Changes in gene expression profiles and cytokine secretions in peripheral monocytes by treatment with small extracellular vesicles derived from a canine lymphoma cell line

Abstract

Interactions between tumor and immune cells within the tumor microenvironment play an important role in tumor progression, and small extracellular vesicles (EVs) derived from these tumor cells have been shown to exert immunomodulatory effects on various immune cells, including macrophages and lymphocytes. Although the immunomodulatory effects of small EVs derived from human cancer cells have been intensively investigated, few studies have investigated the effects of lymphoma-derived small EVs on macrophages in both human and veterinary medicine. Here, we evaluated the effects of canine lymphoma-derived small EVs on canine primary monocytes, which are the major source of macrophages in neoplastic tissues. Comprehensive gene expression analysis of these treated monocytes revealed their distinct activation via the Toll-like receptor (TLR) and NF-κβ signaling pathways. In addition, treatment with lymphoma small EVs increased the secretion of MCP-1, which induces the infiltration and migration of monocytes and lymphocytes in neoplastic and cancer tissues. The results of this study indicate that canine lymphoma small EVs activate monocytes, possibly through the activation of TLR and NF-κβ signaling pathways, and induce monocytes to secrete of MCP-1, which might contribute to immune cell infiltration within the tumor microenvironment.

Keywords: diffuse large B-cell lymphoma; dog; monocyte chemotactic protein-1; nuclear factor-kappa β signaling; toll-like receptor signaling.

Fig. 1.

Extractions of differentially expressed genes (DEGs) and comparisons of gene expression profiles between small extracellular vesicle (EV)-treated and control monocytes. (A) Volcano plot showing DEGs in small EV-treated monocytes compared with control monocytes. Red dots indicate DEGs. (B) Hierarchical clustering using DEGs in small EV-treated monocytes.

Fig. 2.

Comparisons of the expression levels of differentially expressed genes in small extracellular vesicle (EV)-treated and control monocytes using RT-qPCR. Significant differences in relative expression levels were observed in upregulated genes; Interleukin (IL)-1β, IL6, and Tumor necrosis factor (TNF) α, and downregulated genes; Integrin beta 5 (ITGB5), C-X-C motif chemokine ligand 12 (CXCL12), and OTU deubiquitinase 1 (OTUD1). N=3 for all tests. * indicates P<0.05. Data are shown as the mean ± SD.

Fig. 3.

Histograms showing results of gene ontology analyses and pathway analysis using differentially expressed genes extracted from the comparisons of small extracellular vesicle (EV)-treated and control monocytes. (A) Histogram showing the enriched gene ontology biological process terms using differentially expressed genes in small EV-treated monocytes. (B) Histogram showing the enriched gene ontology cellular component terms using differentially expressed genes in small EV-treated monocytes. (C) Histogram showing the enriched gene ontology molecular function terms using differentially expressed genes in small EV-treated monocytes. (D) Histogram showing enriched pathways identified by KEGG pathway enrichment analysis of the differentially expressed genes in small EV-treated monocytes.

Fig. 4.

Histogram showing the enriched pathways in the canonical pathway analysis by Ingenuity Pathway Analysis (IPA) and the differentially expressed genes in small extracellular vesicle (EV)-treated monocytes. Red bar indicates a z-score of >2. Blue bar indicates a z-score of <−2.

Fig. 5.

Comparisons of the concentrations of 10 cytokines in the culture supernatant of control and small extracellular vesicle (EV)-treated monocytes. N=3 for all tests. * indicates P<0.05. Data are shown as the mean + SD.