Production of an interleukin-10 blocking antibody by genetically engineered macrophages increases cancer cell death in human gastrointestinal tumor slice cultures.

Although it can promote effector T-cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (αIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized αIL-10 antibody. The efficacy of αIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by αIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but αIL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an ~5-fold higher rate of tumor cell apoptosis than control.

Hypoxia-inducible lentiviral gene expression in engineered human macrophages.

BACKGROUND: Human immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression is regulated by constitutively active promoters, lentiviral payload gene expression is unregulated, and can result in potentially toxic quantities of proteins. Regulated delivery of lentivirally encoded proteins may allow localized or conditional therapeutic protein expression to support safe delivery of adoptively transferred, genetically modified cells with reduced capacity for systemic toxicities. METHODS: In this study, we engineered human macrophages to express genes regulated by hypoxia responsive elements included in the lentiviral promoter region to drive conditional lentiviral gene expression only under hypoxic conditions. We tested transduced macrophages cultured in hypoxic conditions for the transient induced expression of reporter genes and the secreted cytokine, interleukin-12. Expression of hypoxia-regulated genes was investigated both transcriptionally and translationally, and in the presence of human tumor cells in a slice culture system. Finally, hypoxia-regulated gene expression was evaluated in a subcutaneous humanized-mouse cancer model. RESULTS: Engineered macrophages were shown to conditionally and tranisently express lentivirally encoded gene protein products, including IL-12 in hypoxic conditions in vitro. On return to normoxic conditions, lentiviral payload expression returned to basal levels. Reporter genes under the control of hypoxia response elements were upregulated under hypoxic conditions in the presence of human colorectal carcinoma cells and in the hypoxic xenograft model of glioblastoma, suggesting utility for systemic engineered cell delivery capable of localized gene delivery in cancer. CONCLUSIONS: Macrophages engineered to express hypoxia-regulated payloads have the potential to be administered systemically and conditionally express proteins in tissues with hypoxic conditions. In contrast to immune cells that function or survive poorly in hypoxic conditions, macrophages maintain a proinflammatory phenotype that may support continued gene and protein expression when regulated by conditional hypoxia responsive elements and naturally traffic to hypoxic microenvironments, making them ideal vehicles for therapeutic payloads to hypoxic tissues, such as solid tumors. With the ability to fine-tune delivery of potent proteins in response to endogenous microenvironments, macrophage-based cellular therapies may therefore be designed for different disease settings.

Arming Immune Cell Therapeutics with Polymeric Prodrugs.

Engineered immune cells are an exciting therapeutic modality, which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering is developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed "drugamers." The fluorescein ligand facilitates stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporate prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustains antiproliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system is demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporates a small molecule estrogen analog, CMP8, which stabilizes a degron-tagged transgene to provide temporal regulation of protein activity in engineered T cells. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both antitumor and transgene-activating small molecule prodrugs.

Optimal therapeutic targeting by HDAC inhibition in biopsy-derived treatment-naïve diffuse midline glioma models.

BACKGROUND: Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), have a dismal prognosis, with less than 2% surviving 5 years postdiagnosis. The majority of DIPGs and all DMGs harbor mutations altering the epigenetic regulatory histone tail (H3 K27M). Investigations addressing DMG epigenetics have identified a few promising drugs, including the HDAC inhibitor (HDACi) panobinostat. Here, we use clinically relevant DMG models to identify and validate other effective HDACi and their biomarkers of response. METHODS: HDAC inhibitors were tested across biopsy-derived treatment-naïve in vitro and in vivo DMG models with biologically relevant radiation resistance. RNA sequencing was performed to define and compare drug efficacy and to map predictive biomarkers of response. RESULTS: Quisinostat and romidepsin showed efficacy with low nanomolar half-maximal inhibitory concentration (IC50) values (~50 and ~5 nM, respectively). Comparative transcriptome analyses across quisinostat, romidepsin, and panobinostat showed a greater degree of shared biological effects between quisinostat and panobinostat, and less overlap with romidepsin. However, some transcriptional changes were consistent across all 3 drugs at similar biologically effective doses, such as overexpression of troponin T1 slow skeletal type (TNNT1) and downregulation of collagen type 20 alpha 1 chain (COL20A1), identifying these as potential vulnerabilities or on-target biomarkers in DMG. Quisinostat and romidepsin significantly (P < 0.0001) inhibited in vivo tumor growth. CONCLUSIONS: Our data highlight the utility of treatment-naïve biopsy-derived models; establishes quisinostat and romidepsin as effective in vivo; illuminates potential mechanisms and/or biomarkers of DMG cell lethality due to HDAC inhibition; and emphasizes the need for brain tumor-penetrant versions of potentially efficacious agents.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses.

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma.

BACKGROUND: Targeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose many barriers to immunotherapies, including therapy half-life and persistence, tumor penetrance, and targeting. Therapeutics delivered systemically may not traffic to the tumor site. If cellular therapies or drugs are able to access the tumor site, or can be delivered directly within the tumor, treatments may not persist for the duration necessary to reduce or eliminate tumor burden. An approach that allows durable and titratable local therapeutic protein delivery could improve antitumor efficacy while minimizing toxicities or unwanted on-target, off-tissue effects. METHODS: In this study, human monocyte-derived macrophages were genetically engineered to secrete a bispecific T cell engager (BiTE) specific to the mutated epidermal growth factor variant III (EGFRvIII) expressed by some GBM tumors. We investigated the ability of lentivirally modified macrophages to secrete a functional BiTE that can bind target tumor antigen and activate T cells. Secreted BiTE protein was assayed in a range of T cell functional assays in vitro and in subcutaneous and intracranial GBM xenograft models. Finally, we tested genetically engineered macrophages (GEMs) secreting BiTE and the proinflammatory cytokine interleukin (IL)-12 to amplify T cell responses in vitro and in vivo. RESULTS: Transduced human macrophages secreted a lentivirally encoded functional EGFRvIII-targeted BiTE protein capable of inducing T cell activation, proliferation, degranulation, and killing of antigen-specific tumor cells. Furthermore, BiTE secreting macrophages reduced early tumor burden in both subcutaneous and intracranial mouse models of GBM, a response which was enhanced using macrophages that were dual transduced to secrete both the BiTE protein and single chain IL-12, preventing tumor growth in an aggressive GBM model. CONCLUSIONS: The ability of macrophages to infiltrate and persist in solid tumor tissue could overcome many of the obstacles associated with systemic delivery of immunotherapies. We have found that human GEMs can locally and constitutively express one or more therapeutic proteins, which may help recruit T cells and transform the immunosuppressive tumor microenvironment to better support antitumor immunity.

Progress on Modulating Tumor-Associated Macrophages with Biomaterials.

Tumor-associated macrophages (TAMs) are a complex and heterogeneous population of cells within the tumor microenvironment. In many tumor types, TAMs contribute toward tumor malignancy and are therefore a therapeutic target of interest. Here, three major strategies for regulating TAMs are highlighted, emphasizing the role of biomaterials in these approaches. First, systemic methods for targeting tumor-associated macrophage are summarized and limitations to both passive and active targeting approaches considered. Second, lessons learned from the significant literature on wound healing and macrophage response to implanted biomaterials are discussed with the vision of applying these principles to localized, biomaterial-based modulation of tumor-associated macrophage. Finally, the developing field of engineered macrophages, including genetic engineering and integration with biomaterials or drug delivery systems, is examined. Analysis of major challenges in the field along with exciting opportunities for the future of macrophage-based therapies in oncology are included.

Anti-PD-L1 antibody direct activation of macrophages contributes to a radiation-induced abscopal response in glioblastoma.

BACKGROUND: Most glioblastomas recur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an "abscopal effect," whereby unirradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti-programmed cell death ligand 1 (PD-L1) demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remain unknown. METHODS: We modified an immune-competent, genetically driven mouse glioma model (forced platelet derived growth factor [PDGF] expression + phosphatase and tensin homolog loss) where a portion of the tumor burden is irradiated (PDGF) and another unirradiated luciferase-expressing tumor (PDGF + luciferase) is used as a readout of the abscopal effect following systemic anti-PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of epidermal growth factor receptor variant III (EGFRvIII) (PDGF + EGFRvIII). Statistical tests were two-sided. RESULTS: Following radiation of one lesion, anti-PD-L1 immunotherapy enhanced the abscopal response to the unirradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF + luciferase, n = 8), the abscopal response occurred via anti-PD-L1 driven, extracellular signal-regulated kinase-mediated, bone marrow-derived macrophage phagocytosis of adjacent unirradiated tumor cells, with modest survival implications (median survival 41 days vs radiation alone 37.5 days, P = 0.03). In PDGF-driven gliomas with tumor neoepitope (PDGF + EGFRvIII, n = 8), anti-PD-L1 enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs radiation alone 28 days, P = 0.001). CONCLUSION: Our results indicate that anti-PD-L1 immunotherapy enhances a radiation- induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma.

Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy.

Background: Diffuse intrinsic pontine glioma (DIPG) is a uniformly fatal CNS tumor diagnosed in 300 American children per year. Radiation is the only effective treatment and extends overall survival to a median of 11 months. Due to its location in the brainstem, DIPG cannot be surgically resected. Immunotherapy has the ability to target tumor cells specifically; however, little is known about the tumor microenvironment in DIPGs. We sought to characterize infiltrating immune cells and immunosuppressive factor expression in pediatric low- and high-grade gliomas and DIPG. Methods: Tumor microarrays were stained for infiltrating immune cells. RNA was isolated from snap-frozen tumor tissue and Nanostring analysis performed. DIPG and glioblastoma cells were co-cultured with healthy donor macrophages, T cells, or natural killer (NK) cells, and flow cytometry and cytotoxicity assays performed to characterize the phenotype and function, respectively, of the immune cells. Results: DIPG tumors do not have increased macrophage or T-cell infiltration relative to nontumor control, nor do they overexpress immunosuppressive factors such as programmed death ligand 1 and/or transforming growth factor ß1. H3.3-K27M DIPG cells do not repolarize macrophages, but are not effectively targeted by activated allogeneic T cells. NK cells lysed all DIPG cultures. Conclusions: DIPG tumors have neither a highly immunosuppressive nor inflammatory microenvironment. Therefore, major considerations for the development of immunotherapy will be the recruitment, activation, and retention of tumor-specific effector immune cells.

Effects of tumor grade and dexamethasone on myeloid cells in patients with glioma.

Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

An Uncoupling of Canonical Phenotypic Markers and Functional Potency of Ex Vivo-Expanded Natural Killer Cells.

Recent advances in cellular therapies for patients with cancer, including checkpoint blockade and ex vivo-expanded, tumor-specific T cells, have demonstrated that targeting the immune system is a powerful approach to the elimination of tumor cells. Clinical efforts have also demonstrated limitations, however, including the potential for tumor cell antigenic drift and neoantigen formation, which promote tumor escape and recurrence, as well as rapid onset of T cell exhaustion in vivo. These findings suggest that antigen unrestricted cells, such as natural killer (NK) cells, may be beneficial for use as an alternative to or in combination with T cell based approaches. Although highly effective in lysing transformed cells, to date, few clinical trials have demonstrated antitumor function or persistence of transferred NK cells. Several recent studies describe methods to expand NK cells for adoptive transfer, although the effects of ex vivo expansion are not fully understood. We therefore explored the impact of a clinically validated 12-day expansion protocol using a K562 cell line expressing membrane-bound IL-15 and 4-1BB ligand with high-dose soluble IL-2 on the phenotype and functions of NK cells from healthy donors. Following expansions using this protocol, we found expression of surface proteins that implicate preferential expansion of NK cells that are not fully mature, as is typically associated with highly cytotoxic NK cell subsets. Despite increased expression of markers associated with functional exhaustion in T cells, we found that ex vivo-expanded NK cells retained cytokine production capacity and had enhanced tumor cell cytotoxicity. The preferential expansion of an NK cell subset that is phenotypically immature and functionally pleiotropic suggests that adoptively transferred cells may persist better in vivo when compared with previous methods using this approach. Ex vivo expansion does not quell killer immunoglobulin-like receptor diversity, allowing responsiveness to various factors in vivo that may influence activation and inhibition. Collectively, our data suggest that in addition to robust NK cell expansion that has been described using this method, expanded NK cells may represent an ideal cell therapy that is longer lived, highly potent, and responsive to an array of activating and inhibitory signals.

Immunotherapy for brain tumors: understanding early successes and limitations.

INTRODUCTION: Adverse effects and toxicities related to standard treatments for brain tumors significantly reduce patients' quality of life. Although most immunotherapy approaches for solid tumors have not been successful, several early-phase clinical trials are beginning to reveal a potential role for immunotherapy in the treatment of brain tumors. In particular, methods that activate the innate immune system and induce a polyclonal anti-cancer response have demonstrated that brain tumors are susceptible to immune-mediated tumor destruction. Compared with conventional therapies, modulation of the immune system may improve both survivorship and quality of life during and following treatment. Areas covered: An overview of mechanisms of immunotherapy in the context of current treatments for adult and pediatric brain tumors is provided. Results from recent clinical trials will be discussed, focusing on the favorable safety and efficacy profiles of immunotherapeutics. Expert commentary: Although it is too early to judge the long-term safety of immunotherapy for the treatment of patients with brain tumors, early results suggest that these drugs are well-tolerated and may improve survival and quality of life. Importantly, approaches that activate an anti-tumor immune response lay the framework for iterative development of immunotherapies that can reliably treat patients with brain tumors.

Therapeutic and Prognostic Implications of BRAF V600E in Pediatric Low-Grade Gliomas.

Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.

Severe brachycephalic obstructive airway syndrome is associated with hypercoagulability in dogs.

We evaluated whether dogs with severe brachycephalic obstructive airway syndrome (BOAS) developed a hypercoagulable state similar to people with obstructive sleep apnea. Five dogs with grade 3 BOAS were included as well as 5 healthy control Labrador Retrievers. Venous blood samples were collected from each dog for performance of thromboelastography and determination of hematocrit and platelet count. Groups were compared using a t-test, with p < 0.05 considered significant. Thromboelastography results identified that all BOAS dogs were hypercoagulable compared to the Labradors, having significantly shortened clotting time with increased angle, maximal amplitude, and clot rigidity. BOAS dogs also had evidence of delayed fibrinolysis. These results are consistent with, but more severe than, those previously documented in apparently healthy Bulldogs. Together, these findings support the presence of a hypercoagulable state in brachycephalic dogs, and suggest that this state is amplified by increasing severity of BOAS.

Genetically Engineered Macrophages: A Potential Platform for Cancer Immunotherapy.

In spite of their successes against hematologic malignancies, immunotherapeutic interventions for the treatment of patients with glioblastoma (GBM) have thus far been unsuccessful. This is in part due to the presence of a tumor microenvironment that fosters neoplastic growth and protects the tumor from destruction by the immune system. A novel genetically engineered macrophage-based platform has been developed with the potential to minimize the effects of the suppressive tumor microenvironment and improve innate and adaptive antitumor immune responses. A newly described lentiviral expression system was validated for the generation of transduced monocytes and monocyte-derived macrophages, and transgene expression was shown to be stable over the course of weeks to months, both in vitro and in a mouse xenograft model of GBM. Furthermore, the genetically engineered macrophages (GEMs) neither caused morbidity in animals nor contributed to accelerated tumor growth. The versatility of GEMs is also highlighted by showing that they can be engineered to secrete proteins that either reduce immune suppression, such as the soluble transforming growth factor beta receptor II, or promote immune cell activation, by expressing interleukin 21. There is also the potential to prevent GEM-mediated immune suppression by using the CRISPR system to knock out genes responsible for dysfunction of cytotoxic cells, including interleukin 10 and programmed death-ligand 1. Together, these results suggest that GEMs are an ideal cell type for transforming the tumor microenvironment and enhancing antitumor immunity. Importantly, it is anticipated that these findings will have broad applicability to other types of tumors with microenvironments that currently preclude successful immunotherapeutic approaches.

NKG2D ligand expression in pediatric brain tumors.

Adult brain tumors establish an immunosuppressive tumor microenvironment as a modality of immune escape, with several immunotherapies designed to overcome this barrier. However, the relationship between tumor cells and immune cells in pediatric brain tumor patients is not as well-defined. In this study, we sought to determine whether the model of immune escape observed in adult brain tumors is reflected in patients with pediatric brain tumors by evaluating NKG2D ligand expression on tissue microarrays created from patients with a variety of childhood brain tumor diagnoses, and infiltration of Natural Killer and myeloid cells. We noted a disparity between mRNA and protein expression for the 8 known NKG2D ligands. Surprisingly, high-grade gliomas did not have increased NKG2D ligand expression compared to normal adjacent brain tissue, nor did they have significant myeloid or NK cell infiltration. These data suggest that pediatric brain tumors have reduced NK cell-mediated immune surveillance, and a less immunosuppressive tumor microenvironment as compared to their adult counterparts. These data indicate that therapies aimed to improve NK cell trafficking and functions in pediatric brain tumors may have a greater impact on anti-tumor immune responses and patient survival, with fewer obstacles to overcome.

Developing immunotherapeutic strategies to target brain tumors.

INTRODUCTION: Recent years have seen rapid growth in cancer treatments that enhance the anti-tumor activities of the immune system. Collectively known as immunotherapy, modulation of the immune system has shown success treating some hematological malignancies, but has yet to be successfully applied to the treatment of patients with brain tumors. AREAS COVERED: This review highlights mechanistic insights from murine studies and compiled recent clinical trial data, focusing on the most aggressive brain tumor, glioblastoma (GBM). The field has recently accumulated a critical mass of data, and we discuss past treatment failures in the context of newly developed approaches now entering clinical trials. This article provides an overview of the immunotherapeutic armamentarium currently in development for the treatment of patients with GBM, who are in dire need of safe and effective therapies. Expert commentary: Themes that emerge include the importance of mitigating the effects of an immunosuppressive tumor microenvironment and the potential for innate immune cell activation to enhance cytotoxic anti-tumor activity. Consideration of these studies as a collective may inform the design of new immunotherapies, as well as the immune monitoring protocols for patients participating in clinical trials.

Maternal vitamin B6 deficient or supplemented diets on expression of genes related to GABAergic, serotonergic, or glutamatergic pathways in hippocampus of rat dams and their offspring.

SCOPE: Vitamin B6 plays crucial roles on brain development and its maternal deficiency impacts the gamma-aminobutyric acid (GABA)ergic, serotonergic, glutamatergic, and dopaminergic systems in offspring. However, the molecular mechanisms underlying these neurological changes are not well understood. Thus, we aimed at evaluating which components of those neurotransmitter metabolism and signaling pathways can be modulated by maternal vitamin B6 -deficient or B6 -supplementated diets in the hippocampus of rat dams and their offspring. METHODS AND RESULTS: Female Wistar rats were fed three different diets: control (6 mg vitamin B6 /kg), supplemented (30 mg vitamin B6 /kg) or deficient diet (0 mg vitamin B6 /kg), from 4 weeks before pregnancy through lactation. Newborn pups (10 days old) from rat dams fed vitamin B6 -deficient diet presented hyperhomocysteinemia and had a significant increase in mRNA levels of glutamate decarboxylase 1 (Gad1), fibroblast growth factor 2 (Fgf2), and glutamate-ammonia ligase (Glul), while glutaminase (Gls) and tryptophan hydroxylase 1 (Tph1) mRNAs were downregulated. Vitamin B6 supplementation or deficiency did not change hippocampal global DNA methylation. CONCLUSION: A maternal vitamin B6 -deficient diet affects the expression of genes related to GABA, glutamate, and serotonin metabolisms in offspring by regulating Gad1, Glul, Gls, and Tph1 mRNA expression.

Suppression of Mammary Carcinogenesis Through Early Exposure to Dietary Lipotropes Occurs Primarily In Utero.

The study determined whether feeding during lactation affects the suppressive effect of maternal dietary lipotropes (i.e., methionine, choline, folate, and vitamin B12) on mammary carcinogenesis. Pregnant Sprague-Dawley rats were randomly allocated to the control diet during pregnancy and lactation (CC), lipotropes-fortified diet during pregnancy (LC), lipotropes-fortified diet during pregnancy plus lactation (LL), or lipotropes-fortified diet during lactation (CL). Randomly selected female offspring from each group were injected intraperitoneally with 50 mg/kg body weight of N-nitroso-N-methylurea at 50 days of age to induce mammary tumors. The LC and LL diets significantly increased tumor latency and survival (P < 0.05). Tumor volumes were significantly suppressed in LC and LL offspring as compared with the CC and CL pups (3759.1 ± 563.0 and 3603.7 ± 526.1 vs. 7465.0 ± 941.1 and 5219.3 ± 759.8 mm(3), respectively; P < 0.05). Both LC and LL lowered tumor multiplicity as compared with CC and CL (P < 0.05). The LC and LL diets repressed transcription of histone deacetylase (HDAC) 1 as well as total HDAC enzyme activity as compared with CC and CL diets (P < 0.05). Data suggest that the tumor suppressive effect of maternal dietary lipotropes is primarily in utero and may be linked to regulation of proteins involved in chromatin remodeling.

Immune evasion mediated by tumor-derived lactate dehydrogenase induction of NKG2D ligands on myeloid cells in glioblastoma patients.

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses.