Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs.

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME). As such, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 41 transcriptomically distinct cell types including the characterization of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 tumor-associated macrophage (TAM) populations. Cell-cell interaction analysis predicted that mregDCs and TAMs play key roles in modulating T cell mediated immunity. Furthermore, we completed cross-species cell type gene signature homology analysis and found a high degree of similarity between human and canine OS. The data presented here act as a roadmap of canine OS which can be applied to advance translational immuno-oncology research.

A single-cell RNA sequencing atlas of circulating leukocytes from healthy and osteosarcoma affected dogs.

Translationally relevant animal models are essential for the successful translation of basic science findings into clinical medicine. While rodent models are widely accessible, there are numerous limitations that prevent the extrapolation of findings to human medicine. One approach to overcome these limitations is to use animal models that are genetically diverse and naturally develop disease. For example, pet dogs spontaneously develop diseases that recapitulate the natural progression seen in humans and live in similar environments alongside humans. Thus, dogs represent a useful animal model for many areas of research. Despite the value of the canine model, species specific reagent limitations have hampered in depth characterization of canine immune cells, which constrains the conclusions that can be drawn from canine immunotherapy studies. To address this need, we used single-cell RNA sequencing to characterize the heterogeneity of circulating leukocytes in healthy dogs (n = 7) and osteosarcoma (OS) affected dogs (n = 10). We present a cellular atlas of leukocytes in healthy dogs, then employ the dataset to investigate the impact of primary OS tumors on the transcriptome of circulating leukocytes. We identified 36 unique cell populations amongst dog circulating leukocytes, with a remarkable amount of heterogeneity in CD4 T cell subtypes. In our comparison of healthy dogs and dogs with OS, we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) myeloid-derived suppressor cells (MDSCs), as well as aberrations in gene expression within myeloid cells. Overall, this study provides a detailed atlas of canine leukocytes and investigates how the presence of osteosarcoma alters the transcriptional profiles of circulating immune cells.

Chronic adrenergic stress and generation of myeloid-derived suppressor cells: Implications for cancer immunotherapy in dogs.

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for ß-adrenergic receptor signalling in suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.

Reprogramming the Canine Glioma Microenvironment with Tumor Vaccination plus Oral Losartan and Propranolol Induces Objective Responses.

Purpose: Malignant gliomas have a highly immune suppressive tumor microenvironment (TME) which renders them largely unresponsive to conventional therapeutics. Therefore, the present study evaluated a therapeutic protocol designed overcome the immune barrier by combining myeloid cell targeted immunotherapy with tumor vaccination. Experimental Design: We utilized a spontaneously occurring canine glioma model to investigate an oral TME modifying immunotherapy in conjunction with cancer stem cell (CSC) vaccination. Dogs were treated daily with losartan (monocyte migration inhibitor) and propranolol (myeloid-derived suppressor cell depleting agent) plus anti-CSC vaccination on a bi-weekly then monthly schedule. Tumor volume was monitored by MRI and correlated with patient immune responses. Results: Ten dogs with histologically confirmed gliomas were enrolled into a prospective, open-label clinical trial to evaluate the immunotherapy protocol. Partial tumor regression was observed in 2 dogs, while 6 dogs experienced stable disease, for an overall clinical benefit rate of 80%. Overall survival times (median = 351 days) and progression-free intervals (median = 163 days) were comparable to prior studies evaluating surgical debulking followed by immunotherapy. Dogs with detectable anti-CSC antibody responses had an increased overall survival time relative to dogs that did not generate antibody responses (vaccine responder MST = 500 days; vaccine non-responder MST = 218 days; p = 0.02). Conclusions: These findings suggest that combining myeloid cell targeted oral immunotherapy with tumor vaccination can generate objective tumor responses, even in the absence of conventional therapy. Overall, this approach has promise as a readily implemented therapeutic strategy for use in brain cancer patients.

Programmed Cell Death Ligand 1 (PD-L1) Signaling Regulates Macrophage Proliferation and Activation.

Tumor-associated macrophages (TAMs) express programmed cell death ligand 1 (PD-L1) and contribute to the immune-suppressive tumor microenvironment. Although the role of the PD-L1 and PD-1 interaction to regulate T-cell suppression is established, less is known about PD-L1 signaling in macrophages and how these signals may affect the function of TAMs. We used in vitro and in vivo models to investigate PD-L1 signaling in macrophages and the effects of PD-L1 antibody treatment on TAM responses. Treatment of mouse and human macrophages with PD-L1 antibodies increased spontaneous macrophage proliferation, survival, and activation (costimulatory molecule expression, cytokine production). Similar changes were observed in macrophages incubated with soluble CD80 and soluble PD-1, and in PD-L1-/- macrophages. Macrophage treatment with PD-L1 antibodies upregulated mTOR pathway activity, and RNAseq analysis revealed upregulation of multiple macrophage inflammatory pathways. In vivo, treatment with PD-L1 antibody resulted in increased tumor infiltration with activated macrophages. In tumor-bearing RAG-/- mice, upregulated costimulatory molecule expression by TAMs and reduced tumor growth were observed. Combined PD-1/ PD-L1 antibody treatment of animals with established B16 melanomas cured half of the treated mice, whereas treatment with single antibodies had little therapeutic effect. These findings indicate that PD-L1 delivers a constitutive negative signal to macrophages, resulting in an immune-suppressive cell phenotype. Treatment with PD-L1 antibodies reverses this phenotype and triggers macrophage-mediated antitumor activity, suggesting a distinct effect of PD-L1, but not PD-1, antibody treatment. Cancer Immunol Res; 6(10); 1260-73. ©2018 AACR.