Stem Cell-enriched Cultures Research Articles

STEM-20. ROLE OF CELLULAR PRION PROTEIN IN TEMOZOLOMIDE RESISTANCE OF GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma (GBM), the most aggressive type of brain tumor, is initiated and maintained by a subpopulation of tumorigenic cells called glioblastoma stem cells (GSCs) that contribute to progression and resistance to therapy. In GBM therapy the alkylating agent temozolomide (TMZ) is the most effective chemotherapist, but resistance is frequent, especially in GSCs located in hypoxia niches. One of the mechanisms of resistance to TMZ involves the modulation of MGMT, which promotes cell survival through the induction of antiapoptotic proteins expression and can be regulated by hypoxia inducible factors (HIFs). Recent data from our group demonstrate that blocking the interaction between the cellular prion protein (PrPC) and one of its main ligands, the heat shock organizing protein (HOP) with a HOP peptide (HOP230-245) can inhibit GSCs self-renewal and glioblastomas growth in vivo. PrPC silencing decreases the expression of stem cells markers, self-renewal, and tumorigenesis of GSCs. Remarkably, a PrPC peptide which mimics HOP binding site (PrPC106-126) can promote HIF1α expression, inducing protection against cell death. Considering these data together, this study aims to identify the participation of PrPC in the TMZ resistance of GSCs mediated by HIF1α. In addition, we will evaluate HOP230-245 peptide potential in sensitizing GSCs to TMZ, since it binds to PrPC at the specific site 106-126 being able to compete with other PrPC ligands by this domain and, consequently, inhibit functions modulated by these interactions. Preliminary trials on stem cell enriched glioblastoma lineage cultures suggest that treatment with TMZ associated HOP230-245 increases cell death compared to TMZ or the peptide alone, sensitizing cells to TMZ treatment and increasing their efficiency. Finally, the search for therapeutic-combined strategies targeting GSCs is imperative to improve the efficacy of treatments against this incurable tumor.

P03.07.A MRI signature in preoperative imaging predicts mesenchymal stem cell features and radioresistance in primary glioblastoma stem cell cultures

Abstract Background Recently, a molecular signature describing the mesenchymal / proneural features of primary stem-cell-enriched glioblastoma cultures was identified correlating with invasiveness, radiation sensitivity and patient-outcome. However, generating and characterizing primary cultures is time-consuming and this might hamper translation into clinical concepts. The aim of this study was to evaluate the use of standard preoperative MR imaging (T1ce and T2FLAIR sequences) to predict the molecular signature and thus enable the development of clinical treatment concepts based on the molecular properties of the individual tumors. Material and Methods For 16 patients, for whom primary stem cell enriched cultures had been characterized for their mesenchymal / proneural signature and radiation sensitivity, tumor volume (hyperintense volume in T1ce), necrosis (hypointense volume inside the tumor volume) and edema (hyperintense volume in T2FLAIR) were contoured for volumetric analysis. Volume parameters were used to calculate ratios (edema/tumor and necrosis/tumor) and a MRI signature, which was then correlated with molecular parameters and patient outcome stratified by MGMT promoter methylation. Results As expected, the prognosis of patients with MGMT-promoter-methylated tumors was better (n.s.) compared to those with unmethylated MGMT promoters. Neither molecular nor imaging data were significantly different between these subgroups. In the subgroup of patients with unmethylated MGMT promoter, volumetric imaging parameters correlated with the molecular signature and cellular radiation sensitivity of the stem cell enriched cultures. This association was much weaker in the subgroup with methylated MGMT promoter. Conclusion In the subgroup of patients with unmethylated MGMT promoter, volumetric parameters on preoperative standard MR imaging might hint at the molecular properties of the respective tumor and its radiation sensitivity. This might be a clinically applicable method to stratify treatment according to molecular stem cell subtype without tissue culture and molecular analysis.

CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

Chemotherapy is the mainstay for the treatment of non-small cell lung cancer (NSCLC). However, NSCLC cells are either intrinsically chemoresistant or rapidly develop therapy resistance. Cancer stem cells (CSCs) are widely recognized as the cell population responsible for resistance to systemic therapies, but the molecular responses of CSCs to chemotherapeutic agents are largely unknown. We identified the embryonic protein CRIPTO in stem cell-enriched spheroid cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC) derived from NSCLC surgical specimens. The CRIPTO-positive population had increased clonogenic capacity and expression of stem cell-related factors. Stemness-related properties were also obtained with forced CRIPTO expression, whereas CRIPTO downregulation resulted in cell cycle blockade and CSCs death. Cell populations positive and negative for CRIPTO expression were interconvertible, and interfering with their reciprocal equilibrium resulted in altered homeostasis of cell expansion both in spheroid cultures and in tumor xenografts. Chemotherapy treatment of NSCLC cells resulted in reduction of cell number followed by increased CRIPTO expression and selective survival of CRIPTO-positive cells. In NSCLC tumor xenografts, chemotherapeutic agents induced partial cell death and tumor stabilization followed by CRIPTO overexpression and tumor progression. Altogether, these findings indicate CRIPTO as a marker of lung CSCs possibly implicated in cancer cell plasticity and post-chemotherapy tumor progression.

β-Tubulin Isotype, TUBB4B, Regulates The Maintenance of Cancer Stem Cells.

Recent advancements in cancer research have shown that cancer stem cell (CSC) niche is a crucial factor modulating tumor progression and treatment outcomes. It sustains CSCs by orchestrated regulation of several cytokines, growth factors, and signaling pathways. Although the features defining adult stem cell niches are well-explored, the CSC niche is poorly characterized. Since membrane trafficking proteins have been shown to be essential for the localization of critical proteins supporting CSCs, we investigated the role of TUBB4B, a probable membrane trafficking protein that was found to be overexpressed in the membranes of stem cell enriched cultures, in sustaining CSCs in oral cancer. Here, we show that the knockdown of TUBB4B downregulates the expression of pluripotency markers, depletes ALDH1A1+ population, decreases in vitro sphere formation, and diminishes the tumor initiation potential in vivo. As TUBB4B is not known to have any role in transcriptional regulation nor cell signaling, we suspected that its membrane trafficking function plays a role in constituting a CSC niche. The pattern of its expression in tissue sections, forming a gradient in and around the CSCs, reinforced the notion. Later, we explored its possible cooperation with a signaling protein, Ephrin-B1, the abrogation of which reduces the self-renewal of oral cancer stem cells. Expression and survival analyses based on the TCGA dataset of head and neck squamous cell carcinoma (HNSCC) samples indicated that the functional cooperation of TUBB4 and EFNB1 results in a poor prognosis. We also show that TUBB4B and Ephrin-B1 cohabit in the CSC niche. Moreover, depletion of TUBB4B downregulates the membrane expression of Ephrin-B1 and reduces the CSC population. Our results imply that the dynamics of TUBB4B is decisive for the surface localization of proteins, like Ephrin-B1, that sustain CSCs by their concerted signaling.

Nano-Encapsulation of Mithramycin in Transfersomes and Polymeric Micelles for the Treatment of Sarcomas.

Sarcomas are aggressive tumors which often show a poor response to current treatments. As a promising therapeutic alternative, we focused on mithramycin (MTM), a natural antibiotic with a promising anti-tumor activity but also a relevant systemic toxicity. Therefore, the encapsulation of MTM in nano-delivery systems may represent a way to increase its therapeutic window. Here, we designed novel transfersomes and PLGA polymeric micelles by combining different membrane components (phosphatidylcholine, Span 60, Tween 20 and cholesterol) to optimize the nanoparticle size, polydispersity index (PDI) and encapsulation efficiency (EE). Using both thin film hydration and the ethanol injection methods we obtained MTM-loaded transferosomes displaying an optimal hydrodynamic diameter of 100–130 nm and EE values higher than 50%. Additionally, we used the emulsion/solvent evaporation method to synthesize polymeric micelles with a mean size of 228 nm and a narrow PDI, capable of encapsulating MTM with EE values up to 87%. These MTM nano-delivery systems mimicked the potent anti-tumor activity of free MTM, both in adherent and cancer stem cell-enriched tumorsphere cultures of myxoid liposarcoma and chondrosarcoma models. Similarly to free MTM, nanocarrier-delivered MTM efficiently inhibits the signaling mediated by the pro-oncogenic factor SP1. In summary, we provide new formulations for the efficient encapsulation of MTM which may constitute a safer delivering alternative to be explored in future clinical uses.

Genes Involved in the Transcriptional Regulation of Pluripotency Are Expressed in Malignant Tumors of the Uterine Cervix and Can Induce Tumorigenic Capacity in a Nontumorigenic Cell Line.

Transcription factors OCT4, SOX2, KLF4, C-MYC, and NANOG (OSKM-N) regulate pluripotency and stemness, and their ectopic expression reprograms human and murine fibroblasts that constitute the key of regenerative medicine. To determine their contribution to cell transformation, we analyzed the gene expression profiles of these transcription factors in cervical cancer samples and found that they are preferentially expressed in the tumor component. Also, cancer stem cell-enriched cultures grown as sphere cultures showed overexpression of OSKM-N genes. Importantly, we observed that lentiviral-mediated transduction of these factors confers, to a nontumorigenic immortalized human cell line, properties of cancer stem cells as the ability to form tumors in a mouse model. When we performed a meta-analysis using microarray data from cervical cancer biopsies and normal tissues, we found that the expression of OSKM-N and some target genes allowed separating tumor and normal tissues between samples, which enhanced the importance of OSKM-N in the tumorigenesis. Finally, we analyzed and compared both transcript and protein expression profiles of these factors within a cohort of patients with cervical cancer. To our knowledge, this is the first time that the expression of OSKM-N is described to induce one of the main characteristics of the cancer stem cell, the tumorigenicity. And, more importantly, its exogenous expression in a nontumorigenic cell line is sufficient to induce a tumorigenic phenotype; furthermore, the differential expression of this transcription factor distinguishes tumor tissue and normal tissue in cervical samples.

PDTM-28. AN OTX2-PAX3 SIGNALLING AXIS REGULATES GROUP 3 MEDULLOBLASTOMA CELL FATE

Abstract The majority of Group 3 medulloblastomas (MB) exhibit amplification or increased expression of OTX2. OTX2 is primarily known as an oncogenic driver of tumor growth and cell cycle progression in Group 3 MB; however, its role as a repressor of differentiation is poorly characterized. Therefore, we utilized extensive patient data and mapped Group 3 MB chromatin dynamics in stem cell-enriched cultures to evaluate the divergent role of OTX2 in cell fate decisions in Group 3 MB pathogenesis. Several PAX genes were identified as novel OTX2 targets in Group 3 MB. Examination of patient data revealed that PAX3 and PAX6 expression is significantly reduced in Group 3 MB patients and is associated with significantly reduced survival. Functional evaluation of PAX3 and PAX6 expression showed that PAX3 expression significantly reduced self-renewal capacity of Group 3 MB tumorspheres in vitro and significantly prolonged survival and reduced tumor size in orthotopic xenograft models in vivo. RNA-sequencing of PAX3 and PAX6 gain of function (GOF) tumorspheres revealed mTORC1 signalling was specifically downregulated in PAX3 GOF, indicating this pathway may be critical for the survival and self-renewal differences observed between PAX3/PAX6 GOF models. Treatment of Group 3 MB with mTOR inhibitors reduced self-renewal in vitro and significantly prolonged survival and reduced tumor size in vivo. To further evaluate the role for this signalling axis in the Group 3 MB neural lineage hierarchy, we carried out scRNA-sequencing in tumorspheres from 4 Group 3 MB cell lines. Interestingly, a broad range of OTX2 expression was observed across single cell clusters, suggesting distinct OTX2 regulatory hierarchies are present in Group 3 MB. Collectively, our work demonstrates the multifaceted role of OTX2 as a regulator of cell fate decisions in Group 3 MB and identifies a novel role for mTORC1 signalling in Group 3 MB self-renewal and differentiation.

Cancer Stem Cells from Tumor Cell Lines Activate the DNA Damage Response Pathway after Ionizing Radiation More Efficiently Than Noncancer Stem Cells.

Recently, a subpopulation of tumor cells, called cancer stem cells (CSC), has been characterized, and these have emerged as a major topic in cancer research. CSC are proposed to repair DNA damage more efficiently than the rest of tumor cells, resisting chemotherapy or radiotherapy and causing clinical recurrence and metastasis. We aimed to determine the molecular basis of radioresistance and first compared the response to ionizing radiation (IR) between cancer stem cell-enriched cultures grown as spheres and conventional tumor cell line cultures grown as monolayer, from HeLa and MCF-7 cancer cell lines. To verify that our sphere cultures were enriched in CSC, we evaluated the double staining of CD49f and ALDH activity for HeLa cells by flow cytometry. We then evaluated whether differences could exist in sensor elements in the DNA damage response pathway among these cultures. We found that CSC cultures showed less sensitivity to radiation than conventional tumor cell line cultures. We observed a higher baseline expression of activated response sensor proteins of DNA damage, such as ATM, H2A.X, and PARP1, in untreated CSC cultures. These findings provide the first evidence, to our knowledge, that DNA damage response sensor proteins are present and preferentially activated in CSC, as opposed to the bulk of cells in monolayer cultures. Likewise, they provide the basis for biological differences in response to IR between CSC and other tumor cell populations. Understanding the DNA damage response pathway may provide therapeutic targets to sensitize CSC to cytotoxic therapies to improve current cancer treatments.

Stem cell enriched-epithelial spheroid cultures for rapidly assaying small intestinal radioprotectors and radiosensitizers in vitro

Radiation therapy is one of the main treatment options for many cancer patients. Although high doses of radiation may maximize tumor cell killing, dose escalation is limited by toxicity to neighboring normal tissues. This limitation applies particularly to the small intestine, the second most radiosensitive organ in the body. Identifying small intestinal (SI) radioprotectors could enable dose escalation in the treatment of abdominopelvic malignancies. However, the only assay currently available to identify effects of radiomodulating drugs on the regenerating capacity of SI stem cells is the Withers-Elkind microcolony assay, which requires large numbers of mice, making it a costly and low throughput method. Here, we describe a novel spheroid formation assay (SFA) that utilizes SI stem cell-enriched three-dimensional epithelial spheroid cultures to identify gastrointestinal radiomodulators ex vivo. The SFA is scalable for high throughput screening and can be used to identify both radioprotectors and radiosensitizers.

Abstract 4164: Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. Surgical resection is the only potentially curative treatment; however, only 15%-20% of patients present with tumors that can be resected. There is no consensus regarding standard of care in unresectable cases, however many academic centers use stereotactic body radiotherapy (SBRT) to give tumor-directed radiotherapy (RT). Unfortunately, even this conformal technique can still cause severe gastrointestinal (GI) toxic effects caused by the proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic effects of radiation may enable dose escalation that could achieve more effective local control of disease. We and others have previously shown that a prolonged fast of 24 hours protects mice from lethal doses of etoposide. In this study, we extend and build on our previous finding to demonstrate that a similar 24 hour fast also protects from lethal doses of total abdominal radiation. Histologic analyses, using the Withers-Elkind microcolony assay, show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we developed an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells sufficiently to allow animals to receive potentially curative doses of abdominal radiation that would other wise be lethal. Future work will aim to identifying the mechanism by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection. Citation Format: Marimar de la Cruz Bonilla, Kristina M. Stemler, Tara N. Fujimoto, Sabrina Jeter-Jones, Jessica M. Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4164.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

Progress in corneal wound healing.

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells.

Metformin repositioning as antitumoral agent: selective antiproliferative effects in human glioblastoma stem cells, via inhibition of CLIC1-mediated ion current.

Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown. Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity. The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment.

Abstract 210: Id4 inhibition of twist1-mediated autocrine PDGF signaling inhibits growth and promotes apoptosis of glioblastoma-derived stem cell cultures

Abstract Glioblastoma Multiforme (GBM) is the most common, aggressive, and lethal primary brain tumor. A hallmark of GBM is the resistance of tumor stem cells to chemotherapy resulting in tumor recurrence and progression decreasing the quality of life and causing patient death. The prevention of GBM recurrence after chemotherapy by eliminating cancer stem cells enhances survival in genetically engineered mouse models; however, the signal transduction networks that control cancer stem cell survival and proliferation remain poorly understood. We have discovered that decreased expression of Id4, a dominant negative helix-loop-helix transcription factor, is associated with the proliferation and survival of sphere cultures from GBM-derived cell lines, which are enriched for cancer stem cells. In striking contrast, Id4 expression did not affect proliferation or apoptosis in differentiated, adherent cultures of these same cell lines. Moreover, the cell lines we examined required autocrine PDGF signaling for growth. We found that Id4 inhibited autocrine PDGF signaling in cancer stem cell enriched cultures by suppressing the expression of platelet-derived growth factor receptor α (PDGFRα). In these studies we found that Id4 mediated inhibition of PDGFRα expression was mediated through the antagonistic interaction between Id4 and Twist1, a basic helix-loop-helix transcription factor. This observation is consistent with our previously reported finding that Id4, uniquely among the Id family of proteins, binds to and antagonizes Twist1. Further, using the Rembrandt and TCGA databases, we found that Id4 expression levels directly correlate with glioma patient survival and can predict the level of PDGFRα expression. These data provide strong evidence that Id4 may regulate GBM stem cell survival and thereby contribute to GBM tumor progression. Along with our recent report demonstrating that Id4 can block invasion of GBM by silencing Twist1-mediated MMP2 expression, these findings suggest that pharmacological targeting of the Id4/Twist1 complex might provide opportunities to reduce the lethality of GBM by regulating both GBM invasion and tumor stem cell maintenance. Citation Format: Gilbert J. Rahme, Mark A. Israel. Id4 inhibition of twist1-mediated autocrine PDGF signaling inhibits growth and promotes apoptosis of glioblastoma-derived stem cell cultures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 210. doi:10.1158/1538-7445.AM2014-210

Lipid metabolism enzyme ACSVL3 supports glioblastoma stem cell maintenance and tumorigenicity.

BackgroundTargeting cell metabolism offers promising opportunities for the development of drugs to treat cancer. We previously found that the fatty acyl-CoA synthetase VL3 (ACSVL3) is elevated in malignant brain tumor tissues and involved in tumorigenesis. This study investigates the role of ACSVL3 in the maintenance of glioblastoma multiforme (GBM) stem cell self-renewal and the capacity of GBM stem cells to initiate tumor xenograft formation.MethodsWe examined ACSVL3 expression during differentiation of several GBM stem cell enriched neurosphere cultures. To study the function of ACSVL3, we performed loss-of-function by using small interfering RNAs to target ACSVL3 and examined stem cell marker expression, neurosphere formation and tumor initiation properties.ResultsACSVL3 expression levels were substantially increased in GBM stem cell enriched neurosphere cultures and decreased after differentiation of the neurospheres. Down-regulating ACSVL3 with small inhibiting RNAs decreased the expression of markers and regulators associated with stem cell self-renewal, including CD133, ALDH, Musashi-1 and Sox-2. ACSVL3 knockdown in neurosphere cells led to increased expression of differentiation markers GFAP and Tuj1. Furthermore, ACSVL3 knockdown reduced anchorage-independent neurosphere cell growth, neurosphere-forming capacity as well as self-renewal of these GBM stem cell enriched neurosphere cultures. In vivo studies revealed that ACSVL3 loss-of-function substantially inhibited the ability of neurosphere cells to propagate orthotopic tumor xenografts. A link between ACSVL3 and cancer stem cell phenotype was further established by the findings that ACSVL3 expression was regulated by receptor tyrosine kinase pathways that support GBM stem cell self-renewal and tumor initiation, including EGFR and HGF/c-Met pathways.ConclusionsOur findings indicate that the lipid metabolism enzyme ACSVL3 is involved in GBM stem cell maintenance and the tumor-initiating capacity of GBM stem cell enriched-neurospheres in animals.

Targeting the Cytosolic Innate Immune Receptors RIG-I and MDA5 Effectively Counteracts Cancer Cell Heterogeneity in Glioblastoma

Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.

A Molecular Screening Approach to Identify and Characterize Inhibitors of Glioblastoma Stem Cells

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types.

Pharmacological targeting of the constitutively activated MEK/MAPK-dependent signaling pathway in glioma cells inhibits cell proliferation and migration

Activated mitogen-activated protein kinase MAPK cascade leading to ERK1/2 phosphorylation is expressed in the majority of glial neoplasms and negatively correlates with survival time of patients. Here we show that ERK1/2 kinases are constitutively activated in glioma cell lines and stem cell-enriched primary cultures of glioblastoma. Pharmacological targeting of the activated MEK/ERK1/2 module with the MEK inhibitor U0126 attenuates cell cycle progression (11 out of 11 cell lines), impairs single (7 out of 10) and collective cell migration (9 out of 11) and abolishes single cell emigration from monolayers (4 out of 9). Attacking the activated MEK/ERK1/2 module thus partially blocks the tumorigenic potential of glial cancer cells on different levels and strongly suggests the application of combination molecularly targeted therapies to interfere more efficiently with glial tumor development and progression.

Abstract 4155: The small leucine-rich proteoglycan biglycan localizes to the nucleus of neoplastic cells in glioblastoma biopsies and tumor xenografts

Abstract Global molecular profiling efforts are leading to the identification of prognostic gene expression signatures for gliobastoma. Recently, several extracellular matrix (ECM) components typically expressed in mesenchymal tissues, have been shown to be overexpressed in a subgroup of high grade gliomas from poor prognosis patients, including the highly related small leucine-rich repeat proteoglycans (SLRPs) biglycan and decorin. These proteins were originally isolated from cartilage and are widely expressed in differentiated mesenchymal tissues, but their involvement in glioblastoma pathology is not understood. Biglycan expression in glioblastoma surgical specimens, cancer stem cells, and xenografts was assessed by RNA microarray, real-time PCR, Western blot and immunohistochemistry, and compared to decorin. Over 80% of the glioblastoma surgical specimens expressed both proteoglycans in the pericellular location of endothelial cells. In neoplastic cells, while decorin was observed in the pericellular location of discrete regions of about 40% of the samples, biglycan localized to the nucleus in most tumors analyzed. It has been previously reported that biglycan possess a nuclear localization signal, but in vivo nuclear localization in a glial tumor has not been shown. The unglycosylated core biglycan protein was ubiquitous, while the glycosylated form was less uniformly distributed among the tumor samples. Glioblastoma cancer stem cell enriched neurosphere cultures derived from the tumor specimens did not express biglycan or decorin in vitro. However, biglycan was re-expressed upon tumor formation, as nuclear staining was observed in the orthotopic tumor xenograft initiated by the neurospheres. Using the Rembrandt data base (http://caintegrator-info.nci.nih.gov/rembrandt) we observed a robust inverse correlation of biglycan expression in GBMs with survival. The nuclear localization in GBMs suggests an unconventional role for this proteoglycan in the pathology of this malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4155.

A Hierarchy of Self-Renewing Tumor-Initiating Cell Types in Glioblastoma

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.