Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b+CD14−MHCII− cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b+ CD14−MHCII− cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden

Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1.

Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies

Creation of an NCI comparative brain tumor consortium: informing the translation of new knowledge from canine to human brain tumor patients

On September 14–15, 2015, a meeting of clinicians and investigators in the fields of veterinary and human neuro-oncology, clinical trials, neuropathology, and drug development was convened at the National Institutes of Health campus in Bethesda, Maryland. This meeting served as the inaugural event launching a new consortium focused on improving the knowledge, development of, and access to naturally occurring canine brain cancer, specifically glioma, as a model for human disease. Within the meeting, a SWOT (strengths, weaknesses, opportunities, and threats) assessment was undertaken to critically evaluate the role that naturally occurring canine brain tumors could have in advancing this aspect of comparative oncology aimed at improving outcomes for dogs and human beings. A summary of this meeting and subsequent discussion are provided to inform the scientific and clinical community of the potential for this initiative. Canine and human comparisons represent an unprecedented opportunity to complement conventional brain tumor research paradigms, addressing a devastating disease for which innovative diagnostic and treatment strategies are clearly needed

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-alpha and IFN-gamma. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials

Does Adipose-derived Stromal Cell Adjuvant Therapy for Fragmented Medial Coronoid Process in Dogs Influence Outcome? A Pilot Project

Objective: The primary objective of this study was to identify adverse events associated with multiple intra-articular injections of adipose stromal cell (ASC) therapy and secondarily to objectively assess the therapeutic effect of ASC therapy for treatment of fragmented medial coronoid process (FMCP) in dogs when used as an adjuvant to standard of care (SOC) treatment. Background: Preliminary trials assessing autologous ASC therapy to treat osteoarthritis indicate a positive impact on clinical signs, but assessment of donated, allogeneic ASC therapy is lacking.Evidentiary value: This prospective, randomised, controlled trial in dogs (n=30) provides objective evidence for clinical practitioners regarding ASC therapy in a naturally occurring osteoarthritic disease model.Methods: Dogs diagnosed with FMCP and osteoarthritis were enrolled. All dogs had arthroscopic fragment removal and proximal ulnar osteotomy (PUO) and were assigned into three groups (n=10/group): 1) control group with no further treatment beyond the PUO and fragment removal (SOC), 2) PUO + autologous ASCs and 3) PUO+ allogeneic ASCs. Each dog had force platform gait analysis, Canine Brief Pain Inventory (CBPI) questionnaires, and delayed gadolinium enhanced magnetic resonance imaging scores prior to and six months after therapeutic intervention.Results: No serious adverse events were reported in any participant. 3/10 dogs in the control group, 3/10 autologous ASC group and 7/10 allogeneic ASC group participants were assessed as successful outcomes.Conclusion: This study provides preliminary safety data for the use of intra-articular allogeneic ASC therapy to treat osteoarthritis, and justification for larger clinical studies.Application: Clinical practitioners considering ASC therapy within their practice are provided with additional evidence of autologous ASC therapy for osteoarthritis. Researchers committed to developing and generating effective ASC therapies are provided with safety information for allogeneic ASC, as well as identified biases important for study design. <img src="https://www.veterinaryevidence.org/rcvskmod/icons/pr-icon.jpg" alt="Peer Reviewed" /

CD11b⁺CD14⁻MHCII⁻ cells suppress T cell proliferation and cytokine elaboration.

<p>CD11b⁺CD14⁻MHCII⁻ cells were sorted from peripheral blood sample of dogs with cancer and then co-cultured with autologous PBMCs (A, B) or healthy dog PBMCs (C) in the presence of mitogen for 72 hs. Representative examples from a total of eight dogs are shown. The graphs represent proliferative responses after addition of CD11b⁺CD14⁻MHCII⁻ isolated from a single dog with squamous cell carcinoma (3A), prostatic adenocarcinoma (3B) and osteosarcoma (3C). Non-stimulated PBMCs were used as negative control and PBMCs stimulated in absence of CD11b⁺CD14⁻MHCII⁻ cells were used as positive control for proliferation. PBMCs were also co-incubated with PMNs, to control for presence of additional cells (3C, 3D). Proliferative responses were measured by ³H-thymidine incorporation. CPM, counts per minute. Amount of IFN-γ secretion in the co-culture was determined using canine specific IFN-γ ELISA assay (3D). All experiments were performed in triplicate. Mean ± SEM are shown.</p

Characteristics of dogs with cancer in the study.

<p>Characteristics of dogs with cancer in the study.</p

Percentages of circulating CD11b⁺CD14⁻MHCII⁻ cells in dogs with correlates with clinical tumor stage.

<p>(A) Analysis of average CD11⁺CD14⁻MHCII⁻ population frequency in dogs with advanced stage or metastatic tumors (n = 30) compared with early stage non-metastatic tumors (n = 15) and control dogs (n = 18). There was a significantly higher percentage of CD11b⁺CD14⁻MHCII⁻ cells in dogs with advanced cancer versus early stage non-metastatic tumors and healthy dogs (36.04% vs. 9.40% and10.24%, respectively. B) Average CD11b⁺CD14⁻MHCII⁻ population frequency in the major cancer subtypes: advanced stage or metastatic sarcomas (n = 18), early stage non-metastatic sarcomas (n = 6), advanced stage or metastatic carcinomas (n = 7) early stage non-metastatic carcinomas (n = 7), advanced stage or metastatic mast cell tumors (n = 5) and early stage non-metastatic mast cell tumors (n = 2) compared with control dogs (n = 18). Significantly elevated percentages were detected in all advanced tumors subtypes relative to early stage tumors and healthy dogs. Percentages of CD11b⁺CD14⁺MHCII⁻ cells were not significant between groups (* indicates P<0.001). Mean ± SEM are shown.</p