Decoding the interactions between cancer cells and monocyte-derived macrophages in a co-culture model using flow cytometry and bead-based multiplexing immunoassay panels

Macrophages play a pivotal role in the immune response and in the maintenance of tissue homeostasis. It is well known that many tumors recruit monocytes from circulation and transform them into an immunosuppressive subset called Tumor-Associated Macrophages (TAMs). TAMs are highly plastic and can alter their phenotypes between M1 pro-inflammatory and M2 anti-inflammatory according to their location and surrounding cytokine milieu in the tumor microenvironment. M1 macrophages are associated with the recognition and destruction of cancer cells whereas M2 macrophages are thought to promote proliferation, angiogenesis, and metastasis of cancer cells. In this study, monocytes-derived THP1 macrophages were cultured with conditioned medium collected from various cancer cell lines, such as colon cancer cell line HT29, lung cancer cell line A549 and breast cancer cell line MDA-MB-231, to study the influence of cancer cells and their secreted factors on the activation and differentiation of monocyte-derived macrophages. The phenotypes of the macrophages in the culture were characterized by flow cytometric analysis of macrophage surface markers including CD86, CD206, CD163, CD274, CD11b, CD14 and CD40, and the soluble factors produced by them including the M1 and M2 markers were measured using the LEGENDplex ™ Human Macrophage Panel and Human Tumor-Associated Macrophage Panel. In a parallel study, macrophages were activated and polarized into M1 and M2 phenotypes, followed by co-culture with cancer cells. The proliferation and viability of cancer cells were measured at the end of the incubation by flow cytometry to assess the distinct influences of M1 and M2 macrophages on cancer cell cultures. Our data suggests that cancer cells could release soluble factors to actively influence the phenotypes of monocyte-derived macrophages. Moreover, the study demonstrated that M1 and M2 macrophages may have different impact on cultured cancer cell's viability and proliferation. This in vitro study enhances our understanding of the interplay between cancer cells and tumor-associated macrophages, underscoring the utility of LEGENDplex TM panels in studying tumor-associated macrophages.