Macrophages have been associated with invasive breast cancer, but immunotherapy targeting tumor associated macrophages (TAMs) in the tumor microenvironment has been unsuccessful to date. However, emerging data indicates that macrophages may play a role in early breast cancer and thus be a promising avenue towards prevention.
TAMs are a largely heterogeneous population that play a tumor promoting (M2-like) and tumor suppressive (M1-like) role in the tumor microenvironment, but little data exists on macrophages in high risk normal breast and early breast cancer. We found that frequently used tumor promoting TAM markers such as CD206+, CD86-, iNOS- do not adequately identify tumorigenic TAM subsets in ductal carcinoma in situ (DCIS) from mouse mammary glands. Thus to identify the TAM populations in the mouse mammary gland at early stages of breast cancer we aim to couple a new myeloid specific FACS panel to identify the surface markers of the M1-like (CD45+, CD11b+, CD11c-, MCHII high, F4/80+, Arg1-, CD163-, CD206-, iNOS+), M2-like (CD45+, CD11b+, CD11c-, MCHII low, F4/80+, Arg1+, CD163+, CD206+, iNOS-) TAMs with a functional assay for phagocytosis (M1-like characteristic). We are initially developing the phagocytosis assay using cultured bone marrow derived macrophages. In brief, macrophages were polarised in-vitro towards a pro tumor or an anti-tumor endotype using IFN-γ + LPS or IL-4, respectively or left as a non-polarised population (M0). We then performed a functional assay that to identify which of these populations effectively phagocytosed breast cancer cell lines. CellTracker Red dyed macrophages were cultured with CellTracker Green MCF7 and MDA-231 cancer cell lines and phagocytosis was identified by double positive populations of macrophages. We aim to optimize this assay for a small format (6 well plates) for use on mammary macrophages which are relatively low abundance in the mammary gland.
We envision this functional/phenotypic assessment of macrophages will better allow us to define the M1 and M2-like macrophage populations present in the mouse mammary gland.