SCIENCE TIMES
MicroRNA-Based Metastasis Prediction
T
he ability to predict which primary tumors will metastasize to the brain remains elusive. Her2 and TN breast cancers and lung metastases represent the most common brain metastatic tumors. However, other cancers at varying rates can also spread to the brain. This raises certain questions: Do brain metastases represent common molecular pathways used by cancers at various rates? Can this neural niche predilection could be anticipated with evaluation of primary tumor biology? In a recent article by Zhou et al,1 the primary breast cancer cell line MDA-MB-231 and the noncancerous mammary epithelial MCF-10A line were analyzed. The MDA-MB-231 cell line had increased microRNA (miR)-105 compared with MCF-10A.1 After confirming transferability of exosome-dependent miR-105 to human microvascular endothelial cells, they observed that the transferred miR-105 was the mature, not primary or precursor, form of miR-105. Human microvascular endothelial cells with ectopic expression of miR-105 or treatment with MDA-MB-231–derived exosomes displayed a decrease in zona occludens-1 (ZO-1), a molecular component of tight junctions. This effect was abrogated when exosomes derived from MCF-10A cells were used. A 3-dimensional vascular sprouting assay showed destruction of the barrier provided by endothelial monolayers by miR-105–containing exosomes. To determine the ability of the cancer cells to traverse the endothelial layers, a transendothelial invasion assay was used. MDA-231-HM cells from a metastatic cell line had the highest invasion potential. MDA-231-HM cells treated with MCF-10A exosomes, MDA-MB-231 exosomes and anti-miR-105, or MDA-MB-231 exosomes and ZO-1 had decreased traversal through the human microvascular endothelial cells monolayer. These findings were then evaluated through in vivo studies. Through tail vein injections of exosomes, Zhou et al then examined 2 common sites of breast cancer metastases: brain and lung. Injecting exosomes rich in miR-105 increased miR105 in the metastases sites, reduced ZO-1 expression in endothelial cells, and increased vascular permeability. Overexpressing miR-105 in an MCF-10A–derived tumorigenic line (MCFDCIS) did not affect growth of primary tumor in vivo but did affect metastatic growth, an increase very prominently displayed in brain metastases. However, on inhibition of miR105, there was a decrease in metastatic potential
N18 | VOLUME 77 | NUMBER 2 | AUGUST 2015
through restoration of vascular integrity shown by in vivo vascular permeability assays. To investigate the potential of using miR-105 as a prognostic marker for metastases, Zhou et al first investigated miR-105 in their mouse models and observed high circulating miR-105 at premetastatic and metastases stages. Then they used patient data to analyze serum from patients with stage II and III breast cancer (Figure).1 Exosomes purified from sera from patients who later developed distant metastases contained higher miR-105 than exomes from those who did not develop distant metastases in the years of follow-up. Zhou et al then returned to the 3-dimensional vascular structures. Treatment with patient serum, not normal serum, consequently destructed vascular structures. Patient data (n ¼ 75) also revealed the inverse correlation between miR-105 and ZO-1. These data suggest a biomarker-based use for miR-105 to identify patients who could develop distant metastases. Rahul Jandial, MD, PhD Cecilia Choy, MS City of Hope Duarte, California
REFERENCE 1. Zhou W, Fong MY, Min Y, et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell. 2014;25(4):501-515.
Oncolytic Herpes Simplex Virus Glioblastoma Therapy is Potentiated by Tumor Necrosis Factor-a Inhibition
O
ncolytic viruses are being explored as a potential therapy for the aggressive primary brain cancer glioblastoma multiforme (GBM).1 The oncolytic herpes simplex virus (oHSV) was modified to specifically target, replicate within, and destroy cancer cells by generating an antitumor immune response. Although early clinical trials show promise because oHSV-based therapy is well tolerated, its efficacy may be reduced via early innate immunity against viral infections. Recently, Meisen et al2 reported that apoptosis of infected cells and oHSV destruction are mediated by tumor necrosis factor-a (TNFa) secreted by host microglia and macrophages, whereas TNFa blockade enhanced viral replica-
tion, spread, and efficacy. These data suggest that US Food and Drug Administration–approved TNFa blockers may be repurposed to potentiate oHSV-based anti-GBM therapies. The authors initially found that oHSV treatment activated microglia in mice with U87DEGFR intracranial tumors compared with control saline treatment. Analysis of tumor and nontumor regions showed that oHSV led to a statistically significant increase in microglia major histocompatibility complex class II expression. They also discovered that increased microglia triggered macrophage infiltration into tumor-bearing brains. To determine how microglia/macrophages influenced viral replication, oHSV-infected glioma cells were cocultured with macrophages or microglia. Significant viral titer reduction was found after coculture, with more reduction observed for macrophage/oHSV–glioma cocultures. In response to viral infections, TNFa is known to be upregulated and secreted by macrophages and microglia. Using murinespecific TNFa enzyme-linked immunosorbent assay, Meisen et al found that oHSV infection increased the levels of secreted TNFa by 35.42and 9-fold in macrophage and microglia cocultures, respectively. Increased TNFa was also observed in the brain and serum of mice with oHSV-treated intracranial xenografts. Treatment of oHSV-infected glioma cells in monoculture with TNFa resulted in reduced viral replication. TNFa induced apoptosis in the oHSVinfected cells, indicated by membrane blebbing, cell shrinkage, and loss of adherence. Apoptosis markers caspase 8, caspase 3, and cleaved PARP were significantly expressed only in oHSVinfected cells in the presence of TNFa. These proteins were not significantly activated in uninfected glioma cells treated with TNFa or monocultured infected cells not treated with TNFa. The authors then tested inhibition of TNFa-induced apoptosis by coculturing normal macrophages or TNFa-knockout bone marrow–derived macrophages with oHSVinfected glioma cells. They found that oHSV-infected cells cocultured with the TNFa-knockout macrophages had significantly less membrane blebbing and higher viral titers. The addition of a TNFa-blocking antibody also rescued the reduction in viral replication in infected glioma cells cocultured with microglia. In vivo experiments with TNFa blockade were then done to determine whether viral replication and spread could be enhanced. Mice harboring flank U87DEGFR xenografts
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SCIENCE TIMES
Figure. A, circulating microRNA (miR)-105 in patient serum resulted in vascular destruction. Representative images of the treated vascular structures are shown (left). Inset, vascular sprouts per spheroid (right). *P , .05. B, correlation analyses of tumor miR-105, serum (exosomal) miR-105, and zona occludens-1 (ZO-1) levels in patients with breast cancer. miR-105 levels in tumor cells and ZO-1 levels in tumor cells (tumor ZO-1) or tumor-adjacent vascular structures (vascular ZO-1) determined by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. The Pearson correlation coefficient (r) and P value are shown. C, the scores of tumor miR-105, tumor ZO-1, and vascular ZO-1 staining (mean 6 SD). D, representative images of miR-105 and ZO-1 staining in tumor and normal breast tissue sections. Vascular structures are indicated by arrowheads. E, levels of tumor miR-105 and ZO-1 determined in a breast cancer tissue array. The ISH or IHC scores were compared between primary tumors with and without distant or lymph node (LN) metastases. The correlation results between miR-105 and ZO-1 are presented as mean 6 SD. Modified from Cancer Cell (Zhou W, Fong MY, Min Y, et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer cell. 2014;25(4):501-515), with permission from Elsevier.
NEUROSURGERY
VOLUME 77 | NUMBER 2 | AUGUST 2015 | N19
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited
SCIENCE TIMES
were treated with oHSV and then given TNFablocking antibodies or a control antibody. Increased viral replication in TNFa-blocking antibody–treated mice was observed compared with controls. The survival of mice with intracranial GBM xenografts treated with oHSV and TNFa-blocking antibody was significantly better than that of mice treated with oHSV and control antibody. Importantly, there are many already approved TNFa blockers that may be used with oHSV to treat GBM, and clinical studies to test whether TNFa blockers synergize with oHSV anti-GBM therapy are proposed. Kelli B. Pointer, BS Ray R. Zhang, BS John S. Kuo, MD, PhD University of Wisconsin Madison, Wisconsin
REFERENCES 1. Haseley A, Alvarez-Breckenridge C, Chaudhury AR, Kaur B. Advances in oncolytic virus therapy for glioma. Recent Pat CNS Drug Discov. 2009;4(1): 1-13. 2. Meisen WH, Wohleb ES, Jaime-Ramirez AC, et al. The impact of macrophage and microglia secreted TNFa on oncolytic HSV-1 therapy in the glioblastoma tumor microenvironment [published online ahead of print March 13, 2015]. Clin Cancer Res. doi: 10.1158/1078-0432.CCR-14-3118. http:// clincancerres.aacrjournals.org/content/early/2015/03/ 31/1078-0432.CCR-14-3118.long.
Boosting Dendritic Cell Vaccination for Glioblastoma Using Tetanus Toxoid
I
mmunotherapy is an attractive approach for the treatment of malignant gliomas because redirection of the immune system to recognize and ablate brain-infiltrating tumor cells promises to be a targeted, efficacious, and welltolerated treatment. To achieve this goal, a series of challenges need to be overcome, including selection of tumor-specific antigens, identification of these antigens by the immune system in the form of a vaccine, and subsequent activation of the immune system to recognize and elicit cytotoxic responses against tumor cells in the brain. Although extensive research has been performed in malignant gliomas, these tumors are particularly challenging, given their location in the brain and their potent immunosuppressive environment supported by regulatory T cells, immunosuppressive microglia, and cytokine milieu, among other mechanisms.1-4 With regard to malignant glioma–specific antigens, a series of promising candidates are being investigated at the clinical stage, including endothelial growth factor receptor-vIII junctional epitope (a tumor-specific antigen that results from the deletion of part of the
endothelial growth factor receptor that results in a constitutive activation) and interleukin13Ra2.5,6 Other interesting strategies for selecting tumor antigens include the use of autologous tumor lysate and pulling heat shock proteins from autologous tumor tissue because these proteins are loaded with tumor antigens and favor their presentation to the immune system.7,8 Optimal antigenic presentation to promote an effective antitumoral immunity has been attempted by vaccination with immune-stimulant adjuvants and cytokines. An approach to optimize antigen presentation that is under intense investigation is the use of autologous dendritic cells, the ultimate professional antigen-presenting cells. These cells are harvested from the patient, stimulated, loaded with tumor antigens ex vivo, and then subsequently injected back into the patient in the form of a vaccine. To date, this approach has shown some encouraging results in malignant gliomas.5,6,8-10 However, the determinants of success of this strategy are not understood. Exciting recent work published in Nature by the Sampson laboratory at Duke University used tetanus toxoid preconditioning at the vaccination site as a novel strategy to enhance the efficacy of a dendritic cell–based vaccine for newly diagnosed glioblastoma.11 In this study, Mitchell and colleagues11 performed a clinical trial in which 12 patients were randomized to injection of tetanus toxoid vs unpulsed
Figure. Tetanus preconditioning increases dendritic cell (DC) migration to regional lymph nodes and is associated with improved outcomes for glioblastoma patients treated with DCs loaded with phosphoprotein 65. A, DCs in the tetanus preconditioning group exhibited enhanced migration to regional lymph nodes compared with preconditioning with unpulsed DCs (P , .05). Patients who were preconditioned with tetanus toxoid (Td) exhibited prolonged progression-free (B) and overall (C) survival compared with those receiving unpulsed DCs in a clinical trial (P , .05). Modified from Mitchell et al.11 Reprinted by permission from Macmillan Publishers Ltd: Nature (2015;519 (7543):366-369.), copyright 2015.
N20 | VOLUME 77 | NUMBER 2 | AUGUST 2015
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Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited
MicroRNA-Based Metastasis Prediction
T
he ability to predict which primary tumors will metastasize to the brain remains elusive. Her2 and TN breast cancers and lung metastases represent the most common brain metastatic tumors. However, other cancers at varying rates can also spread to the brain. This raises certain questions: Do brain metastases represent common molecular pathways used by cancers at various rates? Can this neural niche predilection could be anticipated with evaluation of primary tumor biology? In a recent article by Zhou et al,1 the primary breast cancer cell line MDA-MB-231 and the noncancerous mammary epithelial MCF-10A line were analyzed. The MDA-MB-231 cell line had increased microRNA (miR)-105 compared with MCF-10A.1 After confirming transferability of exosome-dependent miR-105 to human microvascular endothelial cells, they observed that the transferred miR-105 was the mature, not primary or precursor, form of miR-105. Human microvascular endothelial cells with ectopic expression of miR-105 or treatment with MDA-MB-231–derived exosomes displayed a decrease in zona occludens-1 (ZO-1), a molecular component of tight junctions. This effect was abrogated when exosomes derived from MCF-10A cells were used. A 3-dimensional vascular sprouting assay showed destruction of the barrier provided by endothelial monolayers by miR-105–containing exosomes. To determine the ability of the cancer cells to traverse the endothelial layers, a transendothelial invasion assay was used. MDA-231-HM cells from a metastatic cell line had the highest invasion potential. MDA-231-HM cells treated with MCF-10A exosomes, MDA-MB-231 exosomes and anti-miR-105, or MDA-MB-231 exosomes and ZO-1 had decreased traversal through the human microvascular endothelial cells monolayer. These findings were then evaluated through in vivo studies. Through tail vein injections of exosomes, Zhou et al then examined 2 common sites of breast cancer metastases: brain and lung. Injecting exosomes rich in miR-105 increased miR105 in the metastases sites, reduced ZO-1 expression in endothelial cells, and increased vascular permeability. Overexpressing miR-105 in an MCF-10A–derived tumorigenic line (MCFDCIS) did not affect growth of primary tumor in vivo but did affect metastatic growth, an increase very prominently displayed in brain metastases. However, on inhibition of miR105, there was a decrease in metastatic potential
N18 | VOLUME 77 | NUMBER 2 | AUGUST 2015
through restoration of vascular integrity shown by in vivo vascular permeability assays. To investigate the potential of using miR-105 as a prognostic marker for metastases, Zhou et al first investigated miR-105 in their mouse models and observed high circulating miR-105 at premetastatic and metastases stages. Then they used patient data to analyze serum from patients with stage II and III breast cancer (Figure).1 Exosomes purified from sera from patients who later developed distant metastases contained higher miR-105 than exomes from those who did not develop distant metastases in the years of follow-up. Zhou et al then returned to the 3-dimensional vascular structures. Treatment with patient serum, not normal serum, consequently destructed vascular structures. Patient data (n ¼ 75) also revealed the inverse correlation between miR-105 and ZO-1. These data suggest a biomarker-based use for miR-105 to identify patients who could develop distant metastases. Rahul Jandial, MD, PhD Cecilia Choy, MS City of Hope Duarte, California
REFERENCE 1. Zhou W, Fong MY, Min Y, et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell. 2014;25(4):501-515.
Oncolytic Herpes Simplex Virus Glioblastoma Therapy is Potentiated by Tumor Necrosis Factor-a Inhibition
O
ncolytic viruses are being explored as a potential therapy for the aggressive primary brain cancer glioblastoma multiforme (GBM).1 The oncolytic herpes simplex virus (oHSV) was modified to specifically target, replicate within, and destroy cancer cells by generating an antitumor immune response. Although early clinical trials show promise because oHSV-based therapy is well tolerated, its efficacy may be reduced via early innate immunity against viral infections. Recently, Meisen et al2 reported that apoptosis of infected cells and oHSV destruction are mediated by tumor necrosis factor-a (TNFa) secreted by host microglia and macrophages, whereas TNFa blockade enhanced viral replica-
tion, spread, and efficacy. These data suggest that US Food and Drug Administration–approved TNFa blockers may be repurposed to potentiate oHSV-based anti-GBM therapies. The authors initially found that oHSV treatment activated microglia in mice with U87DEGFR intracranial tumors compared with control saline treatment. Analysis of tumor and nontumor regions showed that oHSV led to a statistically significant increase in microglia major histocompatibility complex class II expression. They also discovered that increased microglia triggered macrophage infiltration into tumor-bearing brains. To determine how microglia/macrophages influenced viral replication, oHSV-infected glioma cells were cocultured with macrophages or microglia. Significant viral titer reduction was found after coculture, with more reduction observed for macrophage/oHSV–glioma cocultures. In response to viral infections, TNFa is known to be upregulated and secreted by macrophages and microglia. Using murinespecific TNFa enzyme-linked immunosorbent assay, Meisen et al found that oHSV infection increased the levels of secreted TNFa by 35.42and 9-fold in macrophage and microglia cocultures, respectively. Increased TNFa was also observed in the brain and serum of mice with oHSV-treated intracranial xenografts. Treatment of oHSV-infected glioma cells in monoculture with TNFa resulted in reduced viral replication. TNFa induced apoptosis in the oHSVinfected cells, indicated by membrane blebbing, cell shrinkage, and loss of adherence. Apoptosis markers caspase 8, caspase 3, and cleaved PARP were significantly expressed only in oHSVinfected cells in the presence of TNFa. These proteins were not significantly activated in uninfected glioma cells treated with TNFa or monocultured infected cells not treated with TNFa. The authors then tested inhibition of TNFa-induced apoptosis by coculturing normal macrophages or TNFa-knockout bone marrow–derived macrophages with oHSVinfected glioma cells. They found that oHSV-infected cells cocultured with the TNFa-knockout macrophages had significantly less membrane blebbing and higher viral titers. The addition of a TNFa-blocking antibody also rescued the reduction in viral replication in infected glioma cells cocultured with microglia. In vivo experiments with TNFa blockade were then done to determine whether viral replication and spread could be enhanced. Mice harboring flank U87DEGFR xenografts
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Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited
SCIENCE TIMES
Figure. A, circulating microRNA (miR)-105 in patient serum resulted in vascular destruction. Representative images of the treated vascular structures are shown (left). Inset, vascular sprouts per spheroid (right). *P , .05. B, correlation analyses of tumor miR-105, serum (exosomal) miR-105, and zona occludens-1 (ZO-1) levels in patients with breast cancer. miR-105 levels in tumor cells and ZO-1 levels in tumor cells (tumor ZO-1) or tumor-adjacent vascular structures (vascular ZO-1) determined by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. The Pearson correlation coefficient (r) and P value are shown. C, the scores of tumor miR-105, tumor ZO-1, and vascular ZO-1 staining (mean 6 SD). D, representative images of miR-105 and ZO-1 staining in tumor and normal breast tissue sections. Vascular structures are indicated by arrowheads. E, levels of tumor miR-105 and ZO-1 determined in a breast cancer tissue array. The ISH or IHC scores were compared between primary tumors with and without distant or lymph node (LN) metastases. The correlation results between miR-105 and ZO-1 are presented as mean 6 SD. Modified from Cancer Cell (Zhou W, Fong MY, Min Y, et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer cell. 2014;25(4):501-515), with permission from Elsevier.
NEUROSURGERY
VOLUME 77 | NUMBER 2 | AUGUST 2015 | N19
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited
SCIENCE TIMES
were treated with oHSV and then given TNFablocking antibodies or a control antibody. Increased viral replication in TNFa-blocking antibody–treated mice was observed compared with controls. The survival of mice with intracranial GBM xenografts treated with oHSV and TNFa-blocking antibody was significantly better than that of mice treated with oHSV and control antibody. Importantly, there are many already approved TNFa blockers that may be used with oHSV to treat GBM, and clinical studies to test whether TNFa blockers synergize with oHSV anti-GBM therapy are proposed. Kelli B. Pointer, BS Ray R. Zhang, BS John S. Kuo, MD, PhD University of Wisconsin Madison, Wisconsin
REFERENCES 1. Haseley A, Alvarez-Breckenridge C, Chaudhury AR, Kaur B. Advances in oncolytic virus therapy for glioma. Recent Pat CNS Drug Discov. 2009;4(1): 1-13. 2. Meisen WH, Wohleb ES, Jaime-Ramirez AC, et al. The impact of macrophage and microglia secreted TNFa on oncolytic HSV-1 therapy in the glioblastoma tumor microenvironment [published online ahead of print March 13, 2015]. Clin Cancer Res. doi: 10.1158/1078-0432.CCR-14-3118. http:// clincancerres.aacrjournals.org/content/early/2015/03/ 31/1078-0432.CCR-14-3118.long.
Boosting Dendritic Cell Vaccination for Glioblastoma Using Tetanus Toxoid
I
mmunotherapy is an attractive approach for the treatment of malignant gliomas because redirection of the immune system to recognize and ablate brain-infiltrating tumor cells promises to be a targeted, efficacious, and welltolerated treatment. To achieve this goal, a series of challenges need to be overcome, including selection of tumor-specific antigens, identification of these antigens by the immune system in the form of a vaccine, and subsequent activation of the immune system to recognize and elicit cytotoxic responses against tumor cells in the brain. Although extensive research has been performed in malignant gliomas, these tumors are particularly challenging, given their location in the brain and their potent immunosuppressive environment supported by regulatory T cells, immunosuppressive microglia, and cytokine milieu, among other mechanisms.1-4 With regard to malignant glioma–specific antigens, a series of promising candidates are being investigated at the clinical stage, including endothelial growth factor receptor-vIII junctional epitope (a tumor-specific antigen that results from the deletion of part of the
endothelial growth factor receptor that results in a constitutive activation) and interleukin13Ra2.5,6 Other interesting strategies for selecting tumor antigens include the use of autologous tumor lysate and pulling heat shock proteins from autologous tumor tissue because these proteins are loaded with tumor antigens and favor their presentation to the immune system.7,8 Optimal antigenic presentation to promote an effective antitumoral immunity has been attempted by vaccination with immune-stimulant adjuvants and cytokines. An approach to optimize antigen presentation that is under intense investigation is the use of autologous dendritic cells, the ultimate professional antigen-presenting cells. These cells are harvested from the patient, stimulated, loaded with tumor antigens ex vivo, and then subsequently injected back into the patient in the form of a vaccine. To date, this approach has shown some encouraging results in malignant gliomas.5,6,8-10 However, the determinants of success of this strategy are not understood. Exciting recent work published in Nature by the Sampson laboratory at Duke University used tetanus toxoid preconditioning at the vaccination site as a novel strategy to enhance the efficacy of a dendritic cell–based vaccine for newly diagnosed glioblastoma.11 In this study, Mitchell and colleagues11 performed a clinical trial in which 12 patients were randomized to injection of tetanus toxoid vs unpulsed
Figure. Tetanus preconditioning increases dendritic cell (DC) migration to regional lymph nodes and is associated with improved outcomes for glioblastoma patients treated with DCs loaded with phosphoprotein 65. A, DCs in the tetanus preconditioning group exhibited enhanced migration to regional lymph nodes compared with preconditioning with unpulsed DCs (P , .05). Patients who were preconditioned with tetanus toxoid (Td) exhibited prolonged progression-free (B) and overall (C) survival compared with those receiving unpulsed DCs in a clinical trial (P , .05). Modified from Mitchell et al.11 Reprinted by permission from Macmillan Publishers Ltd: Nature (2015;519 (7543):366-369.), copyright 2015.
N20 | VOLUME 77 | NUMBER 2 | AUGUST 2015
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