INTRODUCTION: Poor outcomes for glioblastoma (GBM) are partly due to the inability to deliver therapeutic agents to the tumor because of the blood brain barrier (BBB) as well as ineffective agents. We have evaluated bone marrow-derived mesenchymal stem cells (MSCs) as transport vehicles of biological therapeutics, particularly Delta-24-RGD (D24), an engineered tumor-selective oncolytic adenovirus that replicates and lyses only in human tumor cells. In mice, we have shown that MSCs loaded with D24 (MSCs-D24) home to GBMs after intra-arterial (IA) carotid infusion, resulting in widespread distribution in the tumor and oncolysis. Intravenous infusion is ineffective, rendering IA delivery the only effective systemic option. To understand the applicability of IA infusions of MSCs-D24 requires a large animal, human brain tumor model recapitulating the challenges of clinical use. METHODS: Rabbits underwent stereotactic xenoimplantation of human GBM cell lines. Tumor creation was confirmed on magnetic resonance imaging (MRI), histologic and immunohistochemistry analysis. Selective internal carotid artery MSCs-D24 infusion ipsilateral to the tumor was performed assess efficacy and safety. RESULTS: We report on successful xenoimplantation of human glioblastoma cell lines (U87, GSC17, and GSC8-11) in 28 immunosuppressed rabbits using Mycophenolate Mofetil, Dexamethasone, and Tacrolimus. The implanted rabbits were followed for 35 days prior to tumor assessment with MRI, angiography, and histological analysis. On MRI, the tumors were hyperintense on T2-weighted image and enhanced (evidence of BBB breakdown). On histological analysis, tumors showed phenotypic traits of human GBM and display varying levels of vascularity, an important feature for testing IA therapy. Selective internal carotid artery MSCs-D24 infusion was safe in the model, and MSCs-D24 homed to the implanted tumor at 24 hours. CONCLUSIONS: The intracranial immunosuppressed rabbit human GBM model allows testing of ESIA infusion of novel therapeutics (eg. MSCs-D24) in a clinically relevant fashion.

21 The brain tumor immune microenvironment (TIME) continuously evolves during glioma 22 progression, but only a limited view of a highly complex glioma associated immune contexture 23 across isocitrate dehydrogenase mutation (IDH) classified gliomas is known. Herein, we present 24 an unprecedentedly comprehensive view of myeloid and lymphoid cell type diversity based on 25 our single cell RNA sequencing and spectral cytometry-based interrogation of tumor-associated 26 leukocytes from fifty-five IDH stratified primary and recurrent human gliomas and three non- 27 glioma brains. Our analyses revealed twenty-two myeloid and lymphoid cell types within and 28 across glioma subtypes. Glioma severity correlated with microglial attrition concomitant with a 29 continuum of invading monocyte-derived microglia-like and macrophages amongst other 30 infiltrating conventional T and NK lymphocytes and unconventional mucosa associated invariant 31 T (MAIT) cells. Specifically, certain microglial and monocyte-derived subpopulations were 32 associated with antigen presentation gene modules, akin to cross-presenting dendritic cells 33 (DCs). Furthermore, we identified phagocytosis and antigen presentation gene modules 34 enriched in Triggering receptor expressed on myeloid (TREM)-2 + cells as a putative anti-glioma 35 axis. Accelerated glioma growth was observed in Trem2 deficient mice implanted with CT2A 36 glioma cells affirming the anti-glioma role of TREM2 + myeloid cells. In addition to providing a 37 comprehensive landscape of glioma-specific immune contexture, our investigations discover 38 TREM2 as a novel immunotherapy target for brain malignancies. naïve) and standard of care treated recurrent glioma subtypes to define their immune landscape. In addition to corroborating established myeloid dominant glioma characteristics, we redefine glioma TIME by superimposing our advanced findings across glioma subtypes with largely IDH-wt glioma restrictive studies 17,18,22-24 . Our major findings include the following: i) We observed significant attrition of microglia (MG) accompanied by increased infiltration of classical monocytes (c-Mo), monocyte-derived-microglia-like (Mo-MG or MG-like), -macrophages (MDM) and conventional dendritic cells (cDC)-2 in recurrent IDH-wild type gliomas relative to other glioma subtypes; ii) We demonstrate eleven transcriptionally distinct glioma associated MG states inclusive of tumoricidal, inflammatory and metabolic phenotypes; ii) Infiltration of Tregs, NK cells and mucosa associated invariant T (MAIT) were significantly abundant in recurrent IDH- wild type gliomas; and iv) We identified glioma associated myeloid cells with triggering receptor 83 expressed on myeloid (TREM)-2 cells enriched for phagocytosis and antigen-presentation gene putative anti-glioma axis and demonstrate their anti-glioma functions using a xenograft mouse model. In summary, our reverse translational glioma immunophenotyping reveal an unprecedently advanced landscape of glioma TIME that can be exploited for future immunotherapy applications. We further uncover TREM2 as a novel glioma specific immunomodulatory target with likely implications in other brain malignancies. Using single cell transcriptomic profiling, our study uncovers the cellular and molecular landscape of brain-specific glioma immunity. We observed that both myeloid and lymphoid subpopulations exhibit remarkable cellular diversity depending on IDH status and disease severity. Recent high throughput studies have elegantly demonstrated the spatio-temporal distribution of microglial subsets in humans and mice 46 . These studies established the plasticity of microglial cell states and reveal mechanisms by which MG contribute to limiting or promoting neurodegenerative diseases 46 . We observed attrition of tissue-resident microglial cells with a concomitant increased infiltration of non-MG myeloid cells as a distinct feature in IDH-wt gliomas, which is consistent with recent reports 30,31 . In our investigation, we even report reduction in MG in relapsed IDH-mutant gliomas and the highest MG attrition evident in IWR gliomas. Acute inflammation induces transient loss of embryonically derived tissue-resident macrophages as a result of necroptosis and concomitant replenishment either through self- renewal or monocytic input as has been described in murine spleen, liver and lungs 37 . In line with this, we speculate migration of bone marrow derived myeloid cells as a compensatory mechanism of homeostatic myelopoiesis to fill depleting brain macrophage niches. Our results showing increased proportion of Mo-MG like cells in response to dramatic reduced MG in IWR gliomas provides a likely clue for such replenishment patterns in human gliomas. Our findings 253 with MG provide evidence for multifaceted inflammatory phenotypes characterized by IL1A, TNF, IL6, IL10 and GNLY expression on various MG subsets. Although recent bulk mRNA-seq analysis pointed to cumulative spectral nature of glioma associated MG 31 , we clarify heterogeneity of polarization states and identify an unreported palmitic acid (PA) responsive and widely acknowledged IL-4 responsive gene modules in distinct clusters of MG. A Glucocorticoid induced signature has been reported with SEPP1 high Mo-TAMS cells a 7.2 Mucosal associated invariant T (MAIT) cells. Statistical significance was determined using by Kruskal Wallis test at p*<0.05, p**<0.01, p***<0.001.

Tumor recurrence following radiation therapy for patients with malignant gliomas leads to nearly universally fatal outcomes. Treatment resistant glioma stem cells are thought to propagate and drive growth of these tumors, but their markers and our ability to target them specifically are not well understood. Transcriptome analyses suggest that expression of the type-I integral membrane glycoprotein podoplanin (PDPN) may be a prognostic marker in astrocytic gliomas. Here, we aim to explore whether PDPN is a glioma stem cell marker and understand its biological significance. Transcriptome and clinical outcome analyses was carried out using standard bioinformatics tools on publically available TCGA datasets. Glioma stem cell cultures were cultured in serum-free media. PDPN shRNA constructs were generated using standard cloning techniques and delivered via lentiviral transduction. Animal injection experiments were performed with a stereotactic injection apparatus and conducted with prior approval by the institutional review board. Glioma stem cell neurosphere formation assays were used to test response to irradiation, which was delivered in 2, 4 or 6 Gy doses prior to the assay. We demonstrate here that PDPN expression is an independent prognostic marker in gliomas across multiple independent cohorts comprising both high- and low-grade gliomas. PDPN expression is prominent in glioma stem cells and correlates with DNA hypermethylation-induced RT resistance. Enrichment analyses of PDPN expressing GSCs revealed that PDPN correlates with polycomb repressive complex 2 (PRC2) of which the catalytic subunit enhancer of zeste 2 (EZH2) controls DNA methylation and has been previously shown to protect GSCs from radiation-induced cell death. PDPN identifies the tumor-initiating, treatment-resistant (TITR) glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

Glioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain. In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

Background Mesenchymal stem cells (MSCs) are being studied for the treatment of several neurological disorders. The use of MSCs as delivery modality for targeted viral therapeutic agents for the treatment of intracranial pathologies has not been explored. Delta-24-RGD, a tumor-selective oncolytic adenovirus designed to target malignant glioma cells, has been shown effective in animal models. However, the most efficient viral tissue delivery modality is unclear. Bone marrow human MSCs (BM-hMSC) have been shown to have homing capability toward glioma xenografts and the feasibility of oncolytic virus particles loaded in MSCs being delivered to the tumor vascular bed via selective intra-arterial (IA) infusion is being investigated. To evaluate the feasibility of endovascular IA delivery, catheter compatibility with MSCs was tested in vitro. Methods BM-hMSCs were cultured, transfected with Delta-24, and re-suspended in 1% Human Serum Albumin. hMSC-Delta-24 solution was then injected via three microcatheters of different inner diameters (Marathon, Echelon-14, and Marksman). Cell count and viability after injection through the microcatheters based on injection velocity and catheter configuration were assessed. Transwell assay was performed with the injected cells to test the Delta-24 activity against U87 glioma cells. hMSC-Delta-24 compatibility was also tested with commonly used medications in neuroendovascular therapy (Omnipaque, verapamil, and heparin). Results BM-hMSC cell count prior to infusion was 0.123 × 106 cells/mL, 98.7% viability. There was no significant difference in cell count after infused through any of the catheters under standard conditions, with a mean concentration of 0.126 × 106 cells/mL and 97.9% (±1.7%) viability. Injection velocity ranged from 1.01 to 73.17 cc/min, with no significant difference in cell count or viability. The same result was seen in either tortuous or straight microcatheter configurations. Cell count and viability did not change significantly when the hMSC in solution was stored for up to 5 hours on ice or mixed with Omnipaque, verapamil, and heparin. Notably, anti-glioma activity was maintained after microcatheter infusion. Conclusions BM-hMSCs are compatible with a wide variety of commonly used neuroendovascular microcatheters and medications. Stem cell viability and viral agent activity do not appear to be affected by catheter configuration or injection velocity. Commercially available microcatheters can be used to deliver IA stem cell neurotherapeutics. Disclosures V. Srinivasan: None. J. Gumin: None. K. Camstra: None. S. Chen: None. J. Johnson: None. F. Lang: None. P. Kan: 2; C; Stryker, Medtronic, Cerenovus.

Temozolomide in combination with cranial radiation therapy after surgery is standard of care treatment for glioblastoma. However, cognitive impairment is a serious and common side effect of chemoradiation which significantly reduces patient quality of life. In the current study, we tested the effects of nasally administered human bone marrow-derived mesenchymal stem cells (hMSC) in restoring cognitive function after chemoradiation. Adult C57BL/6J mice were treated with chemotherapy (33 mg/kg temozolomide) and cranial irradiation (20 Gy delivered to the whole brain in 10 daily fractions) or sham. We administered one million hMSC nasally 5 and 7 days after the last chemoradiation treatment. Twenty-one days after the second hMSC administration, puzzle box test (PBT) and novel object/place recognition test (NOPRT) were performed to measure executive functioning and memory, respectively. After completion of behavioral testing, resting-state functional magnetic resonance imaging (fMRI) was acquired to measure functional connectivity. Chemoradiation decreased executive function and impaired memory, as measured by the PBT and the NOPRT. Additionally, chemoradiation caused a decrease in global functional connectivity in the brain, as shown by resting-state fMRI. Both cognitive function and functional connectivity were restored by nasal hMSC treatment.Our data suggest that treatment with hMSC restores cognitive function and functional brain damage that has occurred as a result of chemoradiation. Nasal hMSC treatment may represent a realistic therapeutic strategy for the treatment of chemoradiation-induced cognitive impairment.

Epidermal growth factor receptor (EGFR) family members play pivotal roles in cell proliferation, differentiation and survival. Overexpression and mutations of EGFRs, or aberrant EGFR signaling are commonly associated with the development of various cancers, where constitutive NF-κB activation is often found to promote the expression of various proteins involved in the proliferation, survival, migration and epithelial-to-mesenchymal transition of cancer cells. However, the mechanism of EGFR-induced NF-κB activation is not fully defined. Here, we used a Bimolecular Fluorescence Complementation-based functional genomics method to perform a high throughput screening and identified TMEM43/LUMA as a critical component in EGFR signaling network, mediating EGFR-induced NF-κB activation. Our data show that EGFR recruits TMEM43 following EGF stimulation. TMEM43 interacts with the scaffold protein CARMA3 and its associating complex to induce downstream NF-κB activation, and plays a critical role in controlling cell survival. TMEM43 deficiency significantly affects colony formation, survival of anoikis-induced cell death, migration and invasion of cancer cells in vitro, as well as tumor progression in vivo. Importantly, higher expression of TMEM43 closely correlates with brain tumor malignancy, and suppression of TMEM43 expression in brain tumor cells inhibited their growth both in vitro and in vivo. Altogether, our studies reveal a crucial link of EGF receptor to NF-κB activation and tumor progression.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAEmerging evidence indicates that host immunity is critical for successful cancer virotherapy. We found that oncolytic adenovirus Delta-24-RGD elicited antiglioma immunity in an immunocompetent murine model. Recent studies suggested that the therapeutic efficacy of immune checkpoint blockade in cancer patients could be enhanced by strategies inducing tumor inflammation. We hypothesized that highly immunogenic adenovirus combined with targeting immune co-stimulator would result in better anti-tumor efficiency. Thus, we used the backbone of Delta-24-RGD to express the mouse OX40 ligand to increase the recognition of tumor-associated antigens by immune cells. The resulting Delta-24-RGDOX adenovirus maintained the oncolytic potency of Delta-24-RGD and efficiently expressed OX40 ligand on infected cells. More importantly, compared to Delta-24-RGD, Delta-24-RGDOX induced higher antiglioma activity in immunocompetent glioma models but not in an immunodeficient model, and accordingly mediated greater lymphocyte infiltration at tumor sites and stronger lymphocyte antitumor activity. While blocking or stimulating immune checkpoints with antibodies currently dominates the clinical applications, our data demonstrate that intratumoral injection of oncolyticviruses carrying immune co-stimulatory ligands may constitute a powerful alternative to the use of antibodies and other strategies targeting immune checkpoints in cancer therapy.Note: This abstract was not presented at the meeting.Citation Format: Hong Jiang, Xuejun Fan, Karen Clise-Dwyer, Laura Bover, Joy Gumin, Kathryn E. Ruisaard, Farah J. Mukheef, Frederick F. Lang, Candelaria Gomez-Manzano, Juan Fueyo. Delta-24-RGDOX: making cancer more “visible” to the immune system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 280. doi:10.1158/1538-7445.AM2015-280

Evidence indicates that human cancers are maintained by a population of cells with stem-like properties called cancer stem cells (CSCs). However, the influence of the surrounding stromal cells on the behavior of the CSCs remains poorly understood. We have recently shown that the micro-environment of human gliomas, the most aggressive human brain tumors, contains both glioma stem cells (GSCs) and cells that resemble human bone marrow-derived mesenchymal stem cells (BM-MSCs), called Glioma Associated-MSCs (GA-MSCs). We have also shown that GA-MSCs generate a cytokine-mediated increase in the growth and self-renewal (clonogenicity) of GSCs. However, other paracrine interactions between GA-MSCs and GSCs have not been fully explored. Recent studies have suggested that nano-sized vesicles, termed exosomes, may contribute to intercellular communication within the tumor niche. Therefore, we hypothesized that GA-MSC-derived exosomes increase the tumorigenicity GSCs. Here we show for the first time that exosomes can be isolated from patient-derived GA-MSCs and that these exosomes contain oncogenic microRNAs. Importantly, in vitro delivery of exosomes isolated from GA-MSCs significantly increased both the proliferation and clonogenicity of GSCs. Furthermore, GSC xenografts, treated with GA-MSC-derived exosomes, in the brains of nude mice resulted in a greater tumor burden and significantly decreased animal survival. Delivery of microRNA from GA-MSC-derived exosomes to GSCs, was enough to alter the gene expression profile resulting in the glioma-enhancing effects described. We conclude that GA-MSC-derived exosomes represent an alternative intercellular communication mechanism for the transfer of specific microRNA, which enhances the aggressive nature of Glioblastoma.

The role of cancer stem cells in tumor formation and tumor heterogeneity is currently one of the most researched topics in cancer biology, and microRNAs likely have functional relevance in regulation of critical genes and parameters implicated in glioma stem cell (GSC) behavior and differentiation. To address this, we investigated global role of microRNA in GSCs, focusing on DICER, which regulates double-stranded RNA processing for microRNA biogenesis. Analysis of data from the Cancer Genome Atlas (TCGA) database suggests that high Dicer expression level is correlated with better prognosis of GBM patients. Gene signatures correlated with DICER expression levels suggest DICER/miRNA mediated mechanisms potentially regulate a multitude of cellular pathways in GBMs. Immunohistochemistry analysis of GBM tissue microarray reveals that of 54 tumor samples, 26 (48%) of GBM patients have low or undetectable levels of DICER1 protein, indicating frequent inactivation in high grade glioma. To characterize this functionally, we utilized various in vitro approaches, including exposure to hypoxia, to characterize the GSC properties after knockdown of DICER (GSCs did not have significant variation in endogenous DICER levels). Using three different GSC lines, we found in all cases with Dicer knockdown resulted in increased proliferation of three independent GSC lines with concomitant decrease in levels of stem cell markers (Sox2, Bmi1 etc.), a phenotype observed even upon exposure to hypoxia, a state that is known to preserve and promote the stem-like cell phenotype. Results from self-renewal assays as well as expression profiling show that GSCs with depleted levels of Dicer lose stem cell characteristics and acquire a progenitor-cell like state. Our results highlight the role of DICER as potential regulators of GSC stem-like versus progenitor-like state.

MIR-491 is commonly co-deleted with its adjacent CDKN2A on chromosome 9p21.3 in glioblastoma multiforme (GBM). However, it is not known whether deletion of MIR-491 is only a passenger event or has an important role. Small-RNA sequencing of samples from GBM patients demonstrated that both mature products of MIR-491 (miR-491-5p and -3p) are downregulated in tumors compared with the normal brain. The integration of GBM data from The Cancer Genome Atlas (TCGA), miRNA target prediction and reporter assays showed that miR-491-5p directly targets EGFR, CDK6 and Bcl-xL, whereas miR-491-3p targets IGFBP2 and CDK6. Functionally, miR-491-3p inhibited glioma cell invasion; overexpression of both miR-491-5p and -3p inhibited proliferation of glioma cell lines and impaired the propagation of glioma stem cells (GSCs), thereby prolonging survival of xenograft mice. Moreover, knockdown of miR-491-5p in primary Ink4a-Arf-null mouse glial progenitor cells exacerbated cell proliferation and invasion. Therefore, MIR-491 is a tumor suppressor gene that, by utilizing both mature forms, coordinately controls the key cancer hallmarks: proliferation, invasion and stem cell propagation.

Although studies have suggested that bone marrow human mesenchymal stem cells (BM-hMSC) may be used as delivery vehicles for cancer therapy, it remains unclear whether BM-hMSCs are capable of targeting cancer stem cells, including glioma stem cells (GSC), which are the tumor-initiating cells responsible for treatment failures. Using standard glioma models, we identify TGF-b as a tumor factor that attracts BM-hMSCs via TGF-b receptors (TGFbR) on BM-hMSCs. Using human and rat GSCs, we then show for the first time that intravascularly administered BM-hMSCs home to GSC-xenografts that express TGF-b. In therapeutic studies, we show that BMhMSCs carrying the oncolytic adenovirus Delta-24-RGD prolonged the survival of TGF-b–secreting GSC xenografts and that the efficacy of this strategy can be abrogated by inhibition of TGFbR on BM-hMSCs. These findings reveal theTGF-b/TGFbRaxis as amediator of the tropismof BM-hMSCs forGSCs and suggest that TGF-b predicts patients in whom BM-hMSC delivery will be effective. Cancer Res; 73(7); 2333–44. 2012 AACR.