Computed tomography findings in a cohort of 169 dogs with elbow dysplasia - a retrospective study.

BACKGROUND: Canine elbow dysplasia (CED) is a complex developmental skeletal disorder associated with a number of pathological conditions within the cubital joint. Because CED is a heritable disease, it is important to identify and remove the affected animals from breeding. The first objective of this study was to describe the prevalence of medial coronoid process disease (MCPD) without (MCD) or with (FMCP) fragmented medial coronoid process, osteochondrosis (OC) and/or osteochondritis dissecans (OCD), ununited anconeal process (UAP), radio-ulnar incongruence (INC R-U) and humero-ulnar incongruence (INC H-U) in dogs with the use of CT imaging. The second aim was to determine the influence of demographics on the prevalence of investigated pathologies in dogs with clinical evidence of elbow dysplasia. RESULTS: In this retrospective study, CT data records of 169 dogs of different breeds presented to the small animal veterinary clinic from 2012 to 2018 were included. 69.23% of dogs diagnosed with CED were young (≤ 2 years old). The mean age of dogs presented with INC R-U was 1.68 ± 1.82 years, while in dogs without INC R-U the mean age was 2.64 ± 2.59 years. The mean age of dogs with INC H-U was 1.94 ± 2.06 years, while without INC H-U 3.29 ± 2.09 years. Labrador Retrievers, German Shepherd and Bernese Mountain dogs were most frequently presented with CED-associated lameness. In 122 dogs OA of varying severity was found. CONCLUSION: INC H-U, FMCP and MCD were among the most frequently found components of CED found in the present study. OCD and UAP were the least frequently diagnosed. Dogs presented with INC R-U and INC H-U were significantly younger than dogs without these CED components. Boxers, Dog de Bordeaux, American Staffordshire terriers and mixed-breed dogs were diagnosed later in life than the other breeds. OA of varying severity was found in 72.18% of dogs. Males accounted for more than 75% of the study population.

Neural stem cells secreting bispecific T cell engager to induce selective antiglioma activity.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. No treatment provides durable relief for the vast majority of GBM patients. In this study, we've tested a bispecific antibody comprised of single-chain variable fragments (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this bispecific T cell engager (BiTE) (BiTELLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patients' tumors and, in so doing, exerts anti-GBM activity ex vivo. The interaction of BiTELLON with T cells and IL13Rα2-expressing GBM cells stimulates T cell proliferation and the production of proinflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα). We have modified neural stem cells (NSCs) to produce and secrete the BiTELLON (NSCLLON). When injected intracranially in mice with a brain tumor, NSCLLON show tropism for tumor, secrete BiTELLON, and remain viable for over 7 d. When injected directly into the tumor, NSCLLON provide a significant survival benefit to mice bearing various IL13Rα2+ GBMs. Our results support further investigation and development of this therapeutic for clinical translation.

Mesenchymal Stem Cells Successfully Deliver Oncolytic Virotherapy to Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. EXPERIMENTAL DESIGN: Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. RESULTS: Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). CONCLUSIONS: Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

BET inhibition increases ßIII-tubulin expression and sensitizes metastatic breast cancer in the brain to vinorelbine.

Metastases from primary breast cancer result in poor survival. ßIII-tubulin (TUBB3) has been established as a therapeutic target for breast cancer metastases specifically to the brain. In this study, we conducted a systematic analysis to determine the regulation of TUBB3 expression in breast cancer metastases to the brain and strategically target these metastases using vinorelbine (VRB), a drug approved by the U.S. Food and Drug Administration (FDA). We found that human epidermal growth factor receptor 2 (HER2) signaling regulates TUBB3 expression in both trastuzumab-sensitive and trastuzumab-resistant neoplastic cells. We further discovered that bromodomain and extra-terminal domain (BET) inhibition increases TUBB3 expression, rendering neoplastic cells more susceptible to apoptosis by VRB. Orthotopic xenograft assays using two different breast cancer cell models revealed a reduction in tumor volume with BET inhibition and VRB treatment. In addition, in vivo studies using a model of multiple brain metastasis (BM) showed improved survival with the combination of radiation + BET inhibitor (iBET-762) + VRB (75% long-term survivors, P < 0.05). Using in silico analysis and BET inhibition, we found that the transcription factor myeloid zinc finger-1 (MZF-1) protein binds to the TUBB3 promoter. BET inhibition decreases MZF-1 expression and subsequently increases TUBB3 expression. Overexpression of MZF-1 decreases TUBB3 expression and reduces BM in vivo, whereas its knockdown increases TUBB3 expression in breast cancer cells. In summary, this study demonstrates a regulatory mechanism of TUBB3 and provides support for an application of BET inhibition to sensitize breast cancer metastases to VRB-mediated therapy.

GCN2 is essential for CD8+ T cell survival and function in murine models of malignant glioma.

Amino acid deprivation is a strategy that malignancies utilize to blunt anti-tumor T-cell immune responses. It has been proposed that amino acid insufficiency in T-cells is detected by GCN2 kinase, which through phosphorylation of EIF2α, shuts down global protein synthesis leading to T-cell arrest. The role of this amino acid stress sensor in the context of malignant brain tumors has not yet been studied, and may elucidate important insights into the mechanisms of T-cell survival in this harsh environment. Using animal models of glioblastoma and animals with deficiency in GCN2, we explored the importance of this pathway in T-cell function within brain tumors. Our results show that GCN2 deficiency limited CD8+ T-cell activation and expression of cytotoxic markers in two separate murine models of glioblastoma in vivo. Importantly, adoptive transfer of antigen-specific T-cells from GCN2 KO mice did not control tumor burden as well as wild-type CD8+ T-cells. Our in vitro and in vivo data demonstrated that reduction in amino acid availability caused GCN2 deficient CD8+ T-cells to become rapidly necrotic. Mechanistically, reduced CD8+ T-cell activation and necrosis was due to a disruption in TCR signaling, as we observed reductions in PKCθ and phoshpo-PKCθ on CD8+ T-cells from GCN2 KO mice in the absence of tryptophan. Validating these observations, treatment of wild-type CD8+ T-cells with a downstream inhibitor of GCN2 activation also triggered necrosis of CD8+ T-cells in the absence of tryptophan. In conclusion, our data demonstrate the vital importance of intact GCN2 signaling on CD8+ T-cell function and survival in glioblastoma.

Therapeutic targeting of tumor-associated myeloid cells synergizes with radiation therapy for glioblastoma.

Tumor-associated myeloid cells (TAMCs) are key drivers of immunosuppression in the tumor microenvironment, which profoundly impedes the clinical response to immune-dependent and conventional therapeutic modalities. As a hallmark of glioblastoma (GBM), TAMCs are massively recruited to reach up to 50% of the brain tumor mass. Therefore, they have recently been recognized as an appealing therapeutic target to blunt immunosuppression in GBM with the hope of maximizing the clinical outcome of antitumor therapies. Here we report a nano-immunotherapy approach capable of actively targeting TAMCs in vivo. As we found that programmed death-ligand 1 (PD-L1) is highly expressed on glioma-associated TAMCs, we rationally designed a lipid nanoparticle (LNP) formulation surface-functionalized with an anti-PD-L1 therapeutic antibody (αPD-L1). We demonstrated that this system (αPD-L1-LNP) enabled effective and specific delivery of therapeutic payload to TAMCs. Specifically, encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into PD-L1-targeted LNPs led to a robust depletion of TAMCs and an attenuation of their immunosuppressive functions. Importantly, the delivery efficiency of PD-L1-targeted LNPs was robustly enhanced in the context of radiation therapy (RT) owing to the RT-induced up-regulation of PD-L1 on glioma-infiltrating TAMCs. Accordingly, RT combined with our nano-immunotherapy led to dramatically extended survival of mice in 2 syngeneic glioma models, GL261 and CT2A. The high targeting efficiency of αPD-L1-LNP to human TAMCs from GBM patients further validated the clinical relevance. Thus, this study establishes a therapeutic approach with immense potential to improve the clinical response in the treatment of GBM and warrants a rapid translation into clinical practice.

Myeloid-Derived Suppressive Cells Promote B cell-Mediated Immunosuppression via Transfer of PD-L1 in Glioblastoma.

The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFß and IL10. Local delivery of B cell-depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell-mediated immunosuppression.See related Spotlight on p. 1902.

Pharmacologic modulation of nasal epithelium augments neural stem cell targeting of glioblastoma.

Glioblastoma (GBM) remains the most lethal and untreatable central nervous system malignancy. The challenges to devise novel and effective anti-tumor therapies include difficulty in locating the precise tumor border for complete surgical resection, and rapid regrowth of residual tumor tissue after standard treatment. Repeatable and non-invasive intranasal application of neural stem cells (NSCs) was recently shown to enable clinically relevant delivery of therapy to tumors. Treatment with chemotactic NSCs demonstrated significant survival benefits when coupled with radiation and oncolytic virotherapy in preclinical models of GBM. In order to further augment the clinical applicability of this novel therapeutic platform, we postulate that the FDA-approved compound, methimazole (MT), can be safely utilized to delay the nasal clearance and improve the ability of NSCs to penetrate the olfactory epithelium for robust in vivo brain tumor targeting and therapeutic actions. METHODS: To examine the role of reversible reduction of the olfactory epithelial barrier in non-invasive intranasal delivery, we explored the unique pharmacologic effect of MT at a single dosage regimen. In our proof-of-concept studies, quantitative magnetic resonance imaging (MRI), immunocytochemistry, and survival analysis were performed on glioma-bearing mice treated with a single dose of MT prior to intranasal anti-GBM therapy using an oncolytic virus (OV)-loaded NSCs. RESULTS: Based on histology and in vivo imaging, we found that disrupting the olfactory epithelium with MT effectively delays clearance and allows NSCs to persist in the nasal cavity for at least 24 h. MT pretreatment amplified the migration of NSCs to the tumor. The therapeutic advantage of this enhancement was quantitatively validated by tissue analysis and MRI tracking of NSCs loaded with superparamagnetic iron oxide nanoparticles (SPIOs) in live animals. Moreover, we observed significant survival benefits in GBM-bearing mice treated with intranasal delivery of oncolytic virus-loaded NSCs following MT injection. CONCLUSION: Our work identified a novel pharmacologic strategy to accelerate the clinical application of the non-invasive NSCs-based therapeutic platform to tackle aggressive brain tumors.

Local Application of Autologous Platelet-Rich Fibrin Patch (PRF-P) Suppresses Regulatory T Cell Recruitment in a Murine Glioma Model.

The immunosuppressive microenvironment is one of the major factors promoting the growth of glioblastoma multiforme (GBM). Infiltration of CD4+CD25+Foxp3+ regulatory T cells (Tregs) into the tumor microenvironment plays a significant role in the suppression of the anti-tumor immunity and portends a dismal prognosis for patients. Glioma-mediated secretion of chemo-attractant C-C motif ligand 2 and 22 (CCL2/22) has previously been shown by our group to promote Treg migration in vitro. In this study, we show that a local implantation of platelet-rich fibrin patch (PRF-P) into the brain of GL261 glioma-bearing mice prolonged the survival of affected animals by 42.85% (p = 0.0011). Analysis performed on brain tumor tissue harvested from PRF-P-treated mice revealed a specific decrease in intra-tumoral lymphocytes with a preferential depletion of immunosuppressive Tregs. Importantly, co-culture of GL261 or chemo-attractants (CCL2/22) with PRF-P abrogated Treg migration. Pharmacological blockade of the CCL2/22 interaction with their receptors potentiated the inhibitory effect of PRF-P on Tregs recruitment in culture. Moreover, our findings revealed the soluble CD40 ligand (sCD40L) as a major Treg inhibitory player produced by activated platelets entrapped within the fibrin matrix of the PRF-P. Blockade of sCD40L restored the migratory capacity of Tregs, emphasizing the role of PRF-P in preventing the Treg migration to glioma tissue. Our findings highlight autologous PRF-P as a personalized, Treg-selective suppression platform that can potentially supplement and enhance the efficacy of glioma therapies.

Adoptive Transfer of IL13Rα2-Specific Chimeric Antigen Receptor T Cells Creates a Pro-inflammatory Environment in Glioblastoma.

In order to fully harness the potential of immunotherapy with chimeric antigen receptor (CAR)-modified T cells, pre-clinical studies must be conducted in immunocompetent animal models that closely mimic the immunosuppressive malignant glioma (MG) microenvironment. Thus, the goal of this project was to study the in vivo fate of T cells expressing CARs specific for the MG antigen IL13Rα2 (IL13Rα2-CARs) in immunocompetent MG models. Murine T cells expressing IL13Rα2-CARs with a CD28.ζ (IL13Rα2-CAR.CD28.ζ) or truncated signaling domain (IL13Rα2-CAR.Δ) were generated by retroviral transduction, and their effector function was evaluated both in vitro and in vivo. IL13Rα2-CAR.CD28.ζ T cells' specificity toward IL13Rα2 was confirmed through cytokine production and cytolytic activity. In vivo, a single intratumoral injection of IL13Rα2-CAR.CD28.ζ T cells significantly extended the survival of IL13Rα2-expressing GL261 and SMA560 glioma-bearing mice; long-term survivors were resistant to re-challenge with IL13Rα2-negative and IL13Rα2-positive tumors. IL13Rα2-CAR.CD28.ζ T cells proliferated, produced cytokines (IFNγ, TNF-α), and promoted a phenotypically pro-inflammatory glioma microenvironment by inducing a significant increase in the number of CD4+ and CD8+ T cells and CD8α+ dendritic cells and a decrease in Ly6G+ myeloid-derived suppressor cells (MDSCs). Our data underline the significance of CAR T cell studies in immunocompetent hosts and further validate IL13Rα2-CAR T cells as an efficacious therapeutic strategy for MG.

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease.

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

CCL2 Produced by the Glioma Microenvironment Is Essential for the Recruitment of Regulatory T Cells and Myeloid-Derived Suppressor Cells.

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671-82. ©2016 AACR.

Dysfunction of platelet-derived growth factor receptor α (PDGFRα) represses the production of oligodendrocytes from arylsulfatase A-deficient multipotential neural precursor cells.

The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination.