Detecting monocyte trafficking in an animal model of glioblastoma using R2* and quantitative susceptibility mapping.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in glioblastoma (GBM) disease progression has received increasing attention. Recent advances have shown that TAMs can be re-programmed to exert a pro-inflammatory, anti-tumor effect to control GBMs. However, imaging methods capable of differentiating tumor progression from immunotherapy treatment effects have been lacking, making timely assessment of treatment response difficult. We showed that tracking monocytes using iron oxide nanoparticle (USPIO) with MRI can be a sensitive imaging method to detect therapy response directed at the innate immune system. METHODS: We implanted syngeneic mouse glioma stem cells into C57/BL6 mice and treated the animals with either niacin (a stimulator of innate immunity) or vehicle. Animals were imaged using an anatomical MRI sequence, R2* mapping, and quantitative susceptibility mapping (QSM) before and after USPIO injection. RESULTS: Compared to vehicles, niacin-treated animals showed significantly higher susceptibility and R2*, representing USPIO and monocyte infiltration into the tumor. We observed a significant reduction in tumor size in the niacin-treated group 7 days later. We validated our MRI results with flow cytometry and immunofluoresence, which showed that niacin decreased pro-inflammatory Ly6C high monocytes in the blood but increased CD16/32 pro-inflammatory macrophages within the tumor, consistent with migration of these pro-inflammatory innate immune cells from the blood to the tumor. CONCLUSION: MRI with USPIO injection can detect therapeutic responses of innate immune stimulating agents before changes in tumor size have occurred, providing a potential complementary imaging technique to monitor cancer immunotherapies. MANUSCRIPT HIGHLIGHT: We show that iron oxide nanoparticles (USPIOs) can be used to label innate immune cells and detect the trafficking of pro-inflammatory monocytes into the glioblastoma. This preceded changes in tumor size, making it a more sensitive imaging technique.

Correction to: Niacin-mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system.

The article Niacin­mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system, written by Khalil S. Rawji, Adam M.H. Young, Tanay Ghosh, Nathan J. Michaels, Reza Mirzaei, Janson Kappen, Kathleen L. Kolehmainen, Nima Alaeiilkhchi, Brian Lozinski, Manoj K. Mishra, Annie Pu, Weiwen Tang, Salma Zein, Deepak K. Kaushik, Michael B. Keough, Jason R. Plemel, Fiona Calvert, Andrew J. Knights, Daniel J. Gaffney, Wolfram Tetzlaff, Robin J. M. Franklin and V. Wee Yong, was originally published electronically on the publisher's internet.

Niacin-mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system.

Remyelination following CNS demyelination restores rapid signal propagation and protects axons; however, its efficiency declines with increasing age. Both intrinsic changes in the oligodendrocyte progenitor cell population and extrinsic factors in the lesion microenvironment of older subjects contribute to this decline. Microglia and monocyte-derived macrophages are critical for successful remyelination, releasing growth factors and clearing inhibitory myelin debris. Several studies have implicated delayed recruitment of macrophages/microglia into lesions as a key contributor to the decline in remyelination observed in older subjects. Here we show that the decreased expression of the scavenger receptor CD36 of aging mouse microglia and human microglia in culture underlies their reduced phagocytic activity. Overexpression of CD36 in cultured microglia rescues the deficit in phagocytosis of myelin debris. By screening for clinically approved agents that stimulate macrophages/microglia, we have found that niacin (vitamin B3) upregulates CD36 expression and enhances myelin phagocytosis by microglia in culture. This increase in myelin phagocytosis is mediated through the niacin receptor (hydroxycarboxylic acid receptor 2). Genetic fate mapping and multiphoton live imaging show that systemic treatment of 9-12-month-old demyelinated mice with therapeutically relevant doses of niacin promotes myelin debris clearance in lesions by both peripherally derived macrophages and microglia. This is accompanied by enhancement of oligodendrocyte progenitor cell numbers and by improved remyelination in the treated mice. Niacin represents a safe and translationally amenable regenerative therapy for chronic demyelinating diseases such as multiple sclerosis.

T Cell Exhaustion in Glioblastoma: Intricacies of Immune Checkpoints.

Glioblastoma is an aggressive and incurable primary brain tumor. While the blockade of immune checkpoints leads to reversal of T cell exhaustion in many cancers, the efficacy of this therapy in glioblastoma requires further consideration of the brain microenvironment beyond T cell activity. Neural cells are crucially dependent on glucose for survival, and tumor cells rabidly consume glucose; the glucose-deprived microenvironment further elevates immune checkpoint molecules to benefit tumor growth and exacerbate T cell exhaustion. We review here how immune checkpoints drive exhaustion in T cells while favoring tumor metabolism, and discuss how glucose competition in the unique CNS milieu is an important consideration to improve the outcomes of immune checkpoint blockade in glioblastoma.

Prophylactic DNA vaccine targeting Foxp3+ regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model.

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

Resuscitative therapy with erythropoietin reduces oxidative stress and inflammatory responses of vital organs in a rat severe fixed-volume hemorrhagic shock model.

Hemorrhagic shock (HS) still has a high mortality rate and none of the known resuscitative regimens completely reverse its adverse outcomes. This study investigated the effects of different models of resuscitative therapy on the healing of organ damage in a HS model. Male Wistar rats were randomized into six groups: Sham, without HS induction; HS, without resuscitation; HS+Blood, resuscitation with the shed blood; HS+Blood+NS, resuscitation with blood and normal saline; HS+Blood+RL, resuscitation with blood and Ringer's lactate; EPO, erythropoietin was added to the blood and RL. Blood and urine samples were obtained 3 h after resuscitation. Kidney, liver and brain tissue samples were harvested for multiple organ failure evaluation. Survival rate was the highest in the Sham, EPO and HS+Blood+RL groups compared to others. Plasma creatinine concentration, ALT, AST, urinary NAG activity and renal NGAL mRNA expression significantly increased in the HS+Blood+RL group compared to the Sham group. There was a significant increase in tissue oxidative stress markers and pro-inflammatory cytokines in HS+Blood+RL group compared to the Sham rats. EPO had more protective effects on multiple organ failure compared to the HS+Blood+RL group. EPO, as a resuscitative treatment, attenuated HS-induced organ damage. It seems that it has a potential to be attractive for clinical trials.

Increased efficacy of a dendritic cell-based therapeutic cancer vaccine with adenosine receptor antagonist and CD73 inhibitor.

Dendritic cells are important in initiating immune responses; therefore, a range of dendritic cell-based approaches have been established to induce immune response against cancer cells. However, the presence of immunosuppressive mediators such as adenosine in the tumor microenvironment reduces the efficacy of dendritic cell-based cancer immunotherapy. In this study, we investigated whether blockade of the A2A adenosine receptor with a selective antagonist and a CD73 inhibitor may increase the efficacy of a dendritic cell-based cancer vaccine. According to the findings, this therapeutic combination reduced tumor growth, prolonged survival of tumor-bearing mice, and enhanced specific antitumor immune responses. Thus, we suggest that targeting cancer-derived adenosine improves the outcomes of dendritic cell-based cancer immunotherapy.

Study of the tumor microenvironment during breast cancer progression.

BACKGROUND: Different cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy. METHODS: In this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4+ T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7. RESULTS: The results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-α increased with tumor progression. CONCLUSION: We conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

Evaluation of TLR9 expression on PBMCs and CpG ODN-TLR9 ligation on IFN-α production in SLE patients.

CONTEXT: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoreactive antibodies. Recent findings revealed the importance of innate immune responses, especially Toll-like receptors (TLRs) in the pathogenesis of SLE. OBJECTIVE: In this study, the level of TLR9 expression on peripheral blood mononuclear cells (PBMCs) was analyzed. The levels of produced IFN-α were also measured in supernatant of PBMCs from SLE patients and healthy controls after stimulation with CpG ODN2216 which is a plasmocytoid dendritic cell (pDC)-specific TLR9 ligand. MATERIALS AND METHODS: TLR9 expression was analyzed by real-time polymerase chain reaction (PCR) and flow cytometry in 35 SLE patients and 38 healthy controls and IFN-α concentration was measured in supernatants using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results showed that the TLR9 expression in the mRNA and the protein level was significantly higher in PBMCs from SLE patients. However, IFN-α concentration in patients and controls significantly increased in response to CpG stimulation but this increase was significantly higher in healthy controls compared with SLE patients. Our results do not show any association between taking hydroxychloroquine and reduction in IFN-α production in SLE patients. DISCUSSION AND CONCLUSIONS: Regarding the findings of the study, there is the possibility that TLR9 has played a role in SLE pathogenesis, and consequently it implies that TLRs can be considered to be the therapeutic targets for systemic autoimmunity. We may conclude that PBMCs in patients are functionally impaired in response to TLR ligation via innate response stimulating pathogen-associated molecular patterns (PAMPs).

Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

Low Noncytotoxic Concentrations of 5-Fluorouracil Have No Adverse Effects on Maturation and Function of Bone Marrow-Derived Dendritic Cells in vitro: A Potentially Safe Adjuvant for Dendritic Cell-Based Cancer Therapy.

BACKGROUND: Low, noncytotoxic concentrations of various chemotherapeutic drugs like 5-fluorouracil (5-FU) induce antitumor immune responses by selectively depleting tumor-induced immunosuppressive cells, and could therefore be used in combination with dendritic cell (DC) vaccines in order to enhance their immunotherapeutic efficacy. However, the likely negative influences of low, noncytotoxic doses of 5-FU on bone marrow-derived (BM)-DCs in vitro have not yet been investigated. METHODS: The effects of low, noncytotoxic concentrations of 5-FU on mouse BM-DC differentiation and maturation markers (CD11c, MHC class II and CD80) as well as antigen-presenting capacity and cytokine production (IL-12p70 and IL-10) have been assessed. RESULTS: Different low doses of 5-FU had no significant effect on the expression of DC differentiation and maturation or on costimulatory markers (p = 0.5). Furthermore, suboptimal doses of 5-FU did not affect the immunostimulatory functions of DCs such as antigen presentation (p = 0.6) and cytokine production (p = 0.9). CONCLUSIONS: These data suggest that low doses of 5-FU have no adverse effects on DC maturation and function, and the efficacy of DC-based cancer immunotherapy may be greatly enhanced by combining it with suboptimal doses of 5-FU.

Single-Cell Spatial Analysis Identifies Regulators of Brain Tumor-Initiating Cells.

Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor-initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/ß-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan-LRP6 axis could be a therapeutic target to curb tumor growth. SIGNIFICANCE: Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor-initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.

Enhanced liver X receptor signalling reduces brain injury and promotes tissue regeneration following experimental intracerebral haemorrhage: roles of microglia/macrophages.

BACKGROUND: Inflammation-exacerbated secondary brain injury and limited tissue regeneration are barriers to favourable prognosis after intracerebral haemorrhage (ICH). As a regulator of inflammation and lipid metabolism, Liver X receptor (LXR) has the potential to alter microglia/macrophage (M/M) phenotype, and assist tissue repair by promoting cholesterol efflux and recycling from phagocytes. To support potential clinical translation, the benefits of enhanced LXR signalling are examined in experimental ICH. METHODS: Collagenase-induced ICH mice were treated with the LXR agonist GW3965 or vehicle. Behavioural tests were conducted at multiple time points. Lesion and haematoma volume, and other brain parameters were assessed using multimodal MRI with T2-weighted, diffusion tensor imaging and dynamic contrast-enhanced MRI sequences. The fixed brain cryosections were stained and confocal microscopy was applied to detect LXR downstream genes, M/M phenotype, lipid/cholesterol-laden phagocytes, oligodendrocyte lineage cells and neural stem cells. Western blot and real-time qPCR were also used. CX3CR1CreER: Rosa26iDTR mice were employed for M/M-depletion experiments. RESULTS: GW3965 treatment reduced lesion volume and white matter injury, and promoted haematoma clearance. Treated mice upregulated LXR downstream genes including ABCA1 and Apolipoprotein E, and had reduced density of M/M that apparently shifted from proinflammatory interleukin-1ß+ to Arginase1+CD206+ regulatory phenotype. Fewer cholesterol crystal or myelin debris-laden phagocytes were observed in GW3965 mice. LXR activation increased the number of Olig2+PDGFRα+ precursors and Olig2+CC1+ mature oligodendrocytes in perihaematomal regions, and elevated SOX2+ or nestin+ neural stem cells in lesion and subventricular zone. MRI results supported better lesion recovery by GW3965, and this was corroborated by return to pre-ICH values of functional rotarod activity. The therapeutic effects of GW3965 were abrogated by M/M depletion in CX3CR1CreER: Rosa26iDTR mice. CONCLUSIONS: LXR agonism using GW3965 reduced brain injury, promoted beneficial properties of M/M and facilitated tissue repair correspondent with enhanced cholesterol recycling.

Microglia-T cell conversations in brain cancer progression.

The highly immunosuppressive and heterogeneous milieu of brain malignancies contributes to their dismal prognosis. Regardless of their cellular origin, brain tumors grow in an environment with various specialized organ-resident cells. Although homeostatic microglia contribute to a healthy brain, conversations between disease-associated microglia and T cells compromise their individual and collective capacity to curb malignant growth. We review the mechanisms of T cell-microglia interactions and discuss how their collaboration fosters heterogeneity and immunosuppression in brain cancers. Because of the importance of microglia and T cells in the brain tumor microenvironment, it is crucial to understand their interactions to derive innovative therapeutics.

A Distinct Hibiscus sabdariffa Extract Prevents Iron Neurotoxicity, a Driver of Multiple Sclerosis Pathology.

Iron deposition in the brain begins early in multiple sclerosis (MS) and continues unabated. Ferrous iron is toxic to neurons, yet the therapies used in MS do not counter iron neurotoxicity. Extracts of Hibiscus sabdariffa (HS) are used in many cultures for medicinal purposes. We collected a distinct HS extract and found that it abolished the killing of neurons by iron in culture; medications used in MS were ineffective when similarly tested. Neuroprotection by HS was not due to iron chelation or anthocyanin content. In free radical scavenging assays, HS was equipotent to alpha lipoic acid, an anti-oxidant being tested in MS. However, alpha lipoic acid was only modestly protective against iron-mediated killing. Moreover, a subfraction of HS without radical scavenging activity negated iron toxicity, whereas a commercial hibiscus preparation with anti-oxidant activity could not. The idea that HS might have altered properties within neurons to confer neuroprotection is supported by its amelioration of toxicity caused by other toxins: beta-amyloid, rotenone and staurosporine. Finally, in a mouse model of MS, HS reduced disability scores and ameliorated the loss of axons in the spinal cord. HS holds therapeutic potential to counter iron neurotoxicity, an unmet need that drives the progression of disability in MS.

Reduced efficacy of multiple doses of CpG-matured dendritic cell tumor vaccine in an experimental model.

CpG motifs have been advanced as agents that stimulate the maturation of DCs for immunotherapy. The present study tested the hypothesis that multiple doses of CpG-matured DC vaccine would be efficacious for complete eradication of experimentally-induced tumor. Accordingly, WEHI164 cells were implanted subcutaneously in the flank of BALB/c mice. During DC culture, tumor lysate was added to immature DCs followed by addition of CpG or non-CpG control 4-6h later. A total of three doses of CpG or non-CpG control-matured DCs were injected around tumors. The results showed that multiple doses of CpG-matured DCs led to considerable decrease in cytotoxicity of lymphocytes and significantly increased tumor growth rate compared to a single dose. Further, mice which received three doses of the vaccine also displayed significant FoxP3 in tumor tissue. In conclusion, multiple doses of CpG-matured DCs exhibited decreased anti-tumor immunity in association with increased expression of FoxP3.

Efficacy and Safety of a Traditional Herbal Combination in Patients with Type II Diabetes Mellitus: A Randomized Controlled Trial.

BACKGROUND: Capparis spinosa, Rosa canina, Securidaca securigera, Silybum marianum, Urtica dioica, Trigonella foenum-graecum and Vaccinium arctostaphylos are used traditionally as an herbal combination for treatment of diabetic patients in Iran. Despite the clinical evidence supporting their use in solitary form, no controlled human study has determined the efficacy and safety of their combination in treatment of diabetic patients. METHODS: A total 150 type II diabetic patients of both sexes under the oral anti-hyperglycemic drugs treatment (maximum 10 mg glyburide and 1000 mg metformin daily) were randomly assigned to three groups. The patients in each group received either herbal combination or placebo or metformin capsule daily for three months, without any change in their previous oral anti-hyperglycemic drugs dosage. Herbal combination, placebo and metformin capsules matched by shape and color were prepared in the Institute of Medicinal Plants Karaj, Iran. To assess the efficacy and safety of the treatments, the patients fasting plasma glucose, HbA1c, lipid profile, liver enzymes and renal function were determined at the beginning of the study and after three months. RESULTS: Results showed that after three months, the fasting plasma glucose, HbA1c and cholesterol levels in herbal combination were decreased significantly as compared to placebo group (20% and 12% respectively) and also compared to base line (25% and 15% respectively). The herbal combination was as effective as metformin in reduction of FPG (p = 0.001, p = 0.001) and HbA1c (p = 0.028 and p = 0.050 respectively) compared to placebo. No notable hepatic, renal and gastrointestinal side effects were observed in the trial groups. CONCLUSION: The results suggest that traditional herbal combination may safely improve glycemic control in type II diabetic patients with no significant adverse effect. [Formula: see text].

Fibrinogen in the glioblastoma microenvironment contributes to the invasiveness of brain tumor-initiating cells.

Glioblastomas (GBMs) are highly aggressive, recurrent, and lethal brain tumors that are maintained via brain tumor-initiating cells (BTICs). The aggressiveness of BTICs may be dependent on the extracellular matrix (ECM) molecules that are highly enriched within the GBM microenvironment. Here, we investigated the expression of ECM molecules in GBM patients by mining the transcriptomic databases and also staining human GBM specimens. RNA levels for fibronectin, brevican, versican, heparan sulfate proteoglycan 2 (HSPG2), and several laminins were high in GBMs compared to normal brain, and this was corroborated by immunohistochemistry. While fibrinogen transcript was at normal level in GBM, its protein immunoreactivity was prominent within GBM tissues. These ECM molecules in tumor specimens were in proximity to, and surrounding BTICs. In culture, fibronectin and pan-laminin induced the adhesion of BTICs onto the plastic substratum. However, fibrinogen increased the size of the BTIC spheres by facilitating the adhesive property, motility, and invasiveness of BTICs. These features of elevated invasiveness were corroborated in resected GBM specimens by the close proximity of fibrinogen with matrix metalloproteinase (MMP)-2 and-9, which are proteases implicated in metastasis. Moreover, the effect of fibrinogen-induced invasiveness was attenuated in BTICs where MMP-2 and -9 have been inhibited with siRNAs or pharmacological inhibitors. Our results implicate fibrinogen in GBM as a mediator of the invasive properties of BTICs, and as a target for therapy to reduce BTIC tumorigenecity.

PD-1 independent of PD-L1 ligation promotes glioblastoma growth through the NFκB pathway.

Brain tumor­initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2­containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1­ or PD-L1­blocking therapies.