Viveka Nand Yadav

Mechanisms of Glioma Formation: Iterative Perivascular Glioma Growth and Invasion Leads to Tumor Progression, VEGF-Independent Vascularization, and Resistance to Antiangiogenic Therapy

As glioma cells infiltrate the brain they become associated with various microanatomic brain structures such as blood vessels, white matter tracts, and brain parenchyma. How these distinct invasion patterns coordinate tumor growth and influence clinical outcomes remain poorly understood. We have investigated how perivascular growth affects glioma growth patterning and response to antiangiogenic therapy within the highly vascularized brain. Orthotopically implanted rodent and human glioma cells are shown to commonly invade and proliferate within brain perivascular space. This form of brain tumor growth and invasion is also shown to characterize de novo generated endogenous mouse brain tumors, biopsies of primary human glioblastoma (GBM), and peripheral cancer metastasis to the human brain. Perivascularly invading brain tumors become vascularized by normal brain microvessels as individual glioma cells use perivascular space as a conduit for tumor invasion. Agent-based computational modeling recapitulated biological perivascular glioma growth without the need for neoangiogenesis. We tested the requirement for neoangiogenesis in perivascular glioma by treating animals with angiogenesis inhibitors bevacizumab and DC101. These inhibitors induced the expected vessel normalization, yet failed to reduce tumor growth or improve survival of mice bearing orthotopic or endogenous gliomas while exacerbating brain tumor invasion. Our results provide compelling experimental evidence in support of the recently described failure of clinically used antiangiogenics to extend the overall survival of human GBM patients.

Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity

Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the β-galactoside-binding lectin galectin-1. Conversely, galectin-1-deficient glioma cells could be eradicated by host NK cells before the initiation of an antitumor T-cell response. In vitro experiments demonstrated that galectin-1-deficient GL26-Cit glioma cells are ∼3-fold more sensitive to NK-mediated tumor lysis than galectin-1-expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells. Cancer Res; 74(18); 5079-90. ©2014 AACR.

CXCR4 increases in-vivo glioma perivascular invasion, and reduces radiation induced apoptosis: A genetic knockdown study.

Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro, inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.

Survival and Proliferation of Neural Progenitor–Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism

Purpose: One likely cause of treatment failure in glioblastoma is the persistence of glioma stem-like cells (GSLCs) which are highly resistant to therapies currently employed. We found that CXCL12 has highest expression in glioma cells derived from neural progenitor cells (NPC). The development and molecular signature of NPC-derived glioblastomas were analyzed and the therapeutic effect of blocking CXCL12 was tested. Experimental Design: Tumors were induced by injecting DNA into the lateral ventricle of neonatal mice, using the Sleeping Beauty transposase method. Histology and expression of GSLC markers were analyzed during disease progression. Survival upon treatment with pharmacologic (plerixafor) or genetic inhibition of CXCR4 was analyzed. Primary neurospheres were generated and analyzed for proliferation, apoptosis, and expression of proteins regulating survival and cell-cycle progression. Results: Tumors induced from NPCs display histologic features of human glioblastoma and express markers of GSLC. In vivo, inhibiting the CXCL12/CXCR4 signaling axis results in increased survival of tumor-bearing animals. In vitro, CXCR4 blockade induces apoptosis and inhibits cell-cycle progression, downregulates molecules regulating survival and proliferation, and also blocks the hypoxic induction of HIF-1α and CXCL12. Exogenous administration of CXCL12 rescues the drug-induced decrease in proliferation. Conclusions: This study demonstrates that the CXCL12/CXCR4 axis operates in glioblastoma cells under hypoxic stress via an autocrine-positive feedback mechanism, which promotes survival and cell-cycle progression. Our study brings new mechanistic insight and encourages further exploration of the use of drugs blocking CXCL12 as adjuvant agents to target hypoxia-induced glioblastoma progression, prevent resistance to treatment, and recurrence of the disease. Clin Cancer Res; 23(5); 1250–62. ©2016 AACR.

Single vs. combination immunotherapeutic strategies for glioma

ABSTRACT Introduction: Malignant gliomas are highly invasive tumors, associated with a dismal survival rate despite standard of care, which includes surgical resection, radiotherapy and chemotherapy with temozolomide (TMZ). Precision immunotherapies or combinations of immunotherapies that target unique tumor-specific features may substantially improve upon existing treatments. Areas covered: Clinical trials of single immunotherapies have shown therapeutic potential in high-grade glioma patients, and emerging preclinical studies indicate that combinations of immunotherapies may be more effective than monotherapies. In this review, the authors discuss emerging combinations of immunotherapies and compare efficacy of single vs. combined therapies tested in preclinical brain tumor models. Expert opinion: Malignant gliomas are characterized by a number of factors which may limit the success of single immunotherapies including inter-tumor and intra-tumor heterogeneity, intrinsic resistance to traditional therapies, immunosuppression, and immune selection for tumor cells with low antigenicity. Combination of therapies which target multiple aspects of tumor physiology are likely to be more effective than single therapies. While a limited number of combination immunotherapies are described which are currently being tested in preclinical and clinical studies, the field is expanding at an astounding rate, and endless combinations remain open for exploration.

Ndfip1 Regulates Itch Ligase Activity and Airway Inflammation via UbcH7

The ubiquitin-ligating enzyme (E3) Itch plays a crucial role in the regulation of inflammation, and Itch deficiency leads to severe airway inflammation. However, the molecular mechanisms by which Itch function is regulated remain elusive. In this study, we found that nontypeable Haemophilus influenzae induces the association of Itch with Ndfip1. Both Itch−/− and Ndfip1−/− mice exhibited severe airway inflammation in response to nontypeable Haemophilus influenza, which was associated with elevated expression of proinflammatory cytokines. Ndfip1 enhanced Itch ligase activity and facilitated Itch-mediated Tak1 ubiquitination. Mechanistically, Ndfip1 facilitated recruitment of ubiquitin-conjugating enzyme (E2) UbcH7 to Itch. The N-terminal region of Ndfip1 binds to UbcH7, whereas the PY motif binds to Itch. Hence, Ndfip1 acts as an adaptor for UbcH7 and Itch. Reconstitution of full-length Ndfip1 but not the mutants that fail to interact with either UbcH7 or Itch, restored the defect in Tak1 ubiquitination and inhibited elevated proinflammatory cytokine expression by Ndfip1−/− cells. These results provide new mechanistic insights into how Itch function is regulated during inflammatory signaling, which could be exploited therapeutically in inflammatory diseases.

Molecular ablation of tumor blood vessels inhibits therapeutic effects of radiation and bevacizumab

Background Glioblastoma (GBM) is an aggressive and highly vascular tumor with median survival below 2 years. Despite advances in surgery, radiotherapy, and chemotherapy, survival has improved modestly. To combat glioma vascular proliferation, anti-angiogenic agents targeting vascular endothelial growth factor (VEGF) were introduced. Preclinically these agents were effective, yet they did not improve overall survival in phase III trials. We tested the hypothesis that ganciclovir (GCV)-mediated killing of proliferating endothelial cells expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) would have direct antitumor effects, and whether vessel ablation would affect the antitumor activity of anti-VEGF antibodies and radiotherapy. Methods Proliferating endothelial cells were eliminated using GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK (in Tie2-TK-IRES-GFP mice). Syngeneic NRAS/p53 (NP) gliomas were implanted into the brains of Tie2-TK-IRES-GFP mice. Endothelial proliferation activates the Tie2 promoter and HSV1-TK expression. Administration of GCV kills proliferating tumor endothelial cells and slows tumor growth. The effects of endothelial cell ablation on anti-angiogenic therapy were examined using anti-VEGF antibodies or irradiation. Results GCV administration reduced tumor growth and vascular density, increased tumor apoptosis, and prolonged survival. Anti-VEGF antibodies or irradiation also prolonged survival. Surprisingly, combining GCV with irradiation, or with anti-VEGF antibodies, reduced their individual therapeutic effects. Conclusion GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK, anti-VEGF antibodies, or irradiation all reduced growth of a murine glioma. However, elimination of microvascular proliferation decreased the efficacy of anti-VEGF or irradiation therapy. We conclude that, in our model, the integrity of proliferating vessels is necessary for the antiglioma effects of anti-VEGF and radiation therapy.

Viral gene therapy for central nervous system diseases

From a theoretical proposal in 1980’s, gene therapy involving the use of viral vectors to deliver genes for medical treatment of disease has come a long way. Given the complexity and the blood-brain barrier, delivery to the central nervous system (CNS), and the efficacy thereof on brain diseases has been an enormous challenge for gene therapies and resulting clinical trials for treatment of neurological disorders. In spite of this, several studies over the past 2 decades have focused on achieving this using different viral vectors. In this chapter we review the different viral based vectors (Retro-, Lenti-, Adeno- and Adenoassociated viruses) developed over this time in clinical trials for treating different CNS pathologies including brain tumors, Parkinson’s disease, Alzheimer’s disease, and cancer pain. We describe the successes achieved and the challenges still faced for each of these viral vectors as well as brain disease conditions.

Abstract 4145: Brain-derived endothelial cells stimulate migration of different human, mouse, and rat glioma cell lines in vivo and in vitro

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Glioblastoma multiforme (GBM) is the most lethal of the human brain tumors. Despite intensive chemotherapy, radiotherapy and surgery, patients usually die within 1-2 years of diagnosis. Glioma cells display the remarkable capacity to infiltrate the brain. They diffusely invade the brain by active cell migration either along blood vessels, white matter tracts, interstitially, or surrounding the meninges. We identified a number of glioma cells (including glioma stem cells) from mouse and human which migrate primarily along the vasculature. Molecular determinants that attract glioma cells to blood vessels remain poorly understood; inhibition of glioma growth along blood vessels could be a potential treatment strategy for GBM. In the present study, we studied whether brain-derived endothelial cells alone, or in combination with other brain cell types attract glioma cells. To do so we tested the ability of human and mouse brain-derived endothelial cells to stimulate the migration of human, mouse and rat-derived glioma cell lines in an in vitro migration assay. We tested the migration of different primary human glioma cell lines like HF2303, MGG8, MSP-12, IN859, IN2045 and U251 in response to mouse brain-derived endothelial (MBVE) cells and mixed mouse brain cells (MMB). Of these, HF2303, MSP-12 cells showed significant migration but not MGG8, IN859, IN2045 and U251 cell lines. Further, Gl26-cit, a mouse glioma and CNS1-cit a rat cell line also exhibited significant migration towards MBVE as well as MMB cells. These results are in line with our in vivo data showing that HF2303, and Gl26-cit, but not MGG8 cells, invade along the blood vessels. Differential migration of various human glioma cell lines in response to MBVE cells is probably due to their heterogeneous growth patterns in vivo. Interestingly, our in vitro data showed that the mouse endothelial cells together with mouse astrocytes, another brain cell type, promote the migration of Gl26-cit cells to a greater extent than the mouse endothelial cells alone. In summary, the behavior of glioma cells in vitro is consistent with their migration patterns in vivo. Further, other brain cell types like astrocytes along with MBVE cells enhance migration of glioma cells. Therefore, we are now using this in vitro model to discover the molecular mechanisms responsible for glioma growth towards blood vessels. This work was supported by NIH-NINDS, the Department of Neurosurgery, and very generous support from Phil F. Jenkins. Citation Format: Viveka N. Yadav, Gregory J. Baker, Samanthi Narayanan, Maria G. Castro, Pedro R. Lowenstein. Brain-derived endothelial cells stimulate migration of different human, mouse, and rat glioma cell lines in vivo and in vitro. [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 4145. doi:10.1158/1538-7445.AM2015-4145

Abstract 3651: Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Natural killer (NK) cells safeguard against early tumor formation by seeking out and destroying transformed target cells in a process referred to as NK immunosurveillance. While it is clear that malignant brain tumors such as glioblastoma (GBM) evade NK-mediated tumor suppression, the precise mechanisms by which this occurs remain unknown. We now show that shRNA-mediated knockdown of the β-galactoside-binding lectin, galectin-1 (gal-1), in malignant glioma cells leads to the failure to form lethal intracranial tumors in RAG1-/- mice, a mouse strain devoid of adaptive immunity. However, gal-1 deficient glioma growth is fully restored on implantation into the brain of severely immunocompromised NOD-scid IL2Rg null mice, which lack both adaptive and innate immune function, thus implicating the innate immune response in the early rejection of gal-1 deficient glioma. Immunodepletion of NK cells in RAG1-/- or C57BL/6J mice using anti-asialo GM1 or anti-NK1.1 antibodies permit the growth of large gal-1 deficient gliomas, while macrophage depletion with clodronate liposomes only permits limited tumor growth. This combined result suggests that NK cells and macrophages may work together to achieve gal-1 deficient glioma rejection. Antigen-specific IFN-γ ELISpot assays using splenocytes from immunocompetent C57BL/6J mice indicate that gal-1 deficient glioma is cleared prior to the onset of an adaptive anti-tumor immune response. Flow cytometric analysis of brain tumor-infiltrating immune cells reveal that gal-1 deficient gliomas contain significantly more macrophages and granzyme B+ NK cells compared to gal-1 expressing gliomas. In-vitro experiments further show that gal-1 deficient glioma cells are inherently over 3-times more sensitive to NK-mediated tumor lysis, fail to suppress pro-inflammatory (M1) microglial activation, and secrete pro-inflammatory cytokines IL-1β, IL-12p70, and CXCL2. We conclude that glioma-derived gal-1 is a powerful inhibitor of NK-mediated cytotoxicity in-vivo, and predict that its suppression will be of therapeutic value in the treatment of human malignant brain tumors by dramatically heightening anti-tumor NK immunosurveillance. Citation Format: Gregory J. Baker, Viveka Nand Yadav, Peter Chockley, Robert Doherty, Michael Ritt, Sivaraj Sivaramakrishnan, Maria G. Castro, Pedro R. Lowenstein. Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2014-3651