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Glioblastoma (GBM) is the most aggressive form of brain cancer with an overall survival (OS) less than 16 months. Glioblastoma associated mesenchymal stem like cells (G-MSC) were identified in human patient samples, and higher presence of these cells in patient samples co-relates with lower overall survival. Our lab and others have also identified these cells in orthotopic GL261 glioblastoma murine models. We hypothesize that infiltration of G-MSC plays a key role in creating the highly immunosuppressive environment seen in GBMs. In order to investigate this, we utilized data from the TCGA database to stratify patients into two groups, showing higher or lower expression of the G-MSC markers (CD73, CD90, CD105). Patients expressing higher levels of G-MSC markers showed higher expression of CD4+ cells, as compared to patients with lower G-MSC marker expression. Further, patients with higher G-MSC markers showed high levels of PTGS2, the gene encoding cyclooxygenase 2 (COX2), a known tumor growth promoting and immunosuppressive molecule. We further investigated CD4+ T cell infiltration using GL261 orthotopic implants and observed that CD4+ T cells positively corelated with levels of G-MSC in these tumors. Our studies indicate that levels of G-MSC co-relate with immune cell infiltration and the expression of immunosuppressive factors such as COX2, underlining the importance of these cells in immunosuppression-driven resistance to therapy. These studies will be later utilized to develop rationale combination therapies to improve the overall survival in GBM.

Purpose: Survivin is an inhibitor of apoptosis protein (IAP) that is highly expressed in many cancers and represents an attractive molecule for targeted cancer therapy. Although primarily regarded as an intracellular protein with diverse actions, survivin has also been identified in association with circulating tumor exosomes. Experimental Design: We have reported that active, specific vaccination with a long peptide survivin immunogen leads to the development of survivin-specific CD8-mediated tumor cell lysis and prolongation of survival in tumor-bearing mice. In addition to cellular antitumor responses, circulating anti-survivin antibodies are detected in the serum of mice and human glioblastoma patients following vaccination with the survivin immunogen. Results: Here we demonstrate that survivin is present on the outer cell membrane of a wide variety of cancer cell types, including both murine and human glioma cells. In addition, antibodies to survivin that are derived from the immunogen display antitumor activity against murine GL261 gliomas in both flank and intracranial tumor models and against B16 melanoma as well. Conclusions: In addition to immunogen-induced, CD8-mediated tumor cell lysis, antibodies to the survivin immunogen have antitumor activity in vivo. Cell-surface survivin could provide a specific target for antibody-mediated tumor immunotherapeutic approaches. Clin Cancer Res; 24(11); 2642–52. ©2018 AACR.

The Inhibitor of Apoptosis Protein survivin (svn) is upregulated in nearly all types of cancer and represents a promising therapeutic target. Localization to specific subcellular compartments and interactions with various binding partners allow survivin to play diverse roles in apoptosis resistance and mitosis. Survivin has recently been found in two extracellular compartments: the outer plasma membrane and secreted exosomes. In addition to svn-wt, splice variants svn-dEX3 and svn-2B are also overexpressed in human tumors. Here we show that, similarly to svn-wt, svn-dEX3 and svn-2B can be displayed on the outer plasma membrane, and secreted in exosomes. Additionally, we have identified a novel interaction of all three forms of survivin with secreted tubulin.

Glioblastoma (GBM) is an aggressive brain cancer, with an overall survival of 14.6 months. The tumor microenvironment in GBM plays major roles in immunosuppression and modulation of the response to therapies. GBM patients with higher levels of mesenchymal stem like cells (G-MSC) show poor overall survival as compared to patients with no/lower G-MSC levels. Our lab found that levels of G-MSC corelate with CD4+ T cells in humans and murine models of GBM, and with immunosuppressive molecules like PTGS2, the gene for cyclooxygenase 2. To investigate the mechanism by which G-MSCs promote immunosuppression, we isolated G-MSCs from an orthotopic mouse model of GBM and subjected them to RNASeq analysis to obtain an unbiased picture of transcriptomic changes occurring upon activation. We identified changes in multiple immune modulating pathways involving antigen presentation, leukocyte migration and activation, and immune checkpoints. Our findings indicate that G-MSCs represent a key immune modulating faction in the microenvironment. Further dissection of the role of these cells in immune modulation will aid us in understanding the biology of the brain tumor microenvironment and identifying potential combination therapies.

Historically, intra-arterial (IA) drug administration for malignant brain tumors including glioblastoma multiforme (GBM) was performed as an attempt to improve drug delivery. With the advent of percutaneous neuorovascular techniques and modern microcatheters, intracranial drug delivery is readily feasible; however, the question remains whether IA administration is safe and more effective compared to other delivery modalities such as intravenous (IV) or oral administrations. Preclinical large animal models allow for comparisons between treatment routes and to test novel agents, but can be expensive and difficult to generate large numbers and rapid results. Accordingly, we developed a murine model of IA drug delivery for GBM that is reproducible with clear readouts of tumor response and neurotoxicities. Herein, we describe a novel mouse model of IA drug delivery accessing the internal carotid artery (ICA) to treat ipsilateral implanted GBM tumors that is consistent and reproducible with minimal experience. The intent of establishing this unique platform is to efficiently interrogate targeted anti-tumor agents that may be designed to take advantage of a directed, regional therapy approach for brain tumors.

Background The survival rate for patients with glioblastoma (GBM) remains dismal. New therapies targeting molecular pathways dysregulated in GBM are needed. One such clinical-stage drug candidate, CBL0137, is a curaxin, small molecules which simultaneously downregulate nuclear factor-kappaB (NF-ĸB) and activate p53 by inactivating the chromatin remodeling complex, Facilitates Chromatin Transcription (FACT). Methods We used publicly available databases to establish levels of FACT subunit expression in GBM. In vitro, we evaluated the toxicity and effect of CBL0137 on FACT, p53, and NF-ĸB on U87MG and A1207 human GBM cells. In vivo, we implanted the cells orthotopically in nude mice and administered CBL0137 in various dosing regimens to assess brain and tumor accumulation of CBL0137, its effect on tumor cell proliferation and apoptosis, and on survival of mice with and without temozolomide (TMZ). Results FACT subunit expression was elevated in GBM compared with normal brain. CBL0137 induced loss of chromatin-unbound FACT, activated p53, inhibited NF-ĸB-dependent transcription, and was toxic to GBM cells. The drug penetrated the blood-brain barrier and accumulated in orthotopic tumors significantly more than normal brain tissue. It increased apoptosis and suppressed proliferation in both U87MG and A1207 tumors. Intravenous administration of CBL0137 significantly increased survival in models of early- through late-stage TMZ-responsive and -resistant GBM, with a trend toward significantly increasing the effect of TMZ in TMZ-responsive U87MG tumors. Conclusion CBL0137 targets GBM according to its proposed mechanism of action, crosses the blood-brain barrier, and is efficacious in both TMZ-responsive and -resistant orthotopic models, making it an attractive new therapy for GBM.

Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides with predicted binding to mouse H2-K(b) and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-K(b)-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were screened by flow cytometry for binding of dimeric H2-K(b):Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN(57-64) and SVN(82-89)) generated specific CD8+ T cells. We chose to focus on the SVN(57-64) peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN(53-67)), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell help. We tested the SVN(53-67) 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a significant prolongation of survival. The SVN(53-67) vaccine was significantly more effective than the SVN(57-64) core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated cytokine support within a single peptide.

Osteopontin (OPN) is a pleotrophic molecule that has been associated with multiple disorders of the central nervous system (CNS). Its roles in CNS malignancy are unclear but suggest that higher levels of OPN expression correlate with increased tumor grade and increased migratory capacity of tumor cells. In this study OPN cDNA was cloned into a retroviral vector and used to infect F98 Fischer rat-derived glioma cells and U87 human-derived glioblastoma multiforme (GBM) cells in vitro. Cells expressing high levels of OPN migrated less distance than control cells in vitro. This effect was not RGD mediated, but was reversed in the presence of c-Jun N-terminal kinase (JNK) inhibitor suggesting that JNK1 is an essential component of a negative feedback loop affecting OPN activated signaling cascades. Implantation of tumor cells expressing high levels of OPN into adult Fischer rats and nude rats resulted in morphologically distinct tumors and prolonged host survival relative to controls. We propose that local produced, high level OPN expression limits the malignant character of glioma cells and that the downstream mechanisms involved represent pathways that may have therapeutic value in the treatment of human CNS malignancy.

Survivin is a member of the inhibitor of apoptosis protein family. Gliomas and many other tumors express survivin at high levels; whereas, normal fully differentiated cells generally do not. Therefore, survivin represents a tumor-specific target for cancer vaccine therapy. It has been shown that it is possible to produce a MHC-I-restricted cellular immunologic response to survivin vaccines. To study differences in immunogenicity between murine and human survivin proteins, we vaccinated C57BL/6 mice with bone marrow dendritic cells (BMDC) transfected with expression vectors containing the murine and human survivin genes. Mice vaccinated with BMDCs expressing a truncated human survivin protein developed cytotoxic T lymphocyte to subcutaneous GL261 glioma cells and exhibited prolonged tumor-free survival compared to mice vaccinated with BMDCs transfected with vector alone (P

The role that transforming growth factor beta1 (TGF-beta1) plays in influencing growth of glioma cells is somewhat controversial. To further understand the potential growth-regulatory effects of TGF-beta1,we constructed an animal astroglial tumor model by injecting either wild-type or virally transduced human U-87 glioblastoma cells into nude rat brains. Wild type U-87 cells produced very low amounts of TGF-beta1 and were highly tumorigenic. In contrast, U-87 cells transduced to express high levels of TGF-beta1 showed reduced tumor size in vivo, in a dose-dependent manner. This reduction in tumor size was not due to either decreased vascularity or increased apoptosis. To test whether TGF-beta1 overproduction inhibited tumor growth through an autocrine mechanism, the highest TGF-beta1 producing cells were then double transduced with a vector expressing the kinase-truncated type II TGF-beta receptor. Cells expressing high levels of truncated TGF-beta receptor were less sensitive to TGF-beta1 mediated growth inhibition in vitro and produced more aggressive tumors in vivo. The data suggest that the degree of tumorigenicity of the U-87 high-grade glioblastoma cell line may be associated with correspondingly low level of production of TGF-beta1. These results also would tend to support the possibility that TGF-beta1 may be useful in treating some high-grade gliomas.

The mammalian brain contains neural stem cells (NSCs) that allow continued neurogenesis throughout the life of the animal. However, neurogenesis is known to decline during aging and, to the extent that neurogenesis is required for normal CNS function, this may contribute to neurodegenerative disease. Decreased neurogenesis could result from loss of NSCs or dysfunction at some later step, and distinguishing these possibilities is important for understanding the cause of the decline. However, because of the inability to distinguish NSCs from their rapidly dividing progenyin situ, it has not been possible to quantitatively assess the NSC populations in young and old animals. In this report we show that the G1 phase-specific expression of the replication factor Mcm2 is a useful marker for detecting slowly cycling putative NSCsin situand confirm the identity of these cells using both cytosine β-d-arabinofuranoside (Ara-C) treatment and a double nucleoside analog-labeling technique. The ability to distinguish NSCs from proliferative progenitors has allowed characterization of the expression of several markers including Nestin, Musashi, and GFAP in these different cell types. Furthermore, comparison of the NSC populations in the subventricular zones of young (2-4 months) and old (24-26 months) mice demonstrates an approximately twofold reduction in the older mice. A similar twofold reduction is also observed in the number of neurospheres recovered in culture from old relative to young animals. The reduction in the neural stem cell population documented here is sufficient to account for the reduced level of neurogenesis in old animals.

An experimental animal model of meningeal leukemia was developed in the nude rat, rnu/rnu, using the human-derived acute lymphoblastic leukemia cell line HPB-ALL. Anesthetized rats were placed in a modified stereotaxic frame and then injected intrathecally, at the level of the cisterna magna, with human leukemic cells. Cerebrospinal fluid and tissue samples from brain, spinal cord, heart, liver, kidney, spleen, bone marrow, and cervical lymph nodes were subjected to histopathologic examination and molecular genetic screening by clonotype primer-directed polymerase chain reaction (CPD-PCR). Ninety-three percent of animals (n = 14) developed signs of meningeal irritation leading to death 30 to 63 days postinjection (median, 36.0 days, mean, 38.7); death occurred between 30 and 39 days in 77% of all animals. Leukemic cells progressively infiltrated the pericerebellar and pericerebral subarachnoid space and infiltrated the Virchow-Robin (perivascular) space. The infiltrating meningeal leukemia closely resembled the pathologic presentation in the human condition. By CPD-PCR, leukemic cells were first detected in cerebrospinal fluid (CSF ) on day 4 postinjection, were variably present over the ensuing 17 days, and were consistently detected after day 21. At terminal stages, CPD-PCR tissue surveys showed leukemic DNA in all brains and spinal cords and rarely in cervical lymph nodes, but leukemic DNA was not detected in any other tissue screened. Leukemic meningitis was reliably produced with a predictable survival time. Intrathecal administration of leukemic cells was an efficient means of transmitting leukemic meningitis and it compartmentalized the disease to the central nervous system (CNS), eliminating potential complications of systemic illness. The use of human-derived cell lines may render this model more relevant to the development of future therapeutic strategies to treat leukemia and lymphoma that invade the CNS.

To study the neuropathology and selected tumour markers of malignant gliomas, an animal glioma model was developed using the implantation of U87 human glioblastoma cells into chick chorioallantoic membrane. The immunohistochemical characteristics were studied and compared with an orthotopic rodent model.The U87 cell suspension was inoculated onto the chick chorioallantoic membrane on embryonic day seven and into the brain of nude rats. Brain tumour sections were examined for various known tumour markers by routine haematoxylin and eosin staining and immunohistochemical analyses.The immunohistochemical analyses showed that S100 protein, glial fibrillary acidic protein and synaptophysin expressions, initially present in tissue culture, were lost in both models. Persistent kallikrein, CD68 and vimentin expressions in U87 cells, as well as in both animal tumour models, were detected. The percentage of p53-positive nuclei, which was higher in the tumours grown on the chick chorioallantoic membrane than in rats, did not correlate with the Ki-67 labelling index. Strong cathepsin expression was maintained from the cell culture to both tumour models. CD3-positive cells and numerous leukocytes, but no CD20-positive cells were detected in any of the animal samples, indicating the immunological response of the host to be primarily cellular. Stronger immune reaction for vascular endothelial growth factor in rats correlated with an observed increase in vascular proliferation in these tumours.A simple, fast-growing, cheap and well-defined chick chorioallantoic membrane model of glioma was established, providing a basis for further experimental studies of genetic and protein expression during human glioma tumourigenesis. This model may possibly replace some rodent models for selective studies.

GsMTx4, a peptide inhibitor for mechanosensitive ion channels (MSCs), promoted neurite outgrowth from PC12 cells in the presence of NGF in a dose-dependent manner between 5 and 100 microM peptide. Enhanced neurite growth required12 h of peptide exposure in cells grown with NGF. Adsorption of GsMTx4 to serum proteins in the media lowered the free peptide concentration of 100 microM to a free concentration of 5 microM, a concentration shown to completely inhibit MSCs in the patch clamp assay. Outside-out patches from PC12 cells grown in NGF had mechanically activated cation channels that were reversibly inhibited by GsMTx4. These results are similar to those observed by Gomez and co-workers in Xenopus spinal cord. The inhibition of mechanosensitive channels by GsMTx4 may be a useful approach to accelerate regeneration of neurons in neurodegenerative diseases and spinal cord injury.

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30-50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.