Human Brain Tumor Initiating Cells Research Articles

A NR2E1-interacting peptide of LSD1 inhibits the proliferation of brain tumour initiating cells.

Elimination of brain tumour initiating cells (BTICs) is important for the good prognosis of malignant brain tumour treatment. To develop a novel strategy targeting BTICs, we studied NR2E1(TLX) involved self-renewal mechanism of BTICs and explored the intervention means. NR2E1 and its interacting protein-LSD1 in BTICs were studied by gene interference combined with cell growth, tumour sphere formation, co-immunoprecipitation and chromatin immunoprecipitation assays. NR2E1 interacting peptide of LSD1 was identified by Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) and analysed by in vitro functional assays. The in vivo function of the peptide was examined with intracranial mouse model by transplanting patient-derived BTICs. We found NR2E1 recruits LSD1, a lysine demethylase, to demethylate mono- and di-methylated histone 3 Lys4 (H3K4me/me2) at the Pten promoter and repress its expression, thereby promoting BTIC proliferation. Using Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides, LSD1-197-211 that locates at the LSD1 SWIRM domain, strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTICs can inhibit intracranial tumour formation. Peptide LSD1-197-211 can repress BTICs by interfering the synergistic function of NR2E1 and LSD1 and may be a promising lead peptide for brain tumour therapy in future.

TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF).

One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.

Cx43 in Neural Progenitors Promotes Glioma Invasion in a 3D Culture System.

The environment that envelops the cancer cells intimately affects the malignancy of human cancers. In the case of glioma, an aggressive adult brain cancer, its high rate of recurrence after total resection is responsible for a poor prognosis. Connexin43 (Cx43) is a gap junction protein with a prominent presence in glioma-associated normal brain cells, specifically in the reactive astrocytes. We previously demonstrated that elimination of Cx43 in these astrocytes reduces glioma invasion in a syngeneic mouse model. To further our investigation in human glioma cells, we developed a scaffold-free 3D platform that takes into account both the tumor and its interaction with the surrounding tissue. Using cell-tracking dyes and 3D laser scanning confocal microscopy, we now report that the elimination of Cx43 protein in neural progenitor spheroids reduced the invasiveness of human brain tumor-initiating cells, confirming our earlier observation in an intact mouse brain. By investigating the glioma invasion in a defined multicellular system with a tumor boundary that mimics the intact brain environment, our findings strengthen Cx43 as a candidate target for glioma control.

Demeclocycline Reduces the Growth of Human Brain Tumor-Initiating Cells: Direct Activity and Through Monocytes.

Myeloid cells that infiltrate into brain tumors are deactivated or exploited by the tumor cells. We previously demonstrated that compromised microglia, monocytes, and macrophages in malignant gliomas could be reactivated by amphotericin-B to contain the growth of brain tumorinitiating cells (BTICs). We identified meclocycline as another activator of microglia, so we sought to test whether its better-tolerated derivative, demeclocycline, also stimulates monocytes to restrict BTIC growth. Monocytes were selected for study as they would be exposed to demeclocycline in the circulation prior to entry into brain tumors to become macrophages. We found that demeclocycline increased the activity of monocytes in culture, as determined by tumor necrosis factor-α production and chemotactic capacity. The conditioned medium of demeclocycline-stimulated monocytes attenuated the growth of BTICs generated from human glioblastoma resections, as evaluated using neurosphere and alamarBlue assays, and cell counts. Demeclocycline also had direct effects in reducing BTIC growth. A global gene expression screen identified several genes, such as DNA damage inducible transcript 4, frizzled class receptor 5 and reactive oxygen species modulator 1, as potential regulators of demeclocycline-mediated BTIC growth reduction. Amongst several tetracycline derivatives, only demeclocycline directly reduced BTIC growth. In summary, we have identified demeclocycline as a novel inhibitor of the growth of BTICs, through direct effect and through indirect stimulation of monocytes. Demeclocycline is a candidate to reactivate compromised immune cells to improve the prognosis of patients with gliomas.

Microglia induces Gas1 expression in human brain tumor-initiating cells to reduce tumorigenecity

We reported previously that microglia decreased the growth of human brain tumor-initiating cells (BTICs). Through microarray analyses of BTICs exposed in vitro to microglia, we found the induction of several genes ascribed to have roles in cell cycle arrest, reduced cell proliferation and differentiation. Herein, we tested the hypothesis that one of these genes, growth arrest specific 1 (Gas1), is a novel growth reduction factor that is induced in BTICs by microglia. We found that microglia increased the expression of Gas1 transcript and protein in glioblastoma patient-derived BTIC lines. Using neurosphere assay we show that RNAi-induced reduction of Gas1 expression in BTICs blunted the microglia-mediated BTIC growth reduction. The role of Gas1 in mediating BTIC growth arrest was further validated using orthotopic brain xenografts in mice. When microglia-induced Gas1-expressing BTIC cells (mGas1-BTICs) were implanted intra-cranially in mice, tumor growth was markedly decreased; this was mirrored in the remarkable increase in survival of mGas1-BT025 and mGas1-BT048 implanted mice, compared to mice implanted with non-microglia-exposed BTIC cells. In conclusion, this study has identified Gas1 as a novel factor and mechanism through which microglia arrest the growth of BTICs for anti-tumor property.

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.

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.

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.

Abstract 2512: Bmi1 identifies treatment-refractory stem cells in human glioblastoma

Abstract Glioblastoma (GBM) is an aggressive and fatal primary adult brain tumor. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence. Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We also adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GFP+ GBM cells. Post-chemoradiotherapy, GFP+ cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. To understand the consequences of Bmi1 dysregulation on target gene expression, we performed global RNA-seq profiling on NSPCs and GBMs. GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 protein expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. Conversely, over-expressing Bmi1 in NSPCs induced stem cell properties in vitro, but failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence. Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence. Citation Format: Parvez Vora, Maleeha Qazi, Chitra Venugopal, Minomi Subapanditha, Sujeivan Mahendram, Chirayu Chokshi, Mohini Singh, David Bakhshinyan, Nicole McFarlane, Sheila Singh. Bmi1 identifies treatment-refractory stem cells in human glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2512.

ATPS-92Bmi1 IDENTIFIES TREATMENT-REFRACTORY STEM CELLS IN HUMAN GLIOBLASTOMA

PURPOSE: Glioblastoma (GBM) is a fatal primary adult brain tumor, characterized by extensive cellular heterogeneity. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of genes regulating BTIC self-renewal drives GBM recurrence. METHODS: Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We then adapted the existing treatment protocol for adults with primary GBM for treatment of immunocompromised mice engrafted with GFP+ human GBM cells. Post-chemoradiotherapy, GFP+ tumor cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo loss-of-function experiments, we investigated the regulatory functions of key BTIC genes in tumor formation. RESULTS: GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy in vitro, suggesting the presence of a treatment-refractory BTIC population. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. CONCLUSION: Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence.

In vitro screen of a small molecule inhibitor drug library identifies multiple compounds that synergize with oncolytic myxoma virus against human brain tumor-initiating cells.

Brain tumor-initiating cells (BTICs) are stem-like cells hypothesized to form a disease reservoir that mediates tumor recurrence in high-grade gliomas. Oncolytic virotherapy uses replication-competent viruses to target and kill malignant cells and has been evaluated in clinic for glioma therapy with limited results. Myxoma virus (MyxV) is a safe and highly effective oncolytic virus (OV) in conventional glioma models but, as seen with other OVs, is only modestly effective for patient-derived BTICs. The objective of this study was to determine whether MyxV treatment against human BTICs could be improved by combining chemotherapeutics and virotherapy. A 73-compound library of drug candidates in clinical use or preclinical development was screened to identify compounds that sensitize human BTICs to MyxV treatment in vitro, and synergy was evaluated mathematically in lead compounds using Chou-Talalay analyses. The effects of combination therapy on viral gene expression and viral replication were also assessed. Eleven compounds that enhance MyxV efficacy were identified, and 6 were shown to synergize with the virus using Chou-Talalay analyses. Four of the synergistic compounds were shown to significantly increase viral gene expression, indicating a potential mechanism for synergy. Three highly synergistic compounds (axitinib, a VEGFR inhibitor; rofecoxib, a cyclooxygenase-2 inhibitor; and pemetrexed, a folate anti-metabolite) belong to classes of compounds that have not been previously shown to synergize with oncolytic viruses in vitro. This study has identified multiple novel drug candidates that synergistically improve MyxV efficacy in a preclinical BTIC glioma model.

Treating brain tumor-initiating cells using a combination of myxoma virus and rapamycin

Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor-initiating cells (BTICs). We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts. The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing "advanced" BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo. Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.

Parvovirus H-1 (H-1PV) exerts oncolytic effects in cell culture models of human brain tumor-initiating cells

Background: Oncolytic virotherapy with H-1PV represents a new therapeutic option for malignant brain tumors currently under clinical investigation in a phase I/IIa H-1PV trial on primary progressive or recurrent glioblastoma. Brain tumor-initiating cells such as glioblastoma „stem-like” cells or medulloblastoma-initiating cells are known to be relatively resistant to conventional anti-neoplastic agents and are thought to give rise to clinical tumor recurrence after standard treatment. This prompted us to address the question whether H-1PV is able to eliminate brain tumor-initiating cells in vitro.

Efficacy and Safety/Toxicity Study of Recombinant Vaccinia Virus JX-594 in Two Immunocompetent Animal Models of Glioma

The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered “cured” (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSVΔM51. Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

Abstract 4283: Myxoma virotherapy targeting temozolomide-resistant brain tumor initiating cells in vitro and in vivo

Abstract Introduction: Malignant gliomas (MGs) still has a poor prognosis and remain largely incurable because their extremely invasive nature, even with significant improvements in surgery, chemotherapy and radiotherapy. Although temozolomide (TMZ) improved the survival of some GBM patients, but recently TMZ resistant became a significantly problem. So new treatments could overcome the TMZ resistant desperately needed. Recently, cells with stem cell qualities have been identified in brain malignancies and have demonstrated brain tumor initiating cells (BTICs) responsible for the initiation of brain tumors and recurrence, as well as chemotherapy and radiotherapy resistant. Therefore, to development of new therapeutic strategies targeting BTICs may provide more effective approach. Oncolytic viruses may represent an effective therapeutic approach to target BTICs because their unique and multiple mechanism of action. We have previously reported that Myxoma Virus (MYXV) “cures” most intracranial brain tumors models in CD-1 nude mice, and significantly improved the efficacy in immunocompetent hosts when combined with rapamycin. Here we further investigated the oncolytic potential of MYXV, alone or in combination with rapamycin in vitro and in animal models established using human BTICs, special for TMZ resistant BTIC lines. Experimental procedure: First, we isolated and cultured BTICs from fresh operative glioblastoma (GBM) specimens, and these cells have all the characteristic of BTICs and form tumors in vivo that very closely resemble human infiltrative GBM. Then, we test the sensitivity to TMZ and we used TMZ resistant BTICs to test the susceptibility to MYXV infection in vitro and in vivo. Results: We found that: 1) All we tested TMZ resistant BTICs susceptible to MV infection and killing in vitro, 2) MYXV replication within TMZ resistant BTICs tumor of the brain and intratumoral administration of MYXV significantly prolongs survival of SCID mice bearing invasive BTICs tumor. 3) And more importantly we found that MYXV Combination treatment with rapamycin further improved the efficacy, even significantly prolongs survival of SCID mice bearing “late-stage” invasive and TMZ resistant BTICs tumor, and this result further confirmed using MRI and bioluminescence image before and after treatment. Conclusion: Our results show for the first time that MYXV alone or in combination treatment with rapamycin significantly inhibited tumor growth and prolongs survival in TMZ resistant BTICs bearing SCID mice models. These observations suggest that MYXV alone or in combination therapy with rapamycin could be an effective therapy for extremely infiltrative GBM, as well as the important TMZ adjuvant therapy for GBM in the future. 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 4283.