Losartan Blocks Osteosarcoma-Elicited Monocyte Recruitment, and Combined With the Kinase Inhibitor Toceranib, Exerts Significant Clinical Benefit in Canine Metastatic Osteosarcoma.

PURPOSE: There is increasing recognition that progress in immuno-oncology could be accelerated by evaluating immune-based therapies in dogs with spontaneous cancers. Osteosarcoma (OS) is one tumor for which limited clinical benefit has been observed with the use of immune checkpoint inhibitors. We previously reported the angiotensin receptor blocker losartan suppressed metastasis in preclinical mouse models through blockade of CCL2-CCR2 monocyte recruitment. Here we leverage dogs with spontaneous OS to determine losartan's safety and pharmacokinetics associated with monocyte pharmacodynamic endpoints, and assess its antitumor activity, in combination with the kinase inhibitor toceranib. PATIENTS AND METHODS: CCL2 expression, monocyte infiltration, and monocyte recruitment by human and canine OS tumors and cell lines were assessed by gene expression, ELISA, and transwell migration assays. Safety and efficacy of losartan-toceranib therapy were evaluated in 28 dogs with lung metastatic OS. Losartan PK and monocyte PD responses were assessed in three dose cohorts of dogs by chemotaxis, plasma CCL2, and multiplex cytokine assays, and RNA-seq of losartan-treated human peripheral blood mononuclear cells. RESULTS: Human and canine OS cells secrete CCL2 and elicit monocyte migration, which is inhibited by losartan. Losartan PK/PD studies in dogs revealed that a 10-fold-higher dose than typical antihypertensive dosing was required for blockade of monocyte migration. Treatment with high-dose losartan and toceranib was well-tolerated and induced a clinical benefit rate of 50% in dogs with lung metastases. CONCLUSIONS: Losartan inhibits the CCL2-CCR2 axis, and in combination with toceranib, exerts significant biological activity in dogs with metastatic osteosarcoma, supporting evaluation of this drug combination in patients with pediatric osteosarcoma. See related commentary by Weiss et al., p. 571.

Lysosomal Biogenesis and Implications for Hydroxychloroquine Disposition.

Lysosomes act as a cellular drug sink for weakly basic, lipophilic (lysosomotropic) xenobiotics, with many instances of lysosomal trapping associated with multiple drug resistance. Lysosomotropic agents have also been shown to activate master lysosomal biogenesis transcription factor EB (TFEB) and ultimately lysosomal biogenesis. We investigated the role of lysosomal biogenesis in the disposition of hydroxychloroquine (HCQ), a hallmark lysosomotropic agent, and observed that modulating the lysosomal volume of human breast cancer cell lines can account for differences in disposition of HCQ. Through use of an in vitro pharmacokinetic (PK) model, we characterized total cellular uptake of HCQ within the duration of static equilibrium (1 hour), as well as extended exposure to HCQ that is subject to dynamic equilibrium (>1 hour), wherein HCQ increases the size of the lysosomal compartment through swelling and TFEB-induced lysosomal biogenesis. In addition, we observe that pretreatment of cell lines with TFEB-activating agent Torin1 contributed to an increase of whole-cell HCQ concentrations by 1.4- to 1.6-fold, which were also characterized by the in vitro PK model. This investigation into the role of lysosomal volume dynamics in lysosomotropic drug disposition, including the ability of HCQ to modify its own disposition, advances our understanding of how chemically similar agents may distribute on the cellular level and examines a key area of lysosomal-mediated multiple drug resistance and drug-drug interaction. SIGNIFICANCE STATEMENT: Hydroxychloroquine is able to modulate its own cellular pharmacokinetic uptake by increasing the cellular lysosomal volume fraction through activation of lysosomal biogenesis master transcription factor EB and through lysosomal swelling. This concept can be applied to many other lysosomotropic drugs that activate transcription factor EB, such as doxorubicin and other tyrosine kinase inhibitor drugs, as these drugs may actively increase their own sequestration within the lysosome to further exacerbate multiple drug resistance and lead to potential acquired resistance.

The Angiotensin Receptor Blocker Losartan Suppresses Growth of Pulmonary Metastases via AT1R-Independent Inhibition of CCR2 Signaling and Monocyte Recruitment.

Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, growth, and angiogenesis. Monocyte recruitment to metastases is mediated primarily via the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis represents an attractive therapeutic target for the treatment of metastatic disease. Losartan, a type I angiotensin II receptor (AT1R) antagonist, has been previously shown to have immunomodulatory actions involving monocyte and macrophage activity. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, we investigated the effects of losartan and its primary metabolite on CCL2-mediated monocyte recruitment and CCR2 receptor function using mouse tumor models and in vitro human monocyte cultures. We show, in this study, that losartan and its metabolite potently inhibit monocyte recruitment through the noncompetitive inhibition of CCL2-induced ERK1/2 activation, independent of AT1R activity. Studies in experimental metastasis models demonstrated that losartan treatment significantly reduced the metastatic burden in mice, an effect associated with a significant decrease in CD11b+/Ly6C+-recruited monocytes in the lungs. Collectively, these results indicate that losartan can exert antimetastatic activity by inhibiting CCR2 signaling and suppressing monocyte recruitment and therefore suggest that losartan (and potentially other AT1R blocker drugs) could be repurposed for use in cancer immunotherapy.

Evaluation of immunological parameters in pit bull terrier-type dogs with juvenile onset generalized demodicosis and age-matched healthy pit bull terrier-type dogs.

BACKGROUND: Juvenile onset generalized demodicosis (JOGD) is thought to occur due to immunological abnormalities and is over-represented in pit bull terrier-type dogs. ANIMALS: Twelve pit bull terrier-type dogs with JOGD and 12 age-matched healthy pit bull terrier-type dogs. OBJECTIVE: To investigate immunological differences between age-matched healthy and JOGD pit bull terrier-type dogs by flow cytometry, multiplex, molecular and serological assays. METHODS AND MATERIALS: Flow cytometry quantified B cells expressing MHCII or surface-bound IgG, CD4+ T cells expressing MHCII, CD8 T cells expressing MHCII or CD11a, neutrophil and monocyte markers. Surface expression was quantified by calculating the geometric mean fluorescence index. The Wilcoxon rank sum test was used to compare median results for IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-18, FOXP3, monocyte chemoattractant protein-1, GM-CSF, KC, IgE, IgA, IgG, IgM, C-reactive protein, lymphocyte, neutrophil and monocyte in the groups. IFN-gamma, IP-10, IL-15, IL-31 and TNF-alpha also were measured; however, insufficient dogs (<5) had values that were in range of the assay to allow for statistical evaluation. Significance was defined as P < 0.05. RESULTS: Serum concentrations of IL-2, IL-18 and MCP-1 were significantly higher (P = 0.01, P = 0.01, P = 0.04) in the JOGD group. Also, IgA median value was significantly higher (P = 0.002) in pit bull terrier-type dogs with JOGD. Flow cytometry revealed that T-cell, neutrophil and monocyte markers were not different between groups. CONCLUSIONS: Results suggest an appropriate compensatory immune response by pit bull terrier-type dogs in the JOGD group and do not support the explanation of global immune deficiency in these dogs.

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.

Comparison of proliferative and immunomodulatory potential of adipose-derived mesenchymal stem cells from young and geriatric cats.

Objectives The objective of this study was to compare the ability of adipose-derived mesenchymal stem cells (aMSCs) generated from young vs geriatric cats to proliferate in culture, suppress lymphocyte proliferation and undergo senescence. Methods Adipose tissues from five young (<5 years) and six geriatric (>10 years) cats were harvested and cryopreserved for subsequent aMSC isolation and culture. aMSC proliferation in culture was compared via determination of time until passage two and by 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The immunomodulatory capacity of aMSCs was assessed using lymphocyte proliferation assays, and senescence was evaluated using senescence-associated B-galactosidase (SABG) expression. All assays were performed on aMSCs between passage two and passage three. Results aMSCs from geriatric cats took significantly longer ( P = 0.008) to reach passage two (median 11 days, range 9-22 days) compared with aMSCs from young healthy cats (median 7 days, range 6-8 days). No significant difference was detected between young and geriatric cats in terms of their ability to suppress lymphocyte proliferation. SABG expression was not significantly different between young and geriatric aMSCs. Conclusions and relevance Compared with young feline aMSCs, geriatric aMSCs are significantly impaired in their ability to rapidly proliferate to passage two following initial culture, presenting a concern for autologous therapy. Nonetheless, once the cells are expanded, young and geriatric cat aMSCs appear to be equivalent in terms of their ability to functionally suppress T-cell activation and proliferation.