RESUMO
Despite advances in uncovering the molecular mechanisms that mediate glioma progression and the implementation of novel therapeutic modalities, patients' prognosis remains dismal. This is due to both systemic and local tumor induced immune suppression. We are particularly interested in the role played by infiltrating immunosuppressive myeloid derived suppressor cells (MDSCs) in the glioma tumor microenvironment (TME). This immunosuppressive TME also interferes with the effectiveness of immunotherapies against glioma. Development of multipronged treatment approaches is imperative when aiming to generate a robust anti-glioma immune response. Evaluating the inhibitory potential of MDSCs within the TME is an important aspect for developing effective treatments for glioma. Herein, we discuss methodology to assess the inhibitory effects of MDSCs isolated from the TME using a mouse glioma model.
Assuntos
Neoplasias Encefálicas/imunologia , Glioma/imunologia , Células Supressoras Mieloides/imunologia , Linfócitos T/imunologia , Microambiente Tumoral , Animais , Neoplasias Encefálicas/patologia , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glioma/patologia , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/patologia , Linfócitos T/patologiaRESUMO
Gliomas are the most common malignant brain tumors in the pediatric population. Even though great efforts have been made to understand their distinctive molecular characteristics, there has not been any improvements in the median survival in decades. In children, high-grade glial tumors have a median survival of 9-15 months. It has recently been demonstrated that pediatric high-grade gliomas (pHGG) are biologically and molecularly different from the adult counterparts, which could explain why conventional treatments universally fail. The development of an in vivo pHGG model harboring the specific genetic alterations encountered in pediatric gliomas is imperative in order to study the molecular basis that drives the progression and aggressiveness of these tumors. It would also enable harnessing these results for the development of novel therapeutic approaches. Our lab has implemented a method to induce brain tumors using transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mouse brain. One of the main advantages of this method is that tumors are induced by altering the genome of the host cells, allowing us to recapitulate the salient features of the human disease. In this chapter we describe a method to isolate two cell populations from tumors generated in situ in mice, i.e., one population enriched in tumor cells and another population enriched in CD45+ cells. We also present methodologies as to how tumor infiltrating immune cells can be phenotypically characterized using flow cytometry.
Assuntos
Neoplasias Encefálicas/imunologia , Citometria de Fluxo/métodos , Glioma/imunologia , Sistema Imunitário/citologia , Microambiente Tumoral , Animais , Neoplasias Encefálicas/genética , Criança , Modelos Animais de Doenças , Engenharia Genética/métodos , Glioma/genética , Humanos , Sistema Imunitário/imunologia , Separação Imunomagnética/métodos , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/imunologia , Camundongos , Transposases/genéticaRESUMO
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.
Assuntos
Quimiocina CXCL12/genética , Glioblastoma/genética , Recidiva Local de Neoplasia/genética , Receptores CXCR4/genética , Animais , Apoptose/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Camundongos , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/patologia , Transdução de Sinais , Transposases/genéticaRESUMO
In the last decade, numerous studies of immunotherapy for malignant glioma (glioblastoma multiforme) have brought new knowledge and new hope for improving the prognosis of this incurable disease. Some clinical trials have reached Phase III, following positive outcomes in Phase I and II, with respect to safety and immunological end points. Results are encouraging especially when considering the promise of sustained efficacy by inducing antitumor immunological memory. Progress in understanding the mechanisms of tumor-induced immune suppression led to the development of drugs targeting immunosuppressive checkpoints, which are used in active clinical trials for glioblastoma multiforme. Insights related to the heterogeneity of the disease bring new challenges for the management of glioma and underscore a likely cause of therapeutic failure. An emerging therapeutic strategy is represented by a combinatorial, personalized approach, including the standard of care: surgery, radiation, chemotherapy with added active immunotherapy and multiagent targeting of immunosuppressive checkpoints.