Suppression of canine myeloid cells by soluble factors from cultured canine tumor cells

Abstract

Background: Cancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function.

Methods: These studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines.

Results: We found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells.

Conclusion: These results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.

Fig. 1

Phenotypic and morphological characteristics of canine myeloid cells. (A) The morphology of canine myeloid cell populations by inverted microscopy. (B) Cell surface expression of CD11b and CD11c of these canine myeloid cell populations.

Fig. 2

MHC class II and CD80 expression in the canine DH82 myeloid cell line. Canine DH82 myeloid cells were fully capable of increasing expression of both (A) CD80 and (B) MHC class II in response to 24 h exposure to LPS. Increased expression levels of both CD80 and MHC class II are seen upon LPS (gray) stimulation compared to unstimulated (black) media controls.

Fig. 3

MHC class II expression in canine DCs. Both canine (A) BM-DCs or (B) DH82 cells had decreased numbers of cells expressing MHC class II.

Fig. 4

CD80 expression in canine BM-DCs following 72-h exposure to TCMs. Both the percentage of CD80 positive cells and relative expression levels (as determined by mean fluorescence intensity; MFI) was decreased in DCs exposed to OSA and MEL TCMs.

Fig. 5

Phagocytosis of fluorescently labeled beads by canine myeloid cells exposed to tumor-derived soluble factors (TDSFs). The top panels show both the percentage of cells containing phagocytosed beads where the M2 gate represents the overall percentage of DH82 cells that have phagocytosed beads and are PE positive. Mean fluorescent intensity (MFI), determined from PE positive (M2-gated) DH82 cells, detects the relative PE intensity of individual cells and represents the relative number of fluorescent beads phagocytosed by the DH82 cells. The bottom panels are images of cultured DH82 cells containing intracellular phagocytosed particles taken by an inverted fluorescent microscope.

Fig. 6

Stimulation of canine spleen cell proliferation by various antigens in the presence of DH82 canine myeloid cells cultured in normal media (DH82) and in the presence of canine melanoma TCM (MEL). Cells were cultured (in triplicate) in the presence of either media, Salmonella sub-type typhimurium lipopolysaccharide (LPS), phytohemagglutinin (PHA), and concanavalin A (ConA). Proliferation was suppressed at a 1:5 ratio (melanoma-conditioned myeloid cells versus spleen cells) while suppression was lost at a ratio of 1:10 (p < 0.05). There was significantly increased suppression (17.3 ± 7.9%; average ± SEM) for melanoma-exposed myeloid cells (p < 0.01; t-test where average ± SEM were each calculated using triplicate values. Similar results were obtained from three independent experiments, each having triplicates for each treatment group.

Fig. 7

Failure of DH82 cells to upregulate nitric oxide production following LPS stimulation. Nitric oxide levels remained low across a broad range of LPS concentrations.

Fig. 8

MHC class II expression levels of BM-DCs differentiated in the presence or absence of TCM (OSA or MEL) from two individual (separate) experiments. There was consistent downregulation of MHC class II expression in BM-DCs differentiated in the presence of either OSA or MEL TCM.

Fig. 9

MHC class II expression levels of BM-MΦs differentiated in the presence or absence of TCM (OSA or MEL) from two individual (separate) experiments. (A) BM-MΦs differentiated in the presence of OSA TCM had variable expression patterns of MHC class II between experiments while (B) BM-MΦs differentiated with MEL TCM showed consistently decreased MHC class II expression between experiments.