Authors:

Nathan Newman

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About Paper:

Cancer immunotherapy utilizes the patient's immune system to target cancer cells. However, cancer cells often induce an immunosuppressive tumor microenvironment (TME) that helps them avoid detection and subsequent targeting by the body's immune system. One such mechanism is recruiting M2 macrophages to the site of the malignancy. There are two broad types of macrophages, namely M1 (pro-inflammatory) and M2 (anti-inflammatory). M2 macrophages dampen the antitumor immune response by releasing cytokines and growth factors to inhibit the function of cytotoxic T cells and natural killer (NK) cells. In contrast, M1 macrophages play a crucial role in recognizing and eliminating cancer cells by presenting tumor antigens to T cells, generating an effective antitumor immune response. The main goal of this study is to generate tumor-associated macrophages (TAMs) from canine monocytes and to comprehensively characterize them both functionally and phenotypically. To do this, canine peripheral blood mononuclear cells were isolated from healthy dogs, and monocytes were enriched using anti-CD14 magnetic beads via magnetic-activated cell sorting (MACS). The purified monocytes were cultured in RPMI medium supplemented with specific growth factors to differentiate into M1 (GM- CSF, IFN-γ & LPS) and M2 (M-CSF, IL-4 & IL-10) macrophages. For TAM differentiation, the monocytes were treated with tumor-conditioned media (TCM) derived from canine melanoma cells and supplemented with M-CSF, IL-4 & IL-10. The resulting M1, M2, and TAM populations were characterized based on expression of various immunomarkers, including CD14, CD206, MHCII, CD163, CD80, and CD86. Additionally, gene expression profiles were assessed through qPCR. By using MACS, a high purity sample of monocytes was collected. Murine, human and canine M2 and TAMs are defined by their co- expression of CD163 and CD206. Meanwhile, no such co-expression was detected on M1 macrophages. Therefore, our protocol successfully isolates and identifies canine M1, M2 and TAMs macrophages. We will evaluate the effect of CD206-targeted peptides on canine M2 and TAMs compared to M1 macrophages in the future.

Source:

Auburn University / College of Sciences and Mathematics / 2025

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Co-authors:

Nathan Newman