Given the poor prognosis of patients with malignant brain cancer, new and innovative approaches are urgently needed to make an impact on this devastating disease. To that end, our research focuses on several areas, with the ultimate aim of conducting translational studies in patients with malignant glioma. Each of these areas is discussed below and aims to develop new therapies that target tumor cells and spare normal and healthy brain cells.
VIROTHERAPY
Oncolytic adenoviruses that are conditionally replicative (CRAds) in tumor cells but not in normal cells represent a novel approach for treating cancer. In this setting, tumor cell infection results in viral replication and ultimately, release of viral progeny thereby facilitating viral spread to neighboring neoplastic cells and enhancing distribution throughout the targeted tumor bed. However, early clinical experience in brain tumor patients treated with first generation CRAds in phase I-II trials has been disappointing; although safe, the approach is limited by (1) poor transduction of brain cancer cells; (2) limited viral distribution and spread beyond the site of injection; and (3) anti-adenoviral immune response which is capable of attenuating the desired therapeutic efficacy. Each of these limitations has been a topic of investigation within our laboratory. In essence, we have approached the problems seen in clinical practice by hypothesis-driven research, which in turn has allowed us to overcome these problems in preclinical models and therefore justify the development of novel therapies for use in future clinical trials. The oncolytic potency of replicating agents is determined by their capability of specifically infecting target cells. Most adenoviruses that have been historically used for gene therapy have been based on serotype 5 (AdWT). Unfortunately, expression of the primary receptor for Ad5 (the coxsackie-adenovirus receptor, CAR) is highly variable on cancer cells. In fact, several studies have demonstrated a resistance of malignant glioma to adenoviral vectors, a finding that was subsequently attributed to the quantitative deficiency of CAR on brain tumor cells. In addition, the lack of well defined tumor-selective promoter (TSP) elements that demonstrate selective transcriptional activity in glioma has hampered the construction of conditionally replicative adenoviral vectors with desired replicative specificity. It is clear that addressing these obstacles is critical to realizing the full therapeutic potential of CRAds for GBM.
To bypass the dependence on CAR for adenoviral entry and replication, we created a new generation of novel adenoviral vectors which utilize transcriptional and transductional control of viral replication. First, we tested a variety of tumor specific promoters and identified survivin (S) as an excellent tumor specific promoter for transcriptional control of E1a, a gene essential for CRAd replication (J Neurosurg 104:583, 2006; Cancer Biol Ther 6:679, 2007). Survivin expression in gliomas is associated with poor prognosis, increased rates of recurrence, and resistance to chemo- and radiotherapy. Second, we have identified several receptors that are over-expressed on brain tumor cells and created a series of pseudotyped Ad5 vectors that recognize these receptors (Mol Cancer Ther 5:2408, 2006; J Gene Med 9:151, 2007; Human Gene Ther, 18:118, 2007; J Neurosurg 107:617, 2007; Cancer Biol Ther 7: 786, 2008; J Med Virol 80:1598, 2008; J Gene Med, 11:1005, 2009). Based on the above date data, we then created a novel oncolytic adenoviral vector which utilizes the survivin promoter and binds to heparan sulfate proteoglycans expressed on malignant brain tumors and named this new vector CRAd-S-pk7 (Hum Gene Ther 18:589, 2007). Our studies with CRAd-S-pk7 indicate that the virus provides the highest level of viral replication and tumor oncolysis in glioma cell lines. At the same time, we observed minimal viral replication and toxicity in normal human brain. Injection of CRAd-S-pk7 inhibited xenograft brain tumor growth by more than 300%. Sixty-seven percent of treated mice with intracranial tumors were long term survivors. To further validate the preclinical efficacy of our novel virus, we have shown that survivin is a new and previously uncharacterized radio-inducible promoter which significantly enhances viral replication and tumor cytotoxicity in the context of glioma stem cells (Cancer Res 68:5778, 2008). Most recently, we have also shown that CRAd-S-pk7 exhibits anti-tumor synergy in conjunction with temozolomide (Br J Cancer, 100:1154, 2009), the chemotherapy agent which represents the standard of care for patients with malignant brain tumors. These findings demonstrate the effectiveness of CRAd-S-pk7 in the context of radiotherapy and chemotherapy and provide the rationale for further development of this novel oncolytic virus for glioma virotherapy. An NIH R01 grant supports the development of novel oncolytic vectors in our laboratory.
STEM CELLS
In addition to advancing the field of adenoviral transduction of brain cancer and viral replication within tumor cells, we have also tackled the problem of poor viral distribution and anti-adenoviral immune response. Previous clinical studies have shown that viral vectors spread only a short distance from initial injection sites and often do not reach sites of distant tumor spread. Due to the infiltrative nature of malignant brain tumors, this drawback is significant. To address it, we have taken advantage of stem cells and their ability to migrate to tumors and areas of injury within the central nervous system. We were the first group in the country to show that human mesenchymal stem cells can be effectively loaded with a replication competent virus and effectively deliver it to an experimental glioma model (Stem Cells, 26:831, 2008). In fact, our early work in this area showed that mesenchymal stem cells demonstrate a 46-fold increase in viral delivery vs. local injection of the virus into the tumor cavity alone. Most recently, we have further expanded on these results to show that virus-loaded neural stem cells also achieve enhanced viral distribution throughout intracranial tumors, a finding that is further associated with reduced tumor growth and increased survival (Gene Ther 16:262, 2009). Of significant importance, the use of stem cells as delivery vehicles for intracranial tumors helps to overcome another limitation of oncolytic virotherapy, specifically the anti-viral immune response. When oncolytic vectors are loaded onto stem cells, the virus effectively "hides" from the immune system for an extended period of time. The ability of stem cells to suppress the anti-viral immune response in a permissive and tumor-bearing animal model is the subject of one of our latest manuscript (Mol Ther, 18:1846, 2010).
Finally, to further enhance the therapeutic efficacy of stem cells, we have optimized them to specifically traffic to intracranial tumors via genetic modification with single-chain antibodies against antigens expressed on gliomas (J Tissue Eng Regen Med, 4:247, 2010). We have then shown that genetic modification of stem cells with a single-chain antibody against EGFRvIII successfully increases the retention of these cells in intracranial tumors, thereby additionally inhibiting tumor growth and prolonging survival (PLoS ONE, 18:5(3):e9750, 2010).
Taken together, these are important findings since enhancing tumor cell transduction and improving viral delivery while at the same time attenuating the anti-adenoviral immune response offer the promise of enhanced clinical efficacy. Our latest work in this area supports the development of neural stem cell based cell carriers for oncolytic virotherapy (Mol Pharm , 8:1559, 2011, Mol Therapy, 19:1714, 2011, JNCI 105:968, 2013). Our collaborator, Dr. Karen Aboody from City of Hope is the first scientist in the world to secure an IND for a neural stem cell line to be used in patients. We have obtained an NIH U01 grant to help complete the FDA directed studies in hopes of securing a new IND for a novel clinical trial that will utilize Dr. Aboody's neural stem cells for the delivery of our oncolytic adenovirus - CRAd-S-pk7 - in the setting of newly diagnosed and recurrent malignant glioma.
To learn more, please see www.multivu.com/players/English/7944251-northwestern-medicine-stem-cell-trial/
IMMUNOTHERAPY
In addition to oncolytic virotherapy, our research interests also extend to the field of brain tumor immunotherapy. The success of brain tumor immunotherapy depends on a delicate interaction between the central nervous system and the immune system. Although historically considered an immune privileged site, increasing evidence suggests that the immune system is finely regulated within the CNS. Understanding the immune response in the central nervous system, with an emphasis on antigen presentation and modulation of the immune response to intracranial tumors, offers an opportunity to advance the field of neuro-immunology and develop novel immunotherapeutic protocols for patients with malignant brain tumors.
Over the past several years, we have focused on the identification and characterization of regulatory T cells (CD4+CD25+FOXP3+, Treg) in malignant brain tumors. While active suppression by Tregs has been shown to play an important role in the down-regulation of T cell responses to foreign and self-antigens in the peripheral immune system, there is limited literature regarding the role of Treg in tumors of the CNS. Our group has been actively investigating the role of Tregs in patients with malignant gliomas and we were one of the first investigative teams to show that (1) there is an increase of Treg both in the blood and local tumor environment of patients with glioma; (2) these glioma infiltrating Treg are functional and immunosuppressive; and (3) they express a number of important immuno-modulatory markers (Neuro-Oncology,8:234, 2006). Subsequently, we have also shown that the degree of Treg infiltration correlates with tumor grade (J Neurooncol 83:145, 2006) and that depletion of Tregs prolongs the survival of animals with experimental brain tumors ( J Neurosurg, 105:430, 2006). These results were also recapitulated in human metastatic brain cancer, where we have shown that Treg infiltration is significantly increased throughout the time of metastatic tumor progression (Int J Oncol 34:1533, 2009). Our mechanistic studies suggest that toll-like receptors participate in Treg function, since CpG stimulation appears to decrease the number of Treg in our glioma model (Glia 54:526, 2006). Progression of intracranial glioma further disrupts thymic homeostasis and our studies suggest the gliomas induce T-cell apoptosis in vivo due to the effects of Notch and its ligand, Jagged-1 (Cancer Immunol Immunother, 57:1807, 2008). Of significant translational value, we have just shown that thymus-derived, rather than tumor-induced regulatory T cells predominate in the context of malignant brain tumors (Neuro-Oncology, 13:1308, 2011) and that IDO appears to increase the recruitment of regulatory T cells in gliomas (Clin Cancer Res 18:6110, 2012). Inhibiting IDO, CTLA-4, and PD-L1 significantly prolongs the survival of animals with brain cancer (Clin Cancer Res, 2014, In press).
These are novel discoveries which have not been previously reported in the context of a CNS malignancy and have important implications for the design of immunotherapeutic strategies for brain cancer. We are currently funded via an NIH R01 to delineate the mechanism of Treg infiltration in malignant brain tumors.
NANOTECHNOLOGY AND NANOMEDICINE
Within the last two years, in response to proposals for collaborative research between the University of Chicago and the Argonne National Laboratory, we have begun two research projects with have led to productive and successful outcomes. In the first, we have focused on light-based nanoplatforms while in the second, novel magnetic nanoparticles.
The ability to integrate the advanced properties of nanoscale materials with the unique recognition capability of biomolecules to achieve active transport, imaging and, potentially, specific elimination of brain cancer makes emerging nanoplatforms interesting for the development of rationally designed modalities in the field of neuro-oncology. The semiconductor TiO2 is well-known as a photocatalyst in the degradation of organic substrates and the deactivation of microorganisms and viruses. Under ultraviolet light (UV) excitation, TiO2 nanoparticles of various sizes and morphologies have been reported to exhibit cytotoxicity toward some tumors. We took advantage of this feature and developed a novel TiO2 nanoparticle covalently tethered to a glioma cell via a polyfunctional linker. For the proof-of-principle, our specific targeting of glioma cells was based on the selective expression of the IL13a2 receptor (IL13a2R) on malignant brain tumors. The linker application enabled absorption of a visible part of solar spectrum by the new nanohybrid. The ensuing glioma phototoxicity was then shown to be mediated via reactive oxygen species that initiated programmed cell death (Nano Lett 9:3337, 2009).
In the last several years, we as well as our collaborators, Dr. Valentyn Novosad and Dr. Elena Rozhkova from Argonne National Laboratory, have also investigated a novel concept of targeted cancer cell destruction by using magnetic vortices as mediators of cellular mechanotransduction. This work was featured on the cover issue of (Nature Materials 9:165, 2010). In it, we have shown that the actuation of the magnetic vortices by an alternating magnetic field leads to oscillatory motion of our novel magnetic nanodisks and transduction of a magneto-mechanical stimulus directly to the cell membrane and into sub-cellular compartments, without damaging surrounding normal cells or tissues. This results in membrane damage, and cellular signal transduction and amplification, causing initiation of cell death. Application of extraordinarily weak magnetic fields produces an extremely high degree of spin-vortex induced cytotoxicity. In fact, in our proof-of-concept experiments, we found that an AC magnetic field with frequency as low as few tens of Hz and as small as tens of Oe applied during only 10 min is sufficient to achieve ~90% cancer cell destruction in vitro.
Since this initial discovery, we have joined forces with Prof. Russell Cowburn at the University of Cambridge. Professor Cowburn is a world-leader in the field of nanoscale magnetism and spintronics and has allowed our group to expand and fine-tune the development of our magnetic nanoparticles. We also collaborate very closely with the laboratory of Dr. Yu Cheng, Professor of Nanotechnology at Tongji University, Shanghai, China, including exchange of students and post-doctoral fellows between our two laboratories. For more information, see our newsclip below!
In addition, we have ongoing projects with Prof. Matthew Tirrell, founding Pritzker Director of the Institute for Molecular Engineering, focusing on development of glioma targeting micelles. We also collaborate with Prof. Wenbin Lin, world authority on the topic of siRNA delivery via nanoparticles, and with Prof. Chuan He, whose expertise in chemical conjugation is allowing us to selectively target malignant brain cancer. This rich group of investigators spanning different areas of expertise is allowing us to approach delivery of nanoparticles to gliomas from multiple angels. Our hope is to translate this approach in clinical trials for patients with brain cancer in the nearby future.
METASTATIC BRAIN CANCER
Although the main focus of our laboratory is the study of primary brain cancer, especially malignant glioma, we have also began work in the area of metastatic breast cancer to the brain. Breast cancer represents one of the most common forms of human cancer and the tremendous morbidity and mortality associated with spread of the disease within the central nervous system requires a dedicated research effort. Specifically, we are interested in examining tumor suppressor genes in order to identify a subset of patients which are at an increased risk for development of brain metastasis. In a recent publication, we have shown that women with breast cancer express a protein called KISS1 (Cancer, 118:2096, 2012). We have shown that patients with breast cancer metastasis lose the expression of KISS1, a finding that is associated with a more advanced stage of the disease as well as shorter metastasis free survival. This work has laid the ground work for our future endeavors which aim to elucidate the mechanism by which cancer cells loose KISS1 in order to metastasize to the CNS.
Although the main focus of our laboratory is the study of primary brain cancer, especially malignant glioma, we have also began work in the area of metastatic breast cancer to the brain. Breast cancer represents one of the most common forms of human cancer and the tremendous morbidity and mortality associated with spread of the disease within the central nervous system requires a dedicated research effort. Specifically, we are interested in examining tumor suppressor genes in order to identify a subset of patients which are at an increased risk for development of brain metastasis. In a recent publication, we have shown that women with breast cancer express a protein called KISS1 (Cancer, 118:2096, 2012). We have shown that patients with breast cancer metastasis lose the expression of KISS1, a finding that is associated with a more advanced stage of the disease as well as shorter metastasis free survival. This work has laid the ground work for our future endeavors which aim to elucidate the mechanism by which cancer cells loose KISS1 in order to metastasize to the CNS.
