Group 3 MBs. MB cell populations recovered separately from brains and spines at (i) engraftment; (ii) post-radiation; (iii) post-radiation and chemotherapy; and (iv) recurrence, during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy to relapse. One of the most intriguing observations from our gene expression data was consistent over-expression of proteins belonging to Inhibitor of DNA-binding/differentiation (ID) family, transcription factors with a basic helix-loop-helix motif that act as suppressors cellular differentiation and a longevity associated protein bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) in our refractory population. The persistent upregulation of genes preserving undifferentiated state and cellular longevity further strengthens the hypothesis of stem-cell like cells driving tumor relapse in MB. We then set out to determine whether genes upregulated at relapse correlated with patient outcome in our therapy-adapted patient-derived xenograft model. Interestingly, the upregulation of the top 90 genes in our relapse cohort was predictive of worse overall survival in patients with group 3 MB. In the next set of experiments, through application of cellular barcoding technology we determined how MB BTICs evolve in response to therapy by tracking unique vector DNA sequences integrated at a single copy level into individual cells. Our differential genomic profile of the “treatment-responsive” tumors against those that fail therapy will thus contribute to discovery of novel therapeutic approaches for the most aggressive subgroup of MB. Citation Format: David Bakhshinyan, Thusyanth Vijayakumar, Chitra Venugopal, Mohini Singh, Maleeha Qazi, Sujeivan Mahendram, Sujeivan Mahendram, Branavan Manoranjan, Nicole McFarlane, Ashley Adile, Sheila Singh. Clonal evolution of medulloblastoma BTICs in response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3870. doi:10.1158/1538-7445.AM2017-3870

Abstract 1925: Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance

Abstract Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting. Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2017-1925

Abstract 3639: Therapeutic targeting of tumorigenic EphA2+/EphA3+ brain tumor initiating cells with bi-specific antibody in human glioblastoma

Abstract Glioblastoma (GBM), the most aggressive primary human brain tumor, carrier a dismal prognosis and is increasingly characterized by cellular and genetic intra-tumoral heterogeneity (ITH). Many of the 14 members of the erythropoietin-producing hepatocellular carcinoma receptor (EphR) family and their ephrin ligands are expressed in GBM cells and constitute potential molecular targets for novel therapeutic agents. We hypothesize that multiple members of the EphR family play a critical role in orchestrating the clonal evolution of GBM progression. Individual Eph receptor targeting strategies have shown only modest pre-clinical success, likely because single agent therapy cannot target the degree of ITH in GBM. Using a highly specific human Eph receptor monoclonal antibody (mAb) panel (EphR profiler), we identified five Eph receptors with dysregulated expression in recurrent GBM as compared to primary GBM. With our unique chemoradiotherapy-adapted, patient-derived xenograft model of GBM, we identified EphA2 and EphA3 expression to be upregulated after therapy. Here we show that EphA2 and EphA3 co-expression marks a highly tumorigenic cell population in recurrent GBM with higher in vitro and in vivo self-renewal and proliferation capacity as compared to EphA2+/EphA3-, EphA2-/EphA3+ or EphA2-/EphA3- cells. Lentiviral mediated knockdown of EphA2 and EphA3 blocks this self-renewal and proliferation capacity in recurrent GBM. Through further characterization using mass cytometry (CyTOF) assay, we find that EphA2 and EphA3 is co-expressed with multiple brain tumor initiating cell (BTIC) markers (CD133, CD15, Bmi1, Sox2, Integrin α6 and FoxG1). Considering the important role of EphA2+/EphA3+ cells in GBM tumorigenesis and recurrence, we generated a bi-specific antibody (bsIgG) that co-targets EphA2 and EphA3. In vitro treatment of GBM with bsEphA2/A3 IgG led to pharmacological blockade of phosphorylated EphA2. We then assessed the in vivo efficacy of the bsEphA2/A3 IgG to block GBM tumor growth in our PDX model, and found that treatment with intracranial bsIgG resulted in non-invasive and significantly smaller lesions. The striking reduction in tumor burden in recurrent GBM after co-targeting of EphA2 and EphA3 validates the premise of our therapeutic strategy of targeting multiple EphRs. Discovering the clonal composition of recurrent GBM will enable us to target cellular subpopulations, and this ITH, with selective compounds that inhibit BTIC and Eph receptor activity with minimal off-target effects. Comprehensive Eph receptor profiling of individual patient-derived GBM will allow us to develop a therapeutic strategy for each patient’s tumor, employing polytherapy with mAbs against Eph receptors expressed at recurrence. Citation Format: Maleeha Qazi, Parvez Vora, Chirayu Chokshi, Chitra Venugopal, Max London, Amy Hu, Minomi Subapandtiha, Mohini Singh, Jarrett Adams, Jason Moffat, Sachdev Sidhu, Sheila K. Singh. Therapeutic targeting of tumorigenic EphA2+/EphA3+ brain tumor initiating cells with bi-specific antibody in human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3639. doi:10.1158/1538-7445.AM2017-3639

Abstract 3758: The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of glioblastoma

Abstract Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM remains poor. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence. Chimeric antigen receptors (CARs) and bispecific T-Cell engaging antibodies (BiTEs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from previously generated RW03 and generated a second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR. Following lentiviral packaging, the T cells isolated from PBMCs were transduced with CD133 CAR construct. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden. The results of this study will establish a translational research program that will form the basis of early phase clinical trials of a promising CD133-based therapeutic strategy for patients with GBM. Citation Format: Parvez Vora, Chirayu Chokshi, Maleeha Qazi, Mohini Singh, Chitra Venugopal, Sujeivan Mahendram, Jarrett Adams, David Bakhshinyan, Max London, Jess Singh, Minomi Subapanditha1,, Nicole McFarlane, James Pan, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3758. doi:10.1158/1538-7445.AM2017-3758

Activation of NOTCH Signaling by Tenascin-C Promotes Growth of Human Brain Tumor-Initiating Cells.

Oncogenic signaling by NOTCH is elevated in brain tumor-initiating cells (BTIC) in malignant glioma, but the mechanism of its activation is unknown. Here we provide evidence that tenascin-C (TNC), an extracellular matrix protein prominent in malignant glioma, increases NOTCH activity in BTIC to promote their growth. We demonstrate the proximal localization of TNC and BTIC in human glioblastoma specimens and in orthotopic murine xenografts of human BTIC implanted intracranially. In tissue culture, TNC was superior amongst several extracellular matrix proteins in enhancing the sphere-forming capacity of glioma patient-derived BTIC. Exogenously applied or autocrine TNC increased BTIC growth through an α2β1 integrin-mediated mechanism that elevated NOTCH ligand Jagged1 (JAG1). Microarray analyses and confirmatory PCR and Western analyses in BTIC determined that NOTCH signaling components including JAG1, ADAMTS15, and NICD1/2 were elevated in BITC after TNC exposure. Inhibition of γ-secretase and metalloproteinase proteolysis in the NOTCH pathway, or silencing of α2β1 integrin or JAG1, reduced the proliferative effect of TNC on BTIC. Collectively, our findings identified TNC as a pivotal initiator of elevated NOTCH signaling in BTIC and define the establishment of a TN-α2β1-JAG1-NOTCH signaling axis as a candidate therapeutic target in glioma patients. Cancer Res; 77(12); 3231-43. ©2017 AACR.

Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells.

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.

Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20pM, mAb2C4:gelonin, 20pM, compared to the ADC (6.3nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

OS06.3 Characterisation of human brain tumour initiating cells leading invasion

Abstract Introduction: Glioblastoma (GBM) belongs to the most aggressive cancers. Main pathophysiological feature of GBM is fast and widespread invasion of the brain parenchyma by a subpopulation of progenitor tumour cells, rendering localized therapies ineffective. In addition, brain tumour initiating cells (BTIC) from GBM may repopulate the tumour site after initially effective treatments. Thus, the invasive progenitor tumour cell fraction is a strategic prime target for early and sustained anti-invasion therapies. To date, the dependency on and interaction with the microenvironment of progenitor cells and their adaptive development from the tumour cell fraction has not been thoroughly investigated. Methods: With organotypic brain slice cultures (OBSC), single cell RNA expression analysis and bioinformatics workup, we established a novel integrated approach to investigate the expression profile of invasion leading cells. A micromanipulator adapter was developed to isolate different three subpopulations within human GBM progenitor cells: leader, follower, and stationary, from the OBSCs. In comparison, we use long-term intravital imaging to analyse the behaviour and interaction of the three cellular fractions with each other and the microenvironment in vivo. Imaging through a cranial window in the brain of a living mouse enables us to visualize single tumour cells. Using a photoswitchable vector expressed in BTICs we specifically label and isolate leader, follower, and stationary cells separately to compare leader cell behaviour in vivo, in situ, and in the patient situation. Findings: The in situ OBSC model is a valuable tool to analyse migration and invasion in conditions simulating normal brain tissue including some microenvironmental influences. This allowed us to monitor human BTIC cell invasion in real time. Invasive cells emerge from the initial cell population, driving invasion into the surrounding tissue. Those leader cells differ in morphology and behaviour from stationary cells, which remain at the implantation site. A subsequent microarray analysis of single cells revealed that leader cells have a markedly distinct expression pattern in comparison to follower and stationary cells. This leader cell-specific expression signature is currently verified in fresh patient tumour biopsies, other BTICs analysed in the OBSC as model, and in the cranial window model. Updated results will be presented at the WFNOS meeting. Outlook: We showed that invasive leader cells have a specific functional and molecular profile. A thorough characterization of these cells in respect to their interaction with and response to the microenvironment that may be involved in the process of transforming cells into invasive leader cells is ongoing. We hypothesize that a deep characterization of these events will shed light on the chronology of one of the gliomagenesis milestones, namely invasion.

P01.37 Impact of C/EBPβ isoforms on brain tumor initiating cells

BACKGROUND: The transcription factor C/EBPβ has been identified as a master regulator of the mesenchymal phenotype in malignant glioma. It is transcribed from an intronless gene, but can be translated into three isoforms (LAP, LAP*, LIP). C/EBPβ has been shown to be upregulated in mesenchymal GBMs and its expression correlates to a worse prognosis in patients. Downregulation of C/EBPβ reduces proliferation and migration in vitro and diminishes tumor growth in vivo. In addition, C/EBPβ protein levels are increased in hypoxic and necrotic areas in human GBM tissue samples. However, expression levels and impact of the different isoforms in glioma are unknown. Crosstalk of C/EBPβ and TFGβ has been shown to influence metastatic behavior of breast carcinoma cells. The aim of this project is therefore to investigate the impact of C/EBPβ isoforms LAP and LIP on phenotype, proliferation and migration of brain tumor initiating cells (BTICs) and their possible interaction with transforming growth factor beta (TGFβ). Methods: LAP and LIP isoforms were stably overexpressed in a set of proneural and mesenchymal BTIC lines. Marker expression was investigated by immunocytochemistry and Western blot. Proliferation was monitored by crystal violet staining and migration by spheroid assay. Genome wide transcriptome analysis was performed by next generation RNA sequencing on a HiSeq (Illumina). Results: Overexpression of LAP ­(full-length protein) induced proneural BTICs to switch towards a mesenchymal phenotype, whereas overexpression of LIP had no such effect. Increased LAP led to enhanced migration in the majority of cell lines irrespective of subtype, whereas LIP mostly decreased migration. Rather the reverse effect was observed regarding proliferation. RNASeq analysis revealed highly differential consequences of LAP and LIP overexpression regarding expressed genes and principal component analysis. TGFβ signaling proved to be one of the most significantly altered pathways both in LAP and LIP cells. However, TGFβ treatment had either no effect or inhibited proliferation or migration independent of C/EBPβ isoform levels. Conclusions: We could show for the first time that overexpression of C/EBPβ is sufficient to induce a mesenchymal phenotype in proneural BTICs and that the LAP isoform is responsible for this transdifferentiation. Independent of glioma subtype, LAP and LIP can elicit opposing effects. Nevertheless, both isoforms might contribute to tumor progression, since shifting the balance between LAP and LIP might add to BTICs capability to adapt to microenvironmental cues, e.g. by adopting a more migratory or proliferative phenotype. In contrast to the findings in breast CA, TGFβ acted independently of C/EBPβ isoforms. Ongoing experiments will provide further insight into downstream effector molecules and regulatory mechanisms for each isoform in GBM.

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma.

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo

BackgroundGlioblastoma is the most frequent and most malignant brain tumor with the patients having a median survival of only 14.6 months. Although glioblastoma patients are treated with surgery, radiation and chemotherapy recurrence is inevitable. A stem-like population of radio- and chemoresistant brain tumor-initiating cells combined with the invasive properties of the tumors is believed to be critical for treatment resistance. In the present study, the aim was to investigate the effect of a novel therapeutic strategy using the lysosomotropic detergent siramesine on glioblastomas.MethodsStandard glioma cell lines and patient-derived spheroids cultures with tumor-initiating stem-like cells were used to investigate effects of siramesine on proliferation and cell death. Responsible mechanisms were investigated by inhibitors of caspases and cathepsins. Effects of siramesine on migrating tumor cells were investigated by a flat surface migration assay and by implanting spheroids into organotypic rat brain slice cultures followed by confocal time-lapse imaging. Finally the effect of siramesine was investigated in an orthotopic mouse glioblastoma model. Results obtained in vitro and in vivo were confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains.ResultsThe results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal membrane. Co-treatment of the cell lines with inhibitors of caspases and cathepsins suggested differential involvement in cell death. Siramesine caused tumor cell death and reduced secondary spheroid formation of patient-derived spheroid cultures. In the flat surface migration model siramesine caused tumor cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional spheroid-brain slice culture model or in the mice xenograft model.ConclusionsIn conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal destabilizing drugs in killing glioblastoma cells, but siramesine was without effect in the organotypic spheroid-brain slice culture model and the in vivo xenograft model.

Protein kinase CK2 is important for the function of glioblastoma brain tumor initiating cells.

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α') and two regulatory (β) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133+ BTICs compared to CD133- cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.

Modeling Physiologic Microenvironments in Three-Dimensional Microtumors Maintains Brain Tumor Initiating Cells.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity, including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy, because BTICs are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using the human-derived biomatrix HuBiogel™ and maintained with physiologically relevant microenvironments. BITC percentages were determined using cell surface marker expression, label retention, and neurosphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient-restricted microtumors significantly increased the percentage of both CD133+ and CFSEhigh cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintained neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Electrophoresis of cell membrane heparan sulfate regulates galvanotaxis in glial cells.

Endogenous electric fields modulate many physiological processes by promoting directional migration, a process known as galvanotaxis. Despite the importance of galvanotaxis in development and disease, the mechanism by which cells sense and migrate directionally in an electric field remains unknown. Here, we show that electrophoresis of cell surface heparan sulfate (HS) critically regulates this process. HS was found to be localized at the anode-facing side in fetal neural progenitor cells (fNPCs), fNPC-derived astrocytes and brain tumor-initiating cells (BTICs), regardless of their direction of galvanotaxis. Enzymatic removal of HS and other sulfated glycosaminoglycans significantly abolished or reversed the cathodic response seen in fNPCs and BTICs. Furthermore, Slit2, a chemorepulsive ligand, was identified to be colocalized with HS in forming a ligand gradient across cellular membranes. Using both imaging and genetic modification, we propose a novel mechanism for galvanotaxis in which electrophoretic localization of HS establishes cell polarity by functioning as a co-receptor and provides repulsive guidance through Slit-Robo signaling.

The Role of Neurotrophin Signaling in Gliomagenesis: A Focus on the p75 Neurotrophin Receptor (p75NTR/CD271).

The p75 neurotrophin receptor (p75NTR, a.k.a. CD271), a transmembrane glycoprotein and a member of the tumor necrosis family (TNF) of receptors, was originally identified as a nerve growth factor receptor in the mid-1980s. While p75NTR is recognized to have important roles during neural development, its presence in both neural and nonneural tissues clearly supports the potential to mediate a broad range of functions depending on cellular context. Using an unbiased in vivo selection paradigm for genes underlying the invasive behavior of glioma, a critical characteristic that contributes to poor clinical outcome for glioma patients, we identified p75NTR as a central regulator of glioma invasion. Herein we review the expanding role that p75NTR plays in glioma progression with an emphasis on how p75NTR may contribute to the treatment refractory nature of glioma. Based on the observation that p75NTR is expressed and functional in two critical glioma disease reservoirs, namely, the highly infiltrative cells that evade surgical resection, and the radiation- and chemotherapy-resistant brain tumor-initiating cells (also referred to as brain tumor stem cells), we propose that p75NTR and its myriad of downstream signaling effectors represent rationale therapeutic targets for this devastating disease. Lastly, we provide the provocative hypothesis that, in addition to the well-documented cell autonomous signaling functions, the neurotrophins, and their respective receptors, contribute in a cell nonautonomous manner to drive the complex cellular and molecular composition of the brain tumor microenvironment, an environment that fuels tumorigenesis.

Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation.

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor and associated with a devastating prognosis. Signal transducer and activator of transcription number 3 (STAT3) is an important pathogenic factor in GBM and can be specifically inhibited with Stattic. Metformin inhibits GBM cell proliferation and migration. Evidence from other tumor models suggests that metformin inhibits STAT3, but there is no specific data on brain tumor initiating cells (BTICs).We explored proliferation and migration of 7 BTICs and their differentiated counterparts (TCs) after treatment with Stattic, metformin or the combination thereof. Invasion was measured in situ on organotypic brain slice cultures. Protein expression of phosphorylated and total STAT3, as well as AMPK and mTOR signaling were explored using Western blot. To determine functional relevance of STAT3 inhibition by Stattic and metformin, we performed a stable knock-in of STAT3 in selected BTICs.Inhibition of STAT3 with Stattic reduced proliferation in all BTICs, but only in 4 out of 7 TCs. Migration and invasion were equally inhibited in BTICs and TCs. Treatment with metformin reduced STAT3-phosphorylation in all investigated BTICs and TCs. Combined treatment with Stattic and metformin led to significant additive effects on BTIC proliferation, but not migration or invasion. No additive effects on TCs could be detected. Stable STAT3 knock-in partly attenuated the effects of Stattic and metformin on BTICs.In conclusion, metformin was found to inhibit STAT3-phosphorylation in BTICs and TCs. Combined specific and unspecific inhibition of STAT3 might represent a promising new strategy in the treatment of glioblastoma.

The identification of human pituitary adenoma-initiating cells.

Classified as benign central nervous system (CNS) tumors, pituitary adenomas account for 10% of diagnosed intracranial neoplasms. Although surgery is often curative, patients with invasive macroadenomas continue to experience significant morbidity and are prone to tumor recurrence. Given the identification of human brain tumor-initiating cells (TICs) that initiate and maintain tumor growth while promoting disease progression and relapse in multiple CNS tumors, we investigated whether TICs also drive the growth of human pituitary adenomas. Using a nanoString-based 80-gene custom codeset specific for developmental pathways, we identified a differential stem cell gene expression profile within human pituitary adenomas. Prospective functional characterization of stem cell properties in patient-derived adenomas representing all hormonal subtypes yielded a subtype-dependent self-renewal profile, which was enriched within the CD15+ cell fraction. The tumor-initiating capacity of CD15high adenoma cells was assayed in comparison to CD15low adenomas using in vivo limiting dilutions, which maintained the rare frequency of TICs. Repeated analyses using sorted cell populations for CD15+ TICs compared to CD15- adenoma cells provided further evidence of xenograft tumor formation to support CD15+ cells as putative pituitary adenoma-initiating cells (PAICs). The clinical utility of our findings was established through in silico analyses and comparative gene expression profiling of primary and recurrent pituitary adenomas. CD15 was enriched in recurrent adenomas, which was validated using routine clinical immunohistochemistry in a limited number of samples. Our work reports the first prospective identification of human PAICs using CD15. Patients with CD15high adenomas may therefore benefit from more aggressive surgical interventions and chemo/radiotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0394-4) contains supplementary material, which is available to authorized users.