M2c-like macrophage associated immunomodulatory function on CD44/CD24 phenotype in triple negative breast cancer (TNBC) subgroups

Objective: CD44+/CD24- breast cancer stem cell-like cells are the dominant subgroup of triple negative breast cancer(TNBC) but we have limited understanding of their immunomodulatory effect. This study aims to evaluate M2c-, M2- and M0-like monocyte/macrophage dependent immunofunctional difference on T cells in TNBC and their effect on CD44/CD24 associated phenotype. Material and method: THP-1 (monocytic), phorbol 12-myristate 13-acetate(PMA)-Induced THP-1(M0/macrophage),M2clike THP-1(CD169+CD163+CD206dim) cells differentiation status were confirmed by NGS and flow cytometry. MDA-MB-231 or MDA-MB-468 triple negative breast cancer (TNBC) cells co-cultured with monocyte/macrophage-like cells were purified with FACS according to their CD44/CD24 expression status. To measure these myleoid cells primed TNBC cells were cocultured with PBMCs obtained from healthy donors and CD4+ or CD8+ T cell proliferation was tested with CFSE dilution by flow cytometry. CD80,CD86,PD-L1 and PD-L2 expression on cancer cells and immune cells were tested by flow cytometry for costimulation. Purified different CD44/CD24 cell subpopulation were plated for scratch assays and analyzed with ImageJ. Results and discussion: CD44+/CD24- subpopulation collected from M2c-like THP1cells/cancer cells co-culture condition, displayed less metastatic and more immunomodulatory capacity than THP-1 and PMA-Induced THP-1 conditions. In addition,CD163,CD169 vs CD14 expression were decreased on M2c-like THP-1 cells(CD44+/CD24- cancer cells primed).CD44+CD24+ population was decreased in THP-1 derived monocytic/macrophagelike cells coculture.In co-cultures between MDA-MB-468 cell lines and THP-1 derived monocytic/macrophage-like cells, especially CD44+CD24+ population was decreased.CD4+ or CD8+ T cells proliferation were affected to monocyte/macrophage-like cells primed CD44/CD24 sub-populations.In this study,CD44/CD24 phenotype in TNBC and their effects on monocytic/macrophage cell activation polarization were evaluated.In addition, the metastatic character and T cell responses of this interaction were detected,and the first preliminary data that could help immunotherapy approaches. Keywords: CD4, CD8, T cells, CD44, CD24 Monocyte, Macrophage